Anti-CD25 antibody and medical application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an antibody specifically bound with CD25 or an antigen binding fragment thereof, a pharmaceutical preparation containing the antibody or the antigen binding fragment thereof, a nucleic acid molecule for coding the antibody or the antigen binding fragment thereof, and a carrier and a host cell for expressing the antibody or the antigen binding fragment thereof. The invention also relates to the medical application of the antibody, the antigen binding fragment thereof or the pharmaceutical preparation containing the antibody.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to an antibody or an antigen-binding fragment thereof that specifically binds to CD25 and a pharmaceutical preparation comprising the antibody or the antigen-binding fragment thereof, as well as a nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof, a vector and a host cell for expressing the antibody or the antigen-binding fragment thereof, and the medical use of the antibody, the antigen-binding fragment thereof or the pharmaceutical preparation comprising the same. Background Art

[0002] Interleukin-2 (IL-2) is a cytokine in the chemokine family. It plays an important role in the body's immune response and antiviral infection. It can stimulate the proliferation of T cells that have been activated by specific antigens or mitogens; it can activate T cells and promote cytokine production; it can stimulate NK cell proliferation, enhance NK killing activity and produce cytokines, induce LAK cell production; promote B cell proliferation and antibody secretion; and activate macrophages. The receptor of IL-2 contains three polypeptide chain subunits, namely α (CD25), β (CD122) and γ (CD132) subunits. IL2RA (Interleukin-2 receptor subunit alpha), also known as CD25 and p55, is a 55kDa type I transmembrane protein. It is mainly expressed in tissues such as the spleen and lymph nodes, and on the surface of cells such as Treg cells, basophils, and activated T cells.

[0003] Daclizumab and Basiliximab are both IgG1 antibodies that inhibit the binding of IL-2 to CD25 and are developed to reduce the activation of effector T cells (Teff). Daclizumab is approved by the FDA for the treatment of multiple sclerosis, but the drug has been withdrawn due to serious safety issues. Basiliximab is approved by the FDA for the prevention of organ transplant rejection, but has not yet been approved for the treatment of autoimmune diseases and tumors. In 2019, Sansure's humanized anti-CD25 monoclonal antibody injection, Xenipai, was launched on the market for the prevention of acute rejection caused by kidney transplantation.

[0004] The expression abundance of CD25 on regulatory T (Treg) cells is higher than that on other immune cells. Therefore, it can participate in the regulation of immune tolerance by controlling the activity of Treg cells. Treg cells can inhibit the activation and proliferation of effector T cells. The contact and consumption of Treg cells are beneficial to promoting tumor immunity. However, CD25 also mediates the activation of Teff cells. If the IL-2 signaling pathway is inhibited, Teff cells will also be inhibited, thus affecting the effect of tumor immunity.

[0005] Currently, there are no anti-CD25 antibodies for tumor treatment on the market. The anti-CD25 antibodies that have entered clinical research include the anti-CD25 antibody RG6292 jointly developed by Roche and TUSK and the non-IL-2 blocking anti-CD25 antibody BA1106 developed by Boan Bio, a subsidiary of Luye Pharma Group. Therefore, the development of anti-CD25 antibodies that specifically kill Treg cells and do not inhibit or partially inhibit the activity of Teff cells, thereby relieving immunosuppression, is still an urgent problem to be solved in this field. Summary of the invention

[0006] The present invention provides an isolated anti-CD25 antibody or an antigen-binding fragment thereof, which has good anti-tumor activity, enhanced binding ability with FcγRIIIa, and / or reduced binding ability with FcγRIIb.

[0007] The present invention provides an isolated anti-CD25 antibody or antigen-binding fragment thereof having at least one of the following characteristics:

[0008] 1) Bind to human CD25 protein epitope;

[0009] 2) have cross-immunoreactivity with monkey and / or mouse CD25;

[0010] 3) With a K of 100 nM or less D Binds to CD25 antigen;

[0011] 4) Using ELISA method to determine EC binding to CD25 50 Value ≤ 5ug / mL; preferably, EC binding to CD25 50 Value ≤1ug / mL;

[0012] 5) eliciting an enhanced CDC, ADCC and / or ADCP response; preferably, eliciting an increased ADCC response;

[0013] 6) does not inhibit or does not substantially inhibit the binding of IL-2 to CD25;

[0014] 7) Has no or little influence on the function of Teff cells;

[0015] 8) binds to FcγRIII with a higher affinity than it binds to FcγRI, FcγRIIc and / or FcγRIIb.

[0016] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises HCDR1-HCDR3 comprised by the heavy chain variable region shown in SEQ ID NO:45, 43, 44, 48, 49, 59, 61, 63, 65, 67, 69, 71, 73 and LCDR1-LCDR3 comprised by the light chain variable region shown in SEQ ID NO:47, 46, 50, 51, 52, 53, 60, 62, 64, 66, 68, 70, 72, 74 according to the Kabat, IMGT, Chothia or AbM numbering systems.

[0017] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

[0018] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein:

[0019] 1) the HCDR1 is selected from a HCDR1 having 0, 1, 2, 3, 4 or 5 amino acid differences with the amino acid sequence of any one of SEQ ID NO: 16, SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 22, SEQ ID NO: 30 or SEQ ID NO: 36,

[0020] 2) the HCDR2 is selected from a HCDR2 having 0, 1, 2, 3, 4 or 5 amino acid differences with the amino acid sequence of any one of SEQ ID NO: 17, SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 31 or SEQ ID NO: 37, and

[0021] 3) the HCDR3 is selected from a HCDR3 having 0, 1, 2, 3, 4 or 5 amino acid differences with the amino acid sequence of any one of SEQ ID NO: 18, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO: 32 or SEQ ID NO: 38;

[0022] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein:

[0023] 1) the LCDR1 is selected from LCDR1 having 0, 1, 2, 3, 4 or 5 amino acid differences with the amino acid sequence of any one of SEQ ID NO: 19, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 39 or SEQ ID NO: 42,

[0024] 2) the LCDR2 is selected from a LCDR2 having 0, 1, 2, 3, 4 or 5 amino acid differences with the amino acid sequence of any one of SEQ ID NO:20, SEQ ID NO:5, SEQ ID NO:14, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:34 or SEQ ID NO:40, and

[0025] 3) the LCDR3 is selected from a LCDR3 having 0, 1, 2, 3, 4 or 5 amino acid differences with the amino acid sequence of any one of SEQ ID NO:21, SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:35 or SEQ ID NO:41;

[0026] Preferably, the CDRs are defined according to the Kabat numbering system.

[0027] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

[0028] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein:

[0029] 1) the HCDR1 is selected from a HCDR1 having 0, 1, 2 or 3 amino acid differences with the amino acid sequence of any one of SEQ ID NO: 16, SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 22, SEQ ID NO: 30 or SEQ ID NO: 36,

[0030] 2) the HCDR2 is selected from a HCDR2 having 0, 1, 2 or 3 amino acid differences with the amino acid sequence of any one of SEQ ID NO: 17, SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 31 or SEQ ID NO: 37, and

[0031] 3) the HCDR3 is selected from a HCDR3 having 0, 1, 2 or 3 amino acid differences with the amino acid sequence of any one of SEQ ID NO: 18, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO: 32 or SEQ ID NO: 38;

[0032] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein:

[0033] 1) the LCDR1 is selected from LCDR1 having 0, 1, 2 or 3 amino acid differences with the amino acid sequence of any one of SEQ ID NO: 19, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 39 or SEQ ID NO: 42,

[0034] 2) the LCDR2 is selected from a LCDR2 having 0, 1, 2 or 3 amino acid differences with the amino acid sequence of any one of SEQ ID NO:20, SEQ ID NO:5, SEQ ID NO:14, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:34 or SEQ ID NO:40, and

[0035] 3) the LCDR3 is selected from a LCDR3 having 0, 1, 2 or 3 amino acid differences with the amino acid sequence of any one of SEQ ID NO:21, SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:35 or SEQ ID NO:41;

[0036] Preferably, the CDRs are defined according to the Kabat numbering system.

[0037] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

[0038] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein:

[0039] 1) the HCDR1 is selected from the HCDR1 shown in SEQ ID NO: 16, SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 22, SEQ ID NO: 30 or SEQ ID NO: 36,

[0040] 2) the HCDR2 is selected from the HCDR2 shown in SEQ ID NO: 17, SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 31 or SEQ ID NO: 37, and

[0041] 3) the HCDR3 is selected from the HCDR3 shown in SEQ ID NO: 18, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO: 32 or SEQ ID NO: 38;

[0042] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein:

[0043] 1) the LCDR1 is selected from the LCDR1 shown in SEQ ID NO: 19, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 39 or SEQ ID NO: 42, or the LCDR1 shown below: the 5th amino acid in SEQ ID NO: 33 is replaced by S, and / or the 7th amino acid is replaced by G, and / or the 9th amino acid is replaced by D,

[0044] 2) the LCDR2 is selected from the group consisting of the LCDR2 shown in SEQ ID NO:20, SEQ ID NO:5, SEQ ID NO:14, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:34 or SEQ ID NO:40, or the LCDR2 shown below: the first amino acid in SEQ ID NO:34 is replaced by F, and / or the fourth amino acid is replaced by N, and

[0045] 3) the LCDR3 is selected from the LCDR3 shown in SEQ ID NO:21, SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:35 or SEQ ID NO:41;

[0046] Preferably, the CDRs are defined according to the Kabat numbering system.

[0047] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof, wherein:

[0048] 1) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein:

[0049] The HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively,

[0050] The HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively,

[0051] The HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively,

[0052] The HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively,

[0053] The HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, respectively,

[0054] The HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, respectively, or

[0055] The HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, respectively;

[0056] and 2) the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein:

[0057] The LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively,

[0058] The LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively,

[0059] The LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively,

[0060] The LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 25 and SEQ ID NO: 26, respectively,

[0061] The LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29, respectively,

[0062] The LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, respectively,

[0063] The LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:35, respectively,

[0064] The LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:41, respectively, or

[0065] The LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:42, SEQ ID NO:34 and SEQ ID NO:41, respectively.

[0066] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof, wherein:

[0067] 1) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively,

[0068] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively;

[0069] 2) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively,

[0070] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively;

[0071] 3) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively,

[0072] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively;

[0073] 4) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively,

[0074] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively;

[0075] 5) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, respectively,

[0076] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 25 and SEQ ID NO: 26, respectively;

[0077] 6) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, respectively,

[0078] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29, respectively;

[0079] 7) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, respectively,

[0080] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, respectively;

[0081] 8) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, respectively,

[0082] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:35, respectively;

[0083] 9) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, respectively,

[0084] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:41, respectively; or

[0085] 10) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, respectively,

[0086] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:42, SEQ ID NO:34 and SEQ ID NO:41, respectively.

[0087] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof, wherein:

[0088] 1) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 16, HCDR2 shown in SEQ ID NO: 17, and HCDR3 shown in SEQ ID NO: 18,

[0089] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 20, and LCDR3 shown in SEQ ID NO: 21;

[0090] 2) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO: 3,

[0091] The light chain variable region comprises LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6;

[0092] 3) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8 and HCDR3 shown in SEQ ID NO:9,

[0093] The light chain variable region comprises LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6;

[0094] 4) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 10, HCDR2 shown in SEQ ID NO: 11, and HCDR3 shown in SEQ ID NO: 12,

[0095] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 13, LCDR2 shown in SEQ ID NO: 14, and LCDR3 shown in SEQ ID NO: 15;

[0096] 5) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 22, HCDR2 shown in SEQ ID NO: 23, and HCDR3 shown in SEQ ID NO: 24,

[0097] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 13, LCDR2 shown in SEQ ID NO: 25, and LCDR3 shown in SEQ ID NO: 26;

[0098] 6) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 22, HCDR2 shown in SEQ ID NO: 23, and HCDR3 shown in SEQ ID NO: 24,

[0099] The light chain variable region comprises LCDR1 shown in SEQ ID NO:27, LCDR2 shown in SEQ ID NO:28, and LCDR3 shown in SEQ ID NO:29;

[0100] 7) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 30, HCDR2 shown in SEQ ID NO: 31, and HCDR3 shown in SEQ ID NO: 32,

[0101] The light chain variable region comprises LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:35;

[0102] 8) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 36, HCDR2 shown in SEQ ID NO: 37, and HCDR3 shown in SEQ ID NO: 38,

[0103] The light chain variable region comprises LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:40, and LCDR3 shown in SEQ ID NO:35;

[0104] 9) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 30, HCDR2 shown in SEQ ID NO: 31, and HCDR3 shown in SEQ ID NO: 32,

[0105] The light chain variable region comprises LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:41;

[0106] 10) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 30, HCDR2 shown in SEQ ID NO: 31, and HCDR3 shown in SEQ ID NO: 32,

[0107] The light chain variable region comprises LCDR1 shown in SEQ ID NO:42, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:41; or

[0108] 11) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 30, HCDR2 shown in SEQ ID NO: 31, and HCDR3 shown in SEQ ID NO: 32,

[0109] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 is selected from the sequence of SEQ ID NO: 33 or the 5th amino acid in the sequence of SEQ ID NO: 33 is replaced by S, and / or the 7th amino acid is replaced by G, and / or the 9th amino acid is replaced by D;

[0110] The LCDR2 is selected from the sequence of SEQ ID NO: 34 or the first amino acid in the sequence of SEQ ID NO: 34 is replaced by F, and / or the fourth amino acid is replaced by N;

[0111] The LCDR3 is selected from the sequence SEQ ID NO: 35 or the 5th amino acid in SEQ ID NO: 35 is replaced by S.

[0112] In certain embodiments, in certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof, wherein:

[0113] 1) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively,

[0114] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively;

[0115] 2) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, respectively,

[0116] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, respectively; or

[0117] 3) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, respectively,

[0118] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:42, SEQ ID NO:34 and SEQ ID NO:41, respectively;

[0119] Preferably, the CDRs are defined according to the Kabat numbering system.

[0120] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively,

[0121] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively.

[0122] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, respectively,

[0123] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, respectively.

[0124] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, respectively,

[0125] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:42, SEQ ID NO:34 and SEQ ID NO:41, respectively.

[0126] In certain embodiments, in certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof, wherein:

[0127] 1) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 16, HCDR2 shown in SEQ ID NO: 17, and HCDR3 shown in SEQ ID NO: 18,

[0128] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 20, and LCDR3 shown in SEQ ID NO: 21; or

[0129] 2) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 30, HCDR2 shown in SEQ ID NO: 31, and HCDR3 shown in SEQ ID NO: 32,

[0130] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 is selected from the sequence of SEQ ID NO: 33 or the 5th amino acid in the sequence of SEQ ID NO: 33 is replaced by S, and / or the 7th amino acid is replaced by G, and / or the 9th amino acid is replaced by D;

[0131] The LCDR2 is selected from the sequence of SEQ ID NO: 34 or the first amino acid in the sequence of SEQ ID NO: 34 is replaced by F, and / or the fourth amino acid is replaced by N;

[0132] The LCDR3 is selected from the sequence SEQ ID NO: 35 or the 5th amino acid in SEQ ID NO: 35 is replaced by S;

[0133] Preferably, the CDRs are defined according to the Kabat numbering system.

[0134] In certain embodiments, in certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof, wherein:

[0135] 1) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 16, HCDR2 shown in SEQ ID NO: 17, and HCDR3 shown in SEQ ID NO: 18,

[0136] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 20, and LCDR3 shown in SEQ ID NO: 21;

[0137] 2) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 30, HCDR2 shown in SEQ ID NO: 31, and HCDR3 shown in SEQ ID NO: 32,

[0138] The light chain variable region comprises LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:35; or

[0139] 3) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 30, HCDR2 shown in SEQ ID NO: 31, and HCDR3 shown in SEQ ID NO: 32,

[0140] The light chain variable region comprises LCDR1 shown in SEQ ID NO:42, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:41;

[0141] Preferably, the CDRs are defined according to the Kabat numbering system.

[0142] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 16, HCDR2 shown in SEQ ID NO: 17, and HCDR3 shown in SEQ ID NO: 18,

[0143] The light chain variable region comprises LCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:20, and LCDR3 shown in SEQ ID NO:21.

[0144] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 30, HCDR2 shown in SEQ ID NO: 31, and HCDR3 shown in SEQ ID NO: 32,

[0145] The light chain variable region comprises LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:35.

[0146] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 30, HCDR2 shown in SEQ ID NO: 31, and HCDR3 shown in SEQ ID NO: 32,

[0147] The light chain variable region comprises LCDR1 shown in SEQ ID NO:42, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:41.

[0148] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 30, HCDR2 shown in SEQ ID NO: 31, and HCDR3 shown in SEQ ID NO: 32,

[0149] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 is selected from the sequence of SEQ ID NO: 33 or the 5th amino acid in the sequence of SEQ ID NO: 33 is replaced by S, and / or the 7th amino acid is replaced by G, and / or the 9th amino acid is replaced by D;

[0150] The LCDR2 is selected from the sequence of SEQ ID NO: 34 or the first amino acid in the sequence of SEQ ID NO: 34 is replaced by F, and / or the fourth amino acid is replaced by N;

[0151] The LCDR3 is selected from the sequence SEQ ID NO: 35 or the 5th amino acid in SEQ ID NO: 35 is replaced by S.

[0152] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof, wherein:

[0153] 1) The amino acid sequence of the heavy chain variable region

[0154] as set forth in SEQ ID NO:45 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0155] as set forth in SEQ ID NO:43 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0156] as set forth in SEQ ID NO:44 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0157] as set forth in SEQ ID NO:48 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0158] as set forth in SEQ ID NO:49 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0159] as set forth in SEQ ID NO:59 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0160] as set forth in SEQ ID NO:61 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0161] as set forth in SEQ ID NO:63 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0162] as set forth in SEQ ID NO:65 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0163] as set forth in SEQ ID NO:67 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0164] as set forth in SEQ ID NO:69 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0165] as set forth in SEQ ID NO:71 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, or

[0166] as set forth in SEQ ID NO:73 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto;

[0167] and 2) the amino acid sequence of the light chain variable region

[0168] as set forth in SEQ ID NO:47 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0169] as set forth in SEQ ID NO:46 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0170] as set forth in SEQ ID NO:50 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0171] as set forth in SEQ ID NO:51 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0172] as set forth in SEQ ID NO:52 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0173] as set forth in SEQ ID NO:53 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0174] as set forth in SEQ ID NO:60 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0175] as set forth in SEQ ID NO:62 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0176] as set forth in SEQ ID NO:64 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0177] as set forth in SEQ ID NO:66 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0178] as set forth in SEQ ID NO:68 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0179] as set forth in SEQ ID NO:70 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0180] as set forth in SEQ ID NO:72 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, or

[0181] As set forth in SEQ ID NO:74 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto.

[0182] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof, wherein:

[0183] 1) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:45 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto;

[0184] The amino acid sequence of the light chain variable region is as shown in SEQ ID NO:47 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto;

[0185] 2) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:49 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto;

[0186] The amino acid sequence of the light chain variable region is as shown in SEQ ID NO:51 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto; or

[0187] 3) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:49 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto;

[0188] The amino acid sequence of the light chain variable region is as shown in SEQ ID NO:53 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto.

[0189] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region, wherein the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:75 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto;

[0190] The amino acid sequence of the light chain constant region is as shown in SEQ ID NO:76 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto.

[0191] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region, wherein the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:75, or has 1, 2, 3, 4 or 5 amino acid differences from the amino acid sequence shown in SEQ ID NO:75;

[0192] The amino acid sequence of the light chain constant region is as shown in SEQ ID NO:76, or has 1, 2, 3, 4 or 5 amino acid differences from the amino acid sequence shown in SEQ ID NO:76.

[0193] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region, wherein the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:75; and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:76.

[0194] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises an antibody heavy chain and an antibody light chain, wherein:

[0195] 1) the amino acid sequence of the antibody heavy chain,

[0196] as set forth in SEQ ID NO:54 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, or

[0197] as set forth in SEQ ID NO:56 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto;

[0198] and 2) the amino acid sequence of the antibody light chain,

[0199] as set forth in SEQ ID NO:55 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto,

[0200] as set forth in SEQ ID NO:57 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, or

[0201] As set forth in SEQ ID NO:58, or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto.

[0202] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises an antibody heavy chain and an antibody light chain, wherein:

[0203] 1) the amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO:54 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto;

[0204] The amino acid sequence of the antibody light chain is as shown in SEQ ID NO:55 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto;

[0205] 2) the amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO:56 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto;

[0206] The amino acid sequence of the antibody light chain is as shown in SEQ ID NO:57 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto; or

[0207] 3) the amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO:56 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto;

[0208] The amino acid sequence of the antibody light chain is as shown in SEQ ID NO:58 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto.

[0209] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises an antibody heavy chain and an antibody light chain, wherein:

[0210] The amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO:54 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto;

[0211] The amino acid sequence of the antibody light chain is as shown in SEQ ID NO:55 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto.

[0212] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises an antibody heavy chain and an antibody light chain, wherein:

[0213] The amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO:56 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto;

[0214] The amino acid sequence of the antibody light chain is as shown in SEQ ID NO:57 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto.

[0215] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises an antibody heavy chain and an antibody light chain, wherein:

[0216] The amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO:56 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto;

[0217] The amino acid sequence of the antibody light chain is as shown in SEQ ID NO:58 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto.

[0218] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises an antibody heavy chain and an antibody light chain, wherein:

[0219] 1) the amino acid sequence of the antibody heavy chain,

[0220] as shown in SEQ ID NO:54 or having 1, 2, 3, 4 or 5 amino acid differences therefrom, or

[0221] As shown in SEQ ID NO:56 or having 1, 2, 3, 4 or 5 amino acid differences therefrom;

[0222] and 2) the amino acid sequence of the antibody light chain,

[0223] As shown in SEQ ID NO:55 or having 1, 2, 3, 4 or 5 amino acid differences therefrom,

[0224] as shown in SEQ ID NO:57 or having 1, 2, 3, 4 or 5 amino acid differences therefrom, or

[0225] As shown in SEQ ID NO:58 or having 1, 2, 3, 4 or 5 amino acid differences therefrom.

[0226] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises an antibody heavy chain and an antibody light chain, wherein:

[0227] 1) The amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO: 54 or has 1, 2, 3, 4 or 5 amino acid differences therefrom;

[0228] The amino acid sequence of the antibody light chain is as shown in SEQ ID NO:55 or has 1, 2, 3, 4 or 5 amino acid differences therefrom;

[0229] 2) the amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO:56 or has 1, 2, 3, 4 or 5 amino acid differences therefrom;

[0230] The amino acid sequence of the antibody light chain is as shown in SEQ ID NO:57 or has 1, 2, 3, 4 or 5 amino acid differences therefrom; or

[0231] 3) the amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO:56 or has 1, 2, 3, 4 or 5 amino acid differences therefrom;

[0232] The amino acid sequence of the antibody light chain is as shown in SEQ ID NO: 58 or has 1, 2, 3, 4 or 5 amino acid differences therefrom.

[0233] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises an antibody heavy chain and an antibody light chain, wherein:

[0234] The amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO:54 or has 1, 2, 3, 4 or 5 amino acid differences therefrom;

[0235] The amino acid sequence of the antibody light chain is as shown in SEQ ID NO: 55 or has 1, 2, 3, 4 or 5 amino acid differences therefrom.

[0236] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises an antibody heavy chain and an antibody light chain, wherein:

[0237] The amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO:56 or has 1, 2, 3, 4 or 5 amino acid differences therefrom;

[0238] The amino acid sequence of the antibody light chain is as shown in SEQ ID NO: 57 or has 1, 2, 3, 4 or 5 amino acid differences therefrom.

[0239] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises an antibody heavy chain and an antibody light chain, wherein:

[0240] The amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO:56 or has 1, 2, 3, 4 or 5 amino acid differences therefrom;

[0241] The amino acid sequence of the antibody light chain is as shown in SEQ ID NO: 58 or has 1, 2, 3, 4 or 5 amino acid differences therefrom.

[0242] In certain embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises an antibody heavy chain and an antibody light chain, wherein:

[0243] 1) The amino acid sequence of the antibody heavy chain is shown in SEQ ID NO: 54, and the amino acid sequence of the antibody light chain is shown in SEQ ID NO: 55;

[0244] 2) the amino acid sequence of the antibody heavy chain is shown in SEQ ID NO: 56, and the amino acid sequence of the antibody light chain is shown in SEQ ID NO: 57; or

[0245] 3) The amino acid sequence of the antibody heavy chain is shown in SEQ ID NO:56, and the amino acid sequence of the antibody light chain is shown in SEQ ID NO:58.

[0246] In certain embodiments, the anti-CD25 antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody; the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, scFv or sdAb; the anti-CD25 antibody is selected as an IgG antibody, selected from IgG1, IgG2, IgG2 or IgG4.

[0247] In certain embodiments, the anti-CD25 antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody; the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, scFv or sdAb; and the anti-CD25 antibody is selected from IgG1 or IgG4.

[0248] In certain embodiments, the anti-CD25 antibody is selected from a humanized antibody or a fully human antibody.

[0249] In certain embodiments, the anti-CD25 antibody is selected as an IgG1 or IgG4 antibody.

[0250] In certain embodiments, the anti-CD25 antibody is selected as an IgG1 type antibody.

[0251] In certain embodiments, the anti-CD25 antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, scFv or sdAb.

[0252] In certain embodiments, the anti-CD25 antibody has a defucosylation modification in the Fc region to enhance the binding ability to FcγRIIIa and / or reduce the binding ability to FcγRIIb.

[0253] The present invention provides a polynucleotide molecule encoding the anti-CD25 antibody or antigen-binding fragment thereof described in any one of the above schemes.

[0254] The present invention also provides an expression vector, which comprises the polynucleotide molecule described in any of the above schemes.

[0255] In certain embodiments, the expression vector is a eukaryotic expression vector.

[0256] The present invention also provides a host cell or an engineered cell, which comprises the polynucleotide molecule described in any of the above schemes or the expression vector described in any of the above schemes.

[0257] In certain embodiments, the host cell or engineered cell is selected from bacteria, yeast or mammalian cells.

[0258] In certain embodiments, the host cell or engineered cell is selected from Escherichia coli, Pichia pastoris, Chinese hamster ovary cells or human embryonic kidney 293 cells.

[0259] The present invention also provides a pharmaceutical preparation, which comprises the anti-CD25 antibody or antigen-binding fragment thereof described in any of the above schemes, the polynucleotide molecule described in any of the above schemes, and one or more pharmaceutically acceptable carriers or excipients.

[0260] The present invention provides a pharmaceutical preparation, which comprises the expression vector described in any of the above schemes or the host cell or engineered cell described in any of the above schemes, and one or more pharmaceutically acceptable carriers or excipients.

[0261] In certain embodiments, the pharmaceutical preparation further comprises a second active ingredient; the second active ingredient is selected from a chemical anticancer agent, an anti-tumor antigen or marker antibody, an immune checkpoint inhibitor or an immunomodulatory antibody.

[0262] In certain embodiments, the second active ingredient is selected from an immune checkpoint inhibitor or an immunomodulatory antibody.

[0263] In certain embodiments, the second active ingredient is selected from PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, TIGIT antibody, TIM-3 antibody, B7-H3 antibody, CD73 antibody, LAG3 antibody, CD27 antibody, CD70 antibody, 4-1BB antibody, GITR antibody, OX40 antibody, CD47 antibody, CD39 antibody, ILDR2 antibody, VISTA antibody, BTLA antibody or VTCN-1 antibody.

[0264] In certain embodiments, the second active ingredient is selected from a PD-1 antibody, a PD-L1 antibody, or a CTLA-4 antibody.

[0265] The present invention also provides a pharmaceutical composition, comprising the anti-CD25 antibody or antigen-binding fragment thereof described in any of the above schemes, the polynucleotide molecule described in any of the above schemes, the expression vector described in any of the above schemes, or the host cell or engineered cell described in any of the above schemes, and one or more second active ingredients.

[0266] In certain embodiments, the second active ingredient is selected from a chemical anticancer agent, an anti-tumor antigen or marker antibody, an immune checkpoint inhibitor, or an immunomodulatory antibody.

[0267] In certain embodiments, the second active ingredient is selected from an immune checkpoint inhibitor or an immunomodulatory antibody.

[0268] In certain embodiments, the second active ingredient is selected from PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, TIGIT antibody, TIM-3 antibody, B7-H3 antibody, CD73 antibody, LAG3 antibody, CD27 antibody, CD70 antibody, 4-1BB antibody, GITR antibody, OX40 antibody, CD47 antibody, CD39 antibody, ILDR2 antibody, VISTA antibody, BTLA antibody or VTCN-1 antibody.

[0269] In certain embodiments, the second active ingredient is selected from a PD-1 antibody, a PD-L1 antibody, or a CTLA-4 antibody.

[0270] The present invention also provides a use of the anti-CD25 antibody or antigen-binding fragment thereof described in any of the above schemes, the polynucleotide molecule described in any of the above schemes, and the pharmaceutical preparation described in any of the above schemes in the preparation of a drug for treating and / or preventing CD25-mediated diseases or conditions.

[0271] The present invention also provides use of the expression vector described in any of the above schemes, the host cell or engineered cell described in any of the above schemes, and the pharmaceutical composition described in any of the above schemes in the preparation of a drug for treating and / or preventing CD25-mediated diseases or conditions.

[0272] In certain embodiments, the disease or disorder is an immune disease or cancer.

[0273] In certain embodiments, the cancer is selected from squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, liver cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, leukemia, colorectal cancer, gastrointestinal cancer, kidney cancer, ovarian cancer, endometrial cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, breast cancer, and head and neck cancer.

[0274] The present invention also provides a kit, which comprises the anti-CD25 antibody or antigen-binding fragment thereof described in any of the above schemes, the polynucleotide molecule described in any of the above schemes, the expression vector described in any of the above schemes, the host cell or engineered cell described in any of the above schemes, the pharmaceutical preparation described in any of the above schemes, or the pharmaceutical composition described in any of the above schemes.

[0275] The relevant sequences of some anti-CD25 antibodies of the present invention are shown in Table 1:

[0276] Table 1 Heavy chain / light chain CDR region sequences

[0277] Sequence number Amino acid sequence Sequence number Amino acid sequence SEQ ID NO:1 TSGMGVG SEQ ID NO:22 SDYAWN SEQ ID NO:2 HIWWNDDKYYNPSLKS SEQ ID NO:23 YISYSGTTTYNPSLKS SEQ ID NO:3 GAYDFFDY SEQ ID NO:24 KTFFDY SEQ ID NO:4 RASESVDNYGISFMN SEQ ID NO:25 NAKTLAE SEQ ID NO:5 AASNQGS SEQ ID NO:26 QHHYDIPYT SEQ ID NO:6 QQSKEVPWT SEQ ID NO:27 SAGQDIRNYLN SEQ ID NO:7 SYWMS SEQ ID NO:28 YSSNLHS SEQ ID NO:8 EIRLKSDNYATHYAESVKG SEQ ID NO:29 QQFIKFPLT SEQ ID NO:9 VYSFPY SEQ ID NO:30 SYWIE SEQ ID NO:10 DYALH SEQ ID NO:31 EILPGSGSTNYNEKFKG SEQ ID NO:11 VISTYSGDTNYNQKFKG SEQ ID NO:32 GGVSSSIFDY SEQ ID NO:12 GTVFDY SEQ ID NO:33 KASQRVNNNVA SEQ ID NO:13 RASENIYSFLE SEQ ID NO:34 YASDRYT SEQ ID NO:14 NAKTLAK SEQ ID NO:35 QQDYNSPYT SEQ ID NO:15 QHHYDTPYT SEQ ID NO:36 TYWIE SEQ ID NO:16 SFGMH SEQ ID NO:37 EILPGRGSTNYNEKFKG SEQ ID NO:17 YISSGSSTIYYADTVKG SEQ ID NO:38 GGGSSSIFDY SEQ ID NO:18 WNWYFDY SEQ ID NO:39 KASQSVGNDVA SEQ ID NO:19 KSSQSLLYSSSQRNHLA SEQ ID NO:40 FASNRYT SEQ ID NO:20 WASTGES SEQ ID NO:41 QQDYSSPYT SEQ ID NO:21 QQYYSYPWT SEQ ID NO:42 KASQRVNANVA

[0278] In the present invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art, but in order to better understand the present invention, the definitions of some terms are provided below. When the definitions and explanations of the terms provided by the present invention are inconsistent with the meanings commonly understood by those skilled in the art, the definitions and explanations of the terms provided by the present invention shall prevail. The abbreviations of amino acids (including residues) are standard 3-letter and / or 1-letter codes used in the art to refer to one of 20 commonly used L-amino acids. The singular form used in the present invention also includes its corresponding plural form, unless otherwise clearly stated in the text.

[0279] "Antibody" refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected 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. Based on this, immunoglobulins can be divided into five categories, or 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 type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain by the difference in the constant region. Each of the five types of Ig can have κ chain or λ chain. The sequence of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain varies greatly, which is the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable, which is the constant region (C region). The variable region includes three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). The three hypervariable regions determine the specificity of the antibody and are also called complementarity determining regions (CDRs).

[0280] "CD25" or "CD25 protein" or "CD25 polypeptide" may optionally include any such protein or variant, conjugate or fragment thereof, including but not limited to known or wild-type CD25 as described herein, as well as any naturally occurring splice variant, amino acid variant or isoform.

[0281] "Binding to CD25" or "binding to CD25 protein" means being able to interact with CD25 or an epitope thereof, and the CD25 or an epitope thereof may be of human origin.

[0282] "Antibodies that bind to CD25" or "anti-CD25 antibodies" refer to antibodies that are able to bind to the CD25 subunit of the IL-2 receptor. This subunit is also known as the α subunit of the IL-2 receptor.

[0283] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art.

[0284] "Antigenic epitope" refers to a site on an antigen molecule that can specifically bind to an immunoglobulin or antibody (including antibodies or antigen-binding fragments disclosed herein) binding site and determine antigen specificity. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed by adjacent amino acids are usually retained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with a denaturing solvent. An epitope usually includes at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis, etc. 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, etc.

[0285] For the determination or definition of "CDR", the deterministic depiction of CDR and the identification of residues comprising the binding site of the antibody can be completed by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be achieved by any of the various techniques known to those skilled 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, the contact definition, and the conformational definition. The CDRs of the antibodies or antibody binding fragments thereof described in the present invention can be defined according to any of the above methods. In the embodiments of the present disclosure, the Kabat numbering rules are used to define CDRs, but it will be appreciated by those skilled in the art that CDRs can also be redefined according to any of the Chothia, extended, AbM, IMGT, contact and / or conformational definitions.

[0286] Sequence "homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if every position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The homology percentage between the 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, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. In general, comparison is performed when the two sequences are aligned to obtain the maximum homology percentage.

[0287] "Reversion mutation" refers to a mutant that completely or partially recovers to its original genotype and phenotype after a second mutation. Complete recovery is due to the fact that the mutated base sequence returns to the original base sequence after the second mutation, so it is also called a true return mutation; partial recovery is due to the fact that the second mutation occurs at another site, resulting in partial recovery of the original phenotype. The "reversion mutation" described in the present disclosure includes complete and / or partial recovery.

[0288] The antibodies disclosed herein may be polyclonal, monoclonal, xenogeneic, allogeneic, isogenic or modified forms thereof, wherein monoclonal antibodies are particularly suitable for use in a number of embodiments. In general, the antibodies disclosed herein are recombinant antibodies. As used herein, "recombinant" refers generally to products such as cells or nucleic acids, proteins or vectors, indicating that the cells, nucleic acids, proteins or vectors have been modified by the introduction of heterologous nucleic acids or proteins or by altering natural nucleic acids or proteins, or that the cells are derived from cells so modified. For example, recombinant cells express genes that are not present in the natural (non-recombinant) cell form or express natural genes that are abnormally expressed, underexpressed or not expressed at all.

[0289] "Monoclonal antibody" or "monoclonal antibody" refers to an antibody obtained from a single clonal cell line, which is not limited to a eukaryotic, prokaryotic or phage clonal cell line. Monoclonal antibodies or antigen-binding fragments can be obtained by recombinant techniques such as hybridoma technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other existing technologies.

[0290] "Mouse antibody" or "hybridoma antibody" in the present disclosure is a mouse monoclonal antibody against human CD25 or its epitope prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with CD25 antigen, and then the hybridoma expressing the antibody with the desired sequence or functional properties is isolated. In a specific embodiment of the present disclosure, the mouse anti-human CD25 antibody or its antigen-binding fragment may further comprise a light chain constant region of a mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a mouse IgG1, IgG2, IgG3 or IgG4 or a variant thereof.

[0291] "Chimeric antibodies" are antibodies formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by mouse antibodies. To establish chimeric antibodies, it is necessary to select a hybridoma that secretes mouse-specific monoclonal antibodies, then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of human antibodies as needed, connect the mouse variable region genes with the human constant region genes into chimeric genes, insert them into an expression vector, and finally express the chimeric antibody molecules in a eukaryotic or prokaryotic industrial system. The constant region of a human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or their variants.

[0292] "Humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting the CDR sequence of a mouse into the framework of the variable region of a human antibody. The strong immune response induced by chimeric antibodies due to carrying a large amount of mouse protein components can be overcome. In order to avoid a decrease in activity while reducing immunogenicity, the human antibody variable region can be subjected to minimal reverse mutations to maintain activity. The antibody disclosed herein may be an affinity-matured humanized antibody, and the CDRs of the affinity-matured antibody parent sequence (including all sequences) are at least 80% identical, for example, 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 improved affinity of the antibody for CD25 compared to a parent antibody without the altered amino acids.

[0293] "Human antibodies" include antibodies having variable and constant regions of human germline immunoglobulin sequences. Human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "human antibodies" do not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (i.e., "humanized antibodies").

[0294] "Isolated antibody" refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium. The term "isolated" does not imply the complete absence of such substances or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compound described herein.

[0295] "Antigen-binding fragments" of antibodies ("parent antibodies") include fragments or derivatives of antibodies, typically including at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH or VL or VHH); bivalent or trivalent or tetravalent antibodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments. When the binding activity of the antigen is expressed on a molar concentration basis, the binding fragment or derivative generally retains at least 10% of its antigen-binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen-binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as "conservative variants" or "functional conservative variants" of antibodies). In some embodiments, the anti-CD25 antibodies of the present disclosure encompass bi- and multi-specific antibodies.

[0296] "Single-chain Fv" or "scFv" antibodies refer to antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide generally also comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0297] An "isotype" antibody refers to the class of antibody provided by the heavy chain constant region genes (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where the modifications have been made to alter Fc function, such as to enhance or reduce effector function or binding to Fc receptors.

[0298] "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0299] "Antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refers to a cell-mediated immune defense in which immune system effector cells actively lyse target cells, such as cancer cells, that have antibody-bound cell surface antigens.

[0300] "Complement dependent cytotoxicity" or "CDC" refers to the effector function of IgG and IgM antibodies, which, when bound to surface antigens, initiate the classical complement pathway, including the formation of a membrane attack complex and target cell lysis. The antibodies of the present invention, when bound to CD25, initiate CDC against cancer cells.

[0301] "Defucosylation" of an antibody can be performed by: (1) producing the defucosylated antibody in a host cell with a fucosylation defect (such as a knockout of the FUT8 gene) (Yamane-Ohnuki N et al., Biotechnol Bioeng. 87(5):614-22, 2004); or (2) having S239D, I332E, A330L substitutions (Kabat numbering) or any or all combinations of these variations in the Fc domain of the antibody (Lazar GA et al., Proc Natl Acad Sci US A. 103(11):4005-4010, 2006).

[0302] 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), as well as one inhibitory receptor, FcγRIIb (CD32b). The communication of IgG antibodies with the immune system is controlled and mediated by FcγRs, which transmit information sensed and collected by the antibodies to the immune system, thereby providing a link between the innate and adaptive immune systems, especially in the context of biological treatments (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 elicit a range of functional responses. For example, in humans, FcγRIIIa is the major 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 effectively deplete Tregs as well.

[0303] "Nucleic acid", "polynucleotide", "nucleic acid molecule" and "polynucleotide molecule" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless expressly limited, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Pat. No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0304] "Expression vector" refers to a nucleic acid molecule that can replicate and express a target gene when transformed, transfected or transduced into a host cell. An expression vector comprises one or more phenotypic selection markers and a replication origin to ensure that the vector is maintained and to provide amplification in the host when necessary. One type of expression vector is a "plasmid", which refers to a circular double-stranded DNA loop into which an additional DNA segment can be connected. Another type of expression vector is a viral vector, in which an additional DNA segment can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and free mammalian vectors with bacterial replication origins). After being introduced into the host cell, other vectors (e.g., non-free mammalian vectors) are integrated into the genome of the host cell, and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively connected genes.

[0305] "Host cell" refers to any type of cell system that can be engineered to produce the antibodies disclosed herein. Host cells are selected from prokaryotic cells, yeast or mammalian cells. Prokaryotes include true bacteria, such as Gram-negative and Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as S. typhimurium, himurium), Serratia such as Serratia marcescens and Shigella, and Bacilli such as Bacillus subtilis and Bacillus licheniformis, Pseudomonas such as Pseudomonas aeruginosa and Streptomyces. An exemplary E. coli cloning host is E. coli 294 (ATCC 31,446), but other strains such as E. coli B, E. coli X1776 (ATCC 31,537) and E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative, not restrictive.

[0306] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody encoding vectors. Saccharomyces cerevisiae is the common baker's yeast and the most commonly used yeast among lower eukaryotic host microorganisms. However, many other genera, species and strains are also common and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as, for example, K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida: Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as S. occidentalis, and filamentous fungi, such as, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0307] Examples of mammalian host cell lines that can be used are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or subcloned 293 cells for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL1442); human lung cells (W138, ATCC CCL 75); human hepatoma cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells.

[0308] "1, 2, 3, 4 or 5 amino acid differences" refers to substitution, addition or subtraction of 1, 2, 3, 4 or 5 amino acids based on the original sequence.

[0309] "Cross-reactivity" or "cross-immunoreactivity" refers to the ability of an antibody of the invention to bind to CD25 from a different species. For example, an antibody of the invention that binds to human CD25 may also bind to CD25 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (e.g., SPR and ELISA), or binding or functional interaction with cells that physiologically express CD25. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance analysis, or flow cytometry.

[0310] "Administering," "applying," and "treating" when applied to an animal, a human, an experimental subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administering," "applying," and "treating" may refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "applying," and "treating" also mean in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnosis, a combination composition, or by another cell. "Treatment," when applied to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0311] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments thereof of the present invention or a conjugate thereof, to a subject who has, is suspected of having, or is prone to having one or more diseases or symptoms thereof, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent. Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms. Although the embodiments of the present invention (e.g., treatment methods or products) may be ineffective in alleviating the symptoms of the target disease in a subject, it should alleviate the target disease symptoms in a statistically significant number of subjects according to any statistical test method known in the art, such as Student t test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0312] The "effective amount" of the present invention refers to the dosage of the drug that can prevent, alleviate, delay, inhibit or cure the subject's disease. The size of the dosage is related to the drug administration method, the pharmacokinetics of the drug, the severity of the disease, the individual characteristics of the subject (gender, weight, height, age), etc.

[0313] "Prevention" herein refers to delaying or preventing the onset of symptoms of cancer. Prevention may be absolute (whereby the disease does not occur) or effective only in certain individuals or for a limited period of time.

[0314] "Cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or unintentional mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

[0315] Advantageous Effects of the Invention

[0316] 1. The anti-CD25 antibody of the present invention has a high affinity with human CD25 protein, K D Value less than 5×10 -7 M.

[0317] 2. The anti-CD25 antibody of the present invention does not inhibit or substantially does not inhibit the binding of IL-2 to CD25, and does not affect or substantially does not affect the function of Teff cells.

[0318] 3. The compounds of the present invention have good tumor inhibitory activity and can induce enhanced ADCC, CDC and / or ADCP reactions.

[0319] 4. The anti-CD25 antibodies of the present invention bind to FcγRIIIa with a higher affinity than to FcγRI, FcγRIIc and / or FcγRIIb. BRIEF DESCRIPTION OF THE DRAWINGS

[0320] Figure 1 : The inhibitory activity of antibodies on the interleukin 2 (IL-2) signaling pathway. KA-1292 is an analog of the control antibody RG6292, whose constant region sequence is derived from human wild-type IgG1, and the applicant prepared it by itself according to its patent (US10745485B2); KA-1329 is an analog of the control antibody Daclizumab (Daclizumab, trade name ZINBRYTA, is a marketed drug, and its sequence was obtained from the IMGT website), and the sample was prepared by the applicant.

[0321] Figures 2A - 2C : Binding data of humanized anti-CD25 antibody to Treg cells and Teff cells. Figure 2A Binding data for humanized antibody hu-KD-143-v3; Figure 2B Binding data for humanized antibody hu-KD-161-v2; Figure 2C Binding data for humanized antibody hu-KD-161-v4.

[0322] Figure 3 : ADCC activity of humanized anti-CD25 antibodies. hu-KD-143-v3-aFU, hu-KD-161-v2-aFU and hu-KD-161-v4-aFU are defucosylated hu-KD-143-v3, hu-KD-161-v2 and hu-KD-161-v4, respectively.

[0323] Figure 4 : Tumor suppressor activity of antibodies hu-KD-0143-v3, hu-KD-0161-v4, and hu-KD-0161-v2 in the MC38 mouse model.

[0324] Figure 5 : Percentage change in body weight of tumor-bearing mice after administration in the MC38 in vivo model.

[0325] Figure 6 : Evaluation results of the anti-tumor activity of antibody hu-KD-0143-v3 alone and in combination with PD-1 antibody in the MC38 in vivo model.

[0326] Figure 7: In the MC38 in vivo model, the body weight changes of mice were tested during the drug administration period.

[0327] Figure 8 : Effects of anti-CD25 antibody on immune cell infiltration in tumor tissues. DETAILED DESCRIPTION

[0328] The technical solution of the present invention will be described below in conjunction with specific embodiments, and the above content of the present invention will be further described in detail, but this should not be interpreted as the scope of the above theme of the present invention being limited to the following examples. Unless otherwise specified, the implementation of the present invention will adopt the conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, etc., and all technologies realized based on the above content of the present invention belong to the scope of the present invention.

[0329] Example 1: Generation of anti-CD25 monoclonal antibodies

[0330] 1.1 Immunization of mice

[0331] First, the fusion protein CD25-mFc of the extracellular region of human CD25 and mouse IgG2a-Fc (Genbank accession number: AAH31470.1) was prepared, and the activity of these protein samples was verified by binding with positive CD25 antibodies. Then, following the commonly used hybridoma mouse immunization protocol, the CD25-mFc protein was emulsified with Freund's adjuvant and used to immunize 6 healthy BALB / C mice aged 6 to 8 weeks. After a certain number of immunizations, blood was collected from the mouse orbits, and the serum was centrifuged and collected after standing at 4°C overnight. The serum titer (titer) was detected by ELISA and FACS methods. The two mice with the highest serum titer (mouse #1 and #2, see Table 2) were boosted. After 3 days, the mice were killed, the spleens were removed, and spleen cells were prepared.

[0332] Table 2. Titer detection of 6 mice after secondary immunization

[0333]

[0334] 1.2 Preparation of hybridomas

[0335] Take myeloma cells SP2 / 0 with good growth status (viable cell count> 95%), mix myeloma cells with spleen cells prepared in step 1.1 at a ratio of 1:2, resuspend and wash the cell mixture with electroporation buffer, add appropriate amount of electroporation buffer to resuspend the cells, and add them to the electroporation fusion tank for fusion. Add the cell suspension to the required volume of complete culture medium containing HAT culture medium (SIGMA, Cat#H0262) prepared in advance, plate on a 96-well culture plate, 150-200μL / well, culture and observe in a CO2 incubator at 37℃. After 7-10 days of culture, the HAT culture medium is replaced with HT culture medium (SIGMA, Cat#H0137). After 3-4 days of culture, take the supernatant from each well for ELISA and FACS detection. For specific operations, see steps (1)-(3) of step 1.3.

[0336] 1.3 Screening of hybridoma antibodies

[0337] (1) ELISA to detect the binding of hybridoma antibodies to human CD25-HIS protein

[0338] Dilute human CD25-HIS protein with PBS, coat the plate with 50ng / well, and place at 4℃ overnight. The next day, block the plate with PBS / 1% BSA for 2h, add hybridoma cell supernatant, 50μL / well, and incubate at room temperature for 1 hour. Wash 5 times with PBST. Add secondary antibody, select the dilution multiple according to the titer of the enzyme-labeled secondary antibody, 50μL / well, incubate at 37℃ for 40min, wash 5 times, and pat dry. Add TMB colorimetric solution 50μL / well, color at 37℃ for 15min-30min. Add stop solution 50μL / well. Measure the OD value of each well at a single wavelength of 450nm (multi-function microplate reader, Molecular Devices, spectraMax M3). Perform ELISA combined with repeated screening, and select positive clones for the next step of screening based on the results.

[0339] (2) FACS detection of the binding of hybridoma antibodies to 293T-hu-CD25 cells

[0340] Collect a certain number of well-growing 293T-hu-CD25 cells (KYinno, KC-1472), wash with FACS buffer (1×PBS+2% FBS), and add 2×10^5 cells / 50ul / well to a deep-well plate. Add the supernatant of the hybridoma positive clones screened in step (1), 50ul / well, incubate in a 4℃ refrigerator for 1h, centrifuge, resuspend the cells with FACS buffer, centrifuge, and wash three times. Add fluorescently labeled secondary antibodies and incubate in a 4℃ refrigerator for 40min. Wash three times with FACS buffer by centrifugation, resuspend the cells with 200ulFACS buffer, and detect the fluorescence signal on a cell flow cytometer.

[0341] (3) ELISA to detect the binding between hybridoma antibodies and monkey CD25 protein

[0342] ELISA plates were coated with CD25 / IL2RA Protein, Cynomolgus, Recombinant (His Tag) (purchased from Sino Biological / 90265-C08H), 50 ng / 100 μL / well. The remaining steps were the same as step (1).

[0343] (4) Hybridoma cell subcloning

[0344] Based on the screening results of step (1)-(3) of 1.3, positive clones that cross-bind with human and monkey CD25 are selected for cell subclone culture in HT medium for 7-10 days. The supernatant of monoclonal cells is selected and positive clones are screened by ELISA and FACS methods. The steps are the same as the mother clone screening in step (1)-(3) of 1.3.

[0345] (5) Preparation and screening of subclonal antibodies

[0346] By screening the supernatant of subcloned cells, positive clones were selected, 50 ml culture was performed, and IgG antibodies in the supernatant were purified for EC analysis by ELISA and FACS. 50 At the same time, it is also tested whether these antibodies will affect the binding of interleukin 2 (IL-2) and CD25, thereby affecting its downstream signaling pathway. For specific operations, see steps (6) to (8).

[0347] (6) ELISA analysis to determine EC binding to CD25-HIS protein 50

[0348] Dilute human CD25-HIS protein with PBS, coat the plate with 50ng / well, and leave at 4℃ overnight. The next day, block the plate with PBS / 1% BSA for 2h; dilute the hybridoma antibody to be tested by 3.16 times, 10 gradients, with a starting concentration of 20ug / ml, add 100μL / well, and incubate at room temperature for 1 hour. Wash 5 times with PBST. Add secondary antibody, select the dilution factor according to the titer of the enzyme-labeled secondary antibody, 50μL / well, incubate at 37℃ for 40min, wash 5 times, and pat dry. Add 50μL / well of TMB colorimetric solution, and develop at 37℃ for 15min-30min. Add 50μL / well of stop solution. Measure the OD value of each well at a single wavelength of 450nm (multi-function microplate reader, Molecular Devices, spectraMax M3). Fit the data with the dose-response model in Graphpad Prism software to obtain the EC 50 The results are shown in Table 3.

[0349] (7) FACS analysis to determine EC binding to 293T-hu-CD25 cells 50

[0350] A certain number of well-grown 293T-hu-CD25 cells were collected, washed by centrifugation with FACS buffer (1×PBS+2% FBS), and added to a deep-well plate at 2×10^5 cells / 50ul / well. The hybridoma antibody to be tested was diluted 3.16 times, 10 gradients, and the starting concentration was 20ug / ml. 100μL / well was added and incubated in a 4°C refrigerator for 1h. Centrifuge, resuspend the cells with FACS buffer, centrifuge, and wash three times. Add fluorescently labeled secondary antibodies and incubate in a 4°C refrigerator for 40min. Wash three times by centrifugation with FACS buffer, resuspend the cells with 200ulFACS buffer, and detect the fluorescence signal on a cell flow cytometer. Read the MFI to fit the data in Graphpad Prism software to obtain EC 50 The results are shown in Table 3.

[0351] Table 3 Binding data of mouse antibodies to CD25-HIS protein and 293T-hu-CD25 cells

[0352]

[0353]

[0354] (8) Antibody inhibitory activity on interleukin 2 (IL-2) signaling pathway

[0355] NK92 cells were starved overnight in serum-free medium. On the second day, a small amount of cells were taken and the expression of CD25 on the cell surface was verified with PE-anti-human CD25 antibody. Hybridoma antibodies of different concentrations were prepared with serum-free RPMI-1640, and the antibodies and starved NK92 cells were mixed in a certain proportion and incubated in an incubator for 30 minutes. IL-2 was diluted with full medium and added to each cell / antibody well, and incubated in an incubator for 20 minutes to stimulate the expression of p-STAT5. 4x cell lysis buffer was prepared, 10μL cell lysis buffer was added to each well, and the mixture was incubated at room temperature for more than 30 minutes. 4x p-STAT5 antibody mixture was prepared, 10μL Phospho-STAT5 Eu Cryptate antibody was added to 190μL detection buffer, and 10μL Phospho-STAT5 d2antibody was added to 190μL detection buffer, mix the two antibodies 200AL together, a total of 400μL; add 16μL of the above cell lysate and 4ul of the above 4x antibody (p-STAT5) mixture to each well of a small volume white plate; seal the white plate with sealing film and incubate overnight at room temperature; read the dual luminescence intensity (665nm and 620nm); calculate the HTRF ratio: HTRF Ratio = (Signal 665nm / Signal 620nm) x 10000. Results are shown in Figure 1 .

[0356] CD25 is a subunit of the cytokine IL2 receptor. When IL2 binds to its receptor, it activates its downstream signaling pathway, leading to an increase in STAT5 phosphorylation levels. The experimental results show that compared with the isotype control antibody mIgG, the Daclizumab analog that competes with IL2 for binding can cause a significant decrease in STAT5 phosphorylation levels, while the RG6292 analog that does not compete with IL2 for binding and most of the CD25 antibody clones in this application will not reduce the increase in STAT5 phosphorylation levels caused by IL2, indicating that these antibodies will not compete with IL2 for binding to CD25, and thus will not inhibit IL2's activation signal to T cells.

[0357] Example 2 Sequencing, production and identification of candidate hybridoma antibodies

[0358] (1) Based on the screening results of subclone antibodies, m-KD-0140, m-KD-0142, m-KD-0143, m-KD-0150, m-KD-0151, m-KD-0161, and m-KD-0165, a total of 7 cell lines, were selected for antibody V gene sequencing. The method is briefly as follows:

[0359] Culture the selected monoclonal hybridoma cells, collect the cell precipitates, extract RNA, and prepare ccDNA by reverse transcription. Use a set of mouse V gene primers to perform PCR amplification of the light chain and heavy chain V genes of the antibody, connect the PCR fragments to the T vector, perform sequencing analysis, and obtain sequence information and then analyze and compare to provide a basis for the next step of the experiment. The specific sequences obtained are shown in Tables 4-1 and 4-2.

[0360] Table 4-1: Amino acid sequences of light chain variable regions and heavy chain variable regions of sequence-specific clones

[0361]

[0362]

[0363] Table 4-2: Sequence numbers corresponding to the CDRs of each clone in Table 4-1

[0364]

[0365]

[0366] (CDR regions are defined according to Kabat numbering; KD-0140_V1 and KD-0140_V2 have the same VL, different VH sequences)

[0367] (2) Based on the screening data of hybridoma mouse antibodies, five antibody clones, m-KD-0140_V2, m-KD-0143, m-KD-0151, m-KD-0161, and m-KD-0165, were selected to construct plasmids for chimeric antibodies: using conventional molecular biological techniques, the signal peptide + variable region gene fragments were amplified, and the gene fragment encoding VH was cloned into the vector pComB-VH containing the human IgG1 heavy chain constant region (SEQ ID NO: 75, see Table 4-3 below) gene fragment, and the gene fragment encoding VL was inserted into the vector pComB-VL containing the human antibody Kappa light chain constant region gene fragment (Ck) (SEQ ID NO: 76, see Table 4-3 below). The heavy chain and light chain plasmids of each antibody were simultaneously transfected into HEK293 cells and cultured in a shaker at 37°C. After 6-7 days, the culture supernatant was collected and purified using Protein A affinity chromatography to purify the IgG antibodies of each clone.

[0368] Table 4-3 Heavy chain / light chain constant region sequences

[0369]

[0370] (3) ELISA analysis to determine EC binding to CD25-HIS protein 50

[0371] The EC values ​​of these chimeric antibodies were determined by the same method as in Example 1. 50 , the results are shown in Table 5.

[0372] (4) FACS analysis to determine EC binding to 293T-hu-CD25 cells 50

[0373] The EC values ​​of these chimeric antibodies were determined by the same method as in Example 1. 50 , the results are shown in Table 5.

[0374] Table 5 Determination of binding between chimeric antibodies and CD25

[0375]

[0376]

[0377] The results of the assays demonstrated that these antibody sequences encode the hybridoma antibodies that were screened to specifically bind to CD25. Clones KD-0143, KD-0151, KD-0161, and KD-0163 had stronger binding forces (smaller EC values) than the positive control antibody RG6292 analog. 50 Value), KD-0143 and KD-0161 were selected as the lead sequences for humanization transformation.

[0378] Example 3 Humanization of candidate hybridoma antibodies

[0379] (1) Humanized clones KD-0143 and KD-0161 to reduce the risk of immunogenicity in clinical applications.

[0380] Humanized design: Using CDR Graft, the main steps are: first, through sequence alignment, find a fully human antibody sequence that is similar to the parent antibody (mouse antibody) sequence or has good developability as the receptor sequence; perform CDR transplantation; perform three-dimensional structural modeling of the antibody, and perform back mutations based on the structural model to avoid the decrease in affinity caused by humanization. The heavy and light chains of the designed humanized sequences are shown in Tables 6-1 and 6-2 below. After preparing these humanized antibodies, the optimal humanized sequence is determined through experiments.

[0381] Table 6-1 Heavy chain / light chain variable region sequences of humanized antibodies

[0382]

[0383]

[0384] Table 6-2: Sequence numbers corresponding to the CDRs of each heavy chain / light chain variable region in Table 6-1

[0385]

[0386] (2) Production of humanized antibodies

[0387] The DNA fragment encoding the above antibody sequence was synthesized and cloned into the human IgG1 transient expression vector using conventional molecular biology techniques (the method is the same as that of "Construction of chimeric antibody expression plasmid"). The different versions of heavy chain sequences and light chain sequences were recombined, and the combined plasmids were simultaneously transfected into HEK293 cells and cultured in a shaker at 37°C. After 6-7 days, the culture supernatant was collected and the IgG antibody was purified using Protein A affinity chromatography.

[0388] Based on the binding data of the humanized antibodies, hu-KD-0143-v3 was selected as the humanized version of clone KD-0143; hu-KD-161-v2 and hu-KD-161-v4 were selected as the humanized versions of clone KD-0161.

[0389] The corresponding sequences of hu-KD-0143-v3, hu-KD-161-v2 and hu-KD-161-v4 are shown in Tables 7-1 and 7-2.

[0390] Table 7-1: Correspondence between humanized antibodies and VH and VL

[0391] Clone Corresponding heavy chain variable region code Corresponding light chain variable region code hu-KD-0143-v3 hu-KD-0143VH3 hu-KD-0143VL2 hu-KD-0161-v2 hu-KD-0161VH2 hu-KD-0161VL2 hu-KD-0161-v4 hu-KD-0161VH2 hu-KD-0161VL4

[0392] Table 7-2: Heavy chain / light chain sequences corresponding to humanized antibodies

[0393]

[0394]

[0395] The above three humanized antibodies were expressed in CHO-K1-D57 cells to obtain the defucosylated humanized antibodies hu-KD-143-v3-aFU, hu-KD-161-v2-aFU and hu-KD-161-v4-aFU. CHO-K1-D57 cells are CHO-K1 cells that have been genetically engineered to remove the Fut8 gene responsible for fucosylation modification (ZL201610194325.7). The antibodies prepared using this platform are non-fucosylated and have enhanced ADCC activity.

[0396] (3) Characterization of selected humanized antibodies

[0397] Affinity determination: The binding constant (K) between humanized antibody and CD25-ECD-His protein was determined using a molecular interaction analyzer (Fortebio 96e).D ). Prepare 5ug / ml of the antibody to be tested and 4x gradient dilution of CD25-ECD-His protein sample (human and monkey HIS-tagged CD25 extracellular domain proteins are produced by our company), with the highest concentration being 20ug / ml. First, use the Protein A probe to adsorb the antibody to be tested, then transfer the probe to the buffer (running buffer), and then transfer it to the CD25-ECD-His protein sample well for antigen-antibody binding. After 100 seconds, transfer the probe to the buffer for dissociation. The instrument's own software analyzes the data and calculates the binding constant (K D ). The results are shown in Table 8.

[0398] Table 8 Affinity determination results of humanized antibodies

[0399]

[0400] Regulatory T cells (Treg) have high levels of CD25 expression. Although the CD25 expression level of effector T cells (Teff) is very low, the CD25 level of activated Teff cells increases significantly. In order to avoid killing Teff cells, the KD of CD25 antibodies should not be too high. In this way, it can effectively bind to Treg cells through avidity, but reduce the binding to Teff cells with low CD25 expression.

[0401] The data in Table 8 show that the humanized antibodies hu-KD-143-v3 and hu-KD-161-v2 have similar affinities to the positive control antibody for both human CD25-His and monkey CD25-His, but the affinity of hu-KD-161-v4 is about 3 times weaker.

[0402] The selected humanized antibodies all had a slower dissociation rate (Koff) than the positive control antibody and may exhibit higher Fc-mediated ADCC activity, thereby more effectively eliminating Treg cells.

[0403] (4) Differential binding with activated primary T cells

[0404] Human PBMC cells were co-incubated with CD3 / CD28 antibody magnetic beads at 37°C for 7-10 days. Antibody samples of different concentrations were prepared and co-incubated with a certain amount of activated T cells. Cells were washed, and fluorescently labeled anti-human Fc secondary antibodies were added and co-incubated. Cells were washed, fixed, and perforated with zwitterionic agents. Co-incubated with fluorescently labeled Foxp3 antibodies and CD3 antibodies. Foxp3 is used as a marker for Treg cells. Cells were washed and loaded onto flow cytometer for data analysis. Results are shown in Figure 2A-2C, showing that the humanized antibody hu-KD-143-v3 ( Figure 2A )、hu-KD-161-v2( Figure 2B ) and hu-KD-161-v4( Figure 2C ) At the same concentration, the binding to Treg cells was significantly stronger than that to Teff cells.

[0405] (5) ADCC activity of antibodies

[0406] KARPAS299 cells (Jinyuan Biotechnology JY803) are human anaplastic large cell lymphoma cells that moderately express CD25. KARPAS299 cells in the logarithmic growth phase were adjusted to a cell concentration of 2×10 5 / mL, add to the U-shaped bottom / V-shaped bottom 96-well cell culture plate, 50μL / well. Add 50μL of diluted antibody solution to each well of the 96-well plate inoculated with cells. The highest antibody concentration in the final system is 50000ng / mL, 5-fold dilution, 9 concentrations, and 2 replicates for each concentration. Incubate the above 96-well plate at 4°C for 30 minutes. Take the effector cells in the logarithmic growth phase (Jurkat-NFAT-Luc2-CD16a-V158 reporter gene cell line) (Kangyuan Bochuang, catalog number KC-1507), adjust the cell concentration to 1×10 5 After the 96-well plate containing the antibody and target cells was washed twice with PBS, the effector cells were added at 100 μL / well. The 96-well plate was incubated at 37°C and 5% CO2 for 6 hours. 100 μL of detection reagent (Stable-Lite TM Luciferase Assay System), shake at room temperature (at least 5 min) to fully lyse the cells, transfer 100 μL of lysate to each well of a 96-well all-white plate, and use a multifunctional microplate reader to detect the luminescence value. Figure 3 and Table 9.

[0407] Table 9 ADCC activity of humanized defucosylated antibodies and non-defucosylated antibodies

[0408] Sample (wild type) <![CDATA[IC 50 (ng / mL)]]> Sample (defucosyl) <![CDATA[IC 50 (ng / mL)]]> hu-KD-143-v3 981.5 hu-KD-143-v3-aFU 121.2 hu-KD-161-v2 ～445 hu-KD-161-v2-aFU 89.4 hu-KD-161-v4 1411 hu-KD-161-v4-aFU 327.1

[0409] Example 4 Tumor Suppressor Activity in Mouse Model

[0410] In vivo pharmacodynamic evaluation of CD25 transgenic mouse colon cancer MC38 model

[0411] Human CD25 transgenic mice (hIL2RA, C57B6, Biocytogen) were used. MC38 cells were inoculated subcutaneously in the anterior ribs of 86 female B-hIL2RA mice. The average tumor volume of the mice was 59 mm3 The mice were divided into groups for drug administration, with 6 mice in each group. Each group was intraperitoneally injected with drugs twice a week. The weight and tumor volume of the mice were measured 3 times a week, and the relationship between the changes in the weight and tumor volume of the tumor-bearing mice and the drug administration time was recorded. The tumor growth inhibition rate TGI was calculated. TV (%) and tumor weight and tumor inhibition rate TGI TW (%) and statistical analysis was performed.

[0412] The results showed that hu-KD-0143-v3 had a significant inhibitory effect on tumor growth at a dose of 10 mg / kg, and hu-KD-0161-v4 and hu-KD-0161-v2 also had a certain inhibitory effect on tumor growth at a dose of 10 mg / kg (see Figure 4 ); the tumor-bearing mice in each group had good tolerance, stable weight gain, no other abnormal manifestations, and were generally in good condition (see Figure 5 ).

[0413] Example 5 Tumor Suppressor Activity in Mouse Model

[0414] (1) In vivo pharmacodynamic evaluation of CD25 transgenic mouse colon cancer MC38 model

[0415] Because humanized antibodies do not bind to mouse CD25, human CD25 transgenic mice (hIL2RA, C57B6, Biocytogen) were used to establish a tumor model. MC38 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin. MC38 cells in the exponential growth phase were collected, serum was washed off, and tumor cells were resuspended in serum-free basal medium to prepare a cell suspension: 2×10 6 / mL, and matrix gel was added at a ratio of 1:1. Female hCD25 humanized mice (C57BL / 6N) were subcutaneously inoculated with 0.1mL of MC38 cells in the right rib, and tumor growth was observed regularly. When the tumor volume grew to about 97mm 3 The animals were divided into groups and administered at different times. There were 4 groups in the experiment, namely Group 1 (normal saline control group), Group 2 (hu-KD-0143-v3, 10 mg / kg group), Group 3 (anti-mPD-1, 1 mg / kg group) (antibody source: Bioxcell, #BP0146), and Group 4 (hu-KD-0143-v3 10 mg / kg + anti-mPD-1 1 mg / kg group), with 6 animals in each group; intraperitoneal administration (IP), twice a week (BIW), for a total of 6 times. The efficacy was evaluated based on the tumor inhibition rate (TGI), and the safety was evaluated based on the changes in animal weight and mortality. Statistical analysis was also performed.

[0416] The results showed that hu-KD-0143-v3 had a significant inhibitory effect on tumor growth at a dose of 10 mg / kg, and showed further synergistic effects when used in combination with anti-mPD1 (see Figure 6 , Table 10); the tumor-bearing mice in each group had good tolerance, their body weight increased steadily, there were no other abnormal manifestations, and their general condition was good (see Figure 7 ).

[0417] Table 10 Tumor volume and TGI of each group on the 21st day after administration of MC38 tumor model

[0418] Group <![CDATA[Day 1 (mm 3 ) a > <![CDATA[Day 21 (mm 3 )]]> TGI (%) <![CDATA[P b ]]> Vehicle (normal saline control group) 97.0±2.7 2186.5±426.9 - - hu-KD-0143-v3 97.0±2.2 1173.1±436.0 46.3 0.044 anti-mPD-1 97.3±2.2 1011.6±189.3 53.7 0.021 hu-KD-0143-v3+anti-mPD-1 97.2±2.2 446.0±119.6 79.6 0.002

[0419] a. Mean ± standard error; b. Compared with the Vehicle group.

[0420] (2) Effect of CD25 antibody on immune cell infiltration in tumor tissue

[0421] Following the above experimental step (1), 4 days after the 6th administration of the experimental mice (22 days after grouping), 5 mice were taken from the combined administration group (hu-KD-0143-v3+anti-mPD-1), and 6 mice were taken from each of the other groups. Fresh tumor tissues of mice were taken, and fat, fiber, membrane, and necrotic tissue in the tumor were removed; the tumor was cut into small pieces, and tumor tissue digestion enzyme was added, and the mixture was shaken at low speed at 37°C for 20 to 30 minutes, filtered, washed, and resuspended in PBS to prepare a single cell suspension of tumors for each mouse. Cells were blocked in a 1.5 mL EP tube; an appropriate amount of fluorescently labeled T cell marker antibody was added, the cells were mixed, incubated at 4°C in the dark for 15-20 minutes, 1 mL of washing solution was added to terminate the staining, and the cells were washed by centrifugation; 1 mL of Foxp3 fixation / membrane permeabilization working solution was added, the cells were mixed thoroughly, and the cells were incubated at 4°C in the dark for 60 minutes; the cells were washed by centrifugation with 1× membrane permeabilization working solution; the recommended amount of Foxp3 antibody was added, the cells were mixed thoroughly, and the cells were incubated at 4°C in the dark for 45 minutes, 2 mL of 1× membrane permeabilization working solution was added, and the cells were washed by centrifugation; the cell suspension was resuspended in 300 uL PBS and transferred to a flow cytometry tube, and the abundance of several immune cells was detected by the machine: total immune cells (CD45+, corresponding to Figure 8 A), CD8+T cells (corresponding to Figure 8 C), Tcon cells (CD4+Foxp3-) (corresponding to Figure 8 B) and Treg cells (CD4+Foxp3+) (corresponding to Figure 8 D), the results are shown in Figure 8Compared with the control group, hu-KD-0143-v3 alone and hu-KD-0143-v3 combined with anti-mPD-1 can reduce the proportion of Treg cells in mouse MC38 tumors, increase the tumor infiltration of CD8+T cells, Tcon cells (CD4+Foxp3-) and total immune cells (CD45+), improve the tumor immune microenvironment, and enhance anti-tumor immunity.

Claims

1. An isolated anti-CD25 antibody or antigen-binding fragment thereof having at least one of the following characteristics: 1) Bind to human CD25 protein epitope; 2) have cross-immunoreactivity with monkey and / or mouse CD25; 3) With a K of 100 nM or less D Binds to CD25 antigen; 4) Using ELISA method to determine EC binding to CD25 50 Value ≤ 5ug / mL; preferably, EC binding to CD25 50 Value ≤1ug / mL; 5) eliciting an enhanced CDC, ADCC and / or ADCP response; preferably, eliciting an increased ADCC response; 6) does not inhibit or does not substantially inhibit the binding of IL-2 to CD25; 7) Has no or little influence on the function of Teff cells; 8) binds to FcγRIII with a higher affinity than it binds to FcγRI, FcγRIIc and / or FcγRIIb.

2. The anti-CD25 antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein: 1) the HCDR1 is selected from the HCDR1 shown in SEQ ID NO: 16, SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 22, SEQ ID NO: 30 or SEQ ID NO: 36, 2) the HCDR2 is selected from the HCDR2 shown in SEQ ID NO: 17, SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 31 or SEQ ID NO: 37, 3) the HCDR3 is selected from the HCDR3 shown in SEQ ID NO: 18, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO: 32 or SEQ ID NO: 38; The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein: 1) the LCDR1 is selected from the LCDR1 shown in SEQ ID NO: 19, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 39 or SEQ ID NO: 42, or the LCDR1 shown below: the 5th amino acid in SEQ ID NO: 33 is replaced by S, and / or the 7th amino acid is replaced by G, and / or the 9th amino acid is replaced by D, 2) the LCDR2 is selected from the LCDR2 shown in SEQ ID NO:20, SEQ ID NO:5, SEQ ID NO:14, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:34 or SEQ ID NO:40, or the LCDR2 shown below: the first amino acid in SEQ ID NO:34 is replaced by F, and / or the fourth amino acid is replaced by N, 3) the LCDR3 is selected from the LCDR3 shown in SEQ ID NO:21, SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:35 or SEQ ID NO:41; Preferably, the CDRs are defined according to the Kabat numbering system.

3. The anti-CD25 antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein: 1) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 16, HCDR2 shown in SEQ ID NO: 17, and HCDR3 shown in SEQ ID NO: 18, The light chain variable region comprises LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 20, and LCDR3 shown in SEQ ID NO: 21; 2) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO: 3, The light chain variable region comprises LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6; 3) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 7, HCDR2 shown in SEQ ID NO: 8, and HCDR3 shown in SEQ ID NO: 9, The light chain variable region comprises LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6; 4) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 10, HCDR2 shown in SEQ ID NO: 11, and HCDR3 shown in SEQ ID NO: 12, The light chain variable region comprises LCDR1 shown in SEQ ID NO: 13, LCDR2 shown in SEQ ID NO: 14, and LCDR3 shown in SEQ ID NO: 15; 5) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 22, HCDR2 shown in SEQ ID NO: 23, and HCDR3 shown in SEQ ID NO: 24, The light chain variable region comprises LCDR1 shown in SEQ ID NO: 13, LCDR2 shown in SEQ ID NO: 25, and LCDR3 shown in SEQ ID NO: 26; 6) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 22, HCDR2 shown in SEQ ID NO: 23, and HCDR3 shown in SEQ ID NO: 24, The light chain variable region comprises LCDR1 shown in SEQ ID NO: 27, LCDR2 shown in SEQ ID NO: 28, and LCDR3 shown in SEQ ID NO: 29; 7) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 30, HCDR2 shown in SEQ ID NO: 31, and HCDR3 shown in SEQ ID NO: 32, The light chain variable region comprises LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:35; 8) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 36, HCDR2 shown in SEQ ID NO: 37, and HCDR3 shown in SEQ ID NO: 38, The light chain variable region comprises LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:40, and LCDR3 shown in SEQ ID NO:35; 9) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 30, HCDR2 shown in SEQ ID NO: 31, and HCDR3 shown in SEQ ID NO: 32, The light chain variable region comprises LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:41; 10) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 30, HCDR2 shown in SEQ ID NO: 31, and HCDR3 shown in SEQ ID NO: 32, The light chain variable region comprises LCDR1 shown in SEQ ID NO:42, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:41; or 11) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 30, HCDR2 shown in SEQ ID NO: 31, and HCDR3 shown in SEQ ID NO: 32; The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 is selected from the sequence of SEQ ID NO: 33 or the 5th amino acid in the sequence of SEQ ID NO: 33 is replaced by S, and / or the 7th amino acid is replaced by G, and / or the 9th amino acid is replaced by D; The LCDR2 is selected from the sequence of SEQ ID NO: 34 or the first amino acid in the sequence of SEQ ID NO: 34 is replaced by F, and / or the fourth amino acid is replaced by N; The LCDR3 is selected from the sequence SEQ ID NO: 35 or the 5th amino acid in SEQ ID NO: 35 is replaced by S.

4. The anti-CD25 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: 1) The amino acid sequence of the heavy chain variable region as set forth in SEQ ID NO:45 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:43 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:44 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:48 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:49 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:59 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:61 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:63 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:65 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:67 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:69 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:71 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, or as set forth in SEQ ID NO:73 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto; and 2) the amino acid sequence of the light chain variable region as set forth in SEQ ID NO:47 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:46 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:50 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:51 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:52 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:53 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:60 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:62 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:64 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:66 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:68 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:70 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:72 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, or As set forth in SEQ ID NO:74 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto.

5. The anti-CD25 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein: 1) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:45 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto; The amino acid sequence of the light chain variable region is as shown in SEQ ID NO:47 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto; 2) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:49 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto; The amino acid sequence of the light chain variable region is as shown in SEQ ID NO:51 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto; or 3) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:49 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto; The amino acid sequence of the light chain variable region is as shown in SEQ ID NO:53 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto.

6. The anti-CD25 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising an antibody heavy chain and an antibody light chain, wherein: 1) the amino acid sequence of the antibody heavy chain, as set forth in SEQ ID NO:54 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, or as set forth in SEQ ID NO:56 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto; and 2) the amino acid sequence of the antibody light chain, as set forth in SEQ ID NO:55 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, as set forth in SEQ ID NO:57 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto, or as set forth in SEQ ID NO:58 or having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto; Preferably, 1) the amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO:54 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto; The amino acid sequence of the antibody light chain is as shown in SEQ ID NO:55 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto; 2) the amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO:56 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto; The amino acid sequence of the antibody light chain is as shown in SEQ ID NO:57 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto; or 3) the amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO:56 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto; The amino acid sequence of the antibody light chain is as shown in SEQ ID NO:58 or has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity thereto.

7. The anti-CD25 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein: The anti-CD25 antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody; the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, scFv or sdAb; the anti-CD25 antibody is selected from an IgG antibody selected from IgG1, IgG2, IgG2 or IgG4, preferably, the anti-CD25 antibody is selected from IgG1 or IgG4; Preferably, the anti-CD25 antibody has a defucosylation modification in the Fc region to enhance the binding ability to FcγRIIIa and / or reduce the binding ability to FcγRIIb.

8. A polynucleotide molecule encoding the anti-CD25 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. A pharmaceutical preparation comprising the anti-CD25 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the polynucleotide molecule according to claim 8, and one or more pharmaceutically acceptable carriers or excipients; Optionally, the pharmaceutical preparation further comprises a second active ingredient; the second active ingredient is selected from a chemical anticancer agent, an anti-tumor antigen or marker antibody, an immune checkpoint inhibitor or an immunomodulatory antibody; Preferably, the second active ingredient is selected from an immune checkpoint inhibitor or an immunomodulatory antibody; Preferably, the second active ingredient is selected from PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, TIGIT antibody, TIM-3 antibody, B7-H3 antibody, CD73 antibody, LAG3 antibody, CD27 antibody, CD70 antibody, 4-1BB antibody, GITR antibody, OX40 antibody, CD47 antibody, CD39 antibody, ILDR2 antibody, VISTA antibody, BTLA antibody or VTCN-1 antibody.

10. Use of the anti-CD25 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the polynucleotide molecule according to claim 8, or the pharmaceutical preparation according to claim 9 in the preparation of a medicament for treating and / or preventing a CD25-mediated disease or condition, Preferably, the disease or disorder is an immune disease or cancer; Preferably, the cancer is selected from squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, liver cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, colorectal cancer, gastrointestinal cancer, kidney cancer, ovarian cancer, endometrial cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, breast cancer and head and neck cancer.

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