CN119552247A

CN119552247A - Anti-CD25 antibody and its medical use

Info

Publication number: CN119552247A
Application number: CN202411204936.6A
Authority: CN
Inventors: 董建波; 朱晓东; 刘伟
Original assignee: Xuanzhu Biopharmaceutical Co Ltd
Current assignee: Xuanzhu Biopharmaceutical Co Ltd
Priority date: 2023-09-01
Filing date: 2024-08-30
Publication date: 2025-03-04

Abstract

The invention belongs to the technical field of medicines, and in particular relates to an antibody or an antigen binding fragment thereof specifically binding to CD25, a pharmaceutical preparation containing the antibody or the antigen binding fragment thereof, 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 medical application of the antibody, the antigen binding fragment thereof or the pharmaceutical preparation containing the antibody or the antigen binding fragment thereof.

Description

Anti-CD 25 antibody and medical application thereof

Technical Field

Background

Interlukin-2 (IL-2) is a cytokine of chemokine family, has important functions on immune response and antiviral infection of organism, can stimulate T cell proliferation activated by specific antigen or Mitogen (Mitogen), can activate T cell, promote cytokine production, stimulate NK cell proliferation, enhance NK killing activity and cytokine production, induce LAK cell production, promote B cell proliferation and secretion of antibody, activate macrophage. The receptor for IL-2 comprises 3 polypeptide chain subunits, designated the α (CD 25), β (CD 122) and γ (CD 132) subunits, respectively. IL2RA (Interlukin-2 receptor subunit alpha), also known as CD25, p55, is a type I transmembrane protein of 55 kDa. Mainly expressed on the surfaces of tissues such as spleen and lymph nodes, treg cells, basophils, activated T cells and the like.

Both dalizumab (Daclizumab) and banizumab (Basiliximab) are IgG 1-type antibodies that inhibit IL-2 binding to CD25, and were developed to reduce effector T cell (Teff) activation. Daclizumab is approved by the FDA for the treatment of multiple sclerosis, but the drug has been withdrawn due to serious safety concerns. The use of banizumab is FDA approved for the prevention of organ transplant rejection, but has not been approved in the treatment of autoimmune diseases and tumors. In 2019, humanized anti-CD 25 monoclonal antibody injection of Sansheng Guojian was marketed as Jianziping, and the indication was to prevent acute rejection caused by kidney transplantation.

CD25 is expressed in abundance on regulatory T (Treg) cells in comparison with other immune cells, so that the expression abundance of the CD25 on regulatory T (Treg) cells is higher than that of other immune cells, the expression abundance of the CD25 on regulatory T (Treg) cells can participate in the regulation of immune tolerance by controlling the activity of the Treg cells, the activation and proliferation of effector T cells can be inhibited by the Treg cells, and the contact and consumption of the Treg cells are beneficial to promoting tumor immunity. However, CD25 also mediates activation of the Teff cells, which would be inhibited if the IL-2 signaling pathway were inhibited, thereby affecting the efficacy of tumor immunity.

Currently no anti-CD 25 antibodies against tumor therapy are marketed, and anti-CD 25 antibodies that have entered clinical research include anti-CD 25 antibody RG6292 developed in combination by roche and TUSK, and non-IL-2 blocking anti-CD 25 antibody BA1106 by boan, a subsidiary under the green leaf pharmaceutical set the League flag. Thus, there remains a need in the art to develop anti-CD 25 antibodies that specifically kill Treg cells without inhibiting or partially inhibiting Teff cell activity, thereby relieving immunosuppression.

Disclosure of Invention

The present invention provides an isolated anti-CD 25 antibody or antigen binding fragment thereof that has good anti-tumor activity, has increased binding to fcyriiia, and/or decreased binding to fcyriib.

The present invention provides an isolated anti-CD 25 antibody or antigen binding fragment thereof having at least one of the following characteristics:

1) Binding to an epitope of human CD25 protein;

2) Has cross-immunoreactivity with monkey and/or murine CD 25;

3) Binding to CD25 antigen with K _D of 100nM or less;

4) The EC ₅₀ value of CD25 binding is less than or equal to 5ug/mL, preferably the EC ₅₀ value of CD25 binding is less than or equal to 1ug/mL, as determined by ELISA;

5) Initiating enhanced CDC, ADCC and/or ADCP reactions, preferably, initiating increased ADCC reactions;

6) Does not inhibit or substantially does not inhibit the binding of IL-2 to CD 25;

7) Does not affect or substantially affects the function of the Teff cells;

8) Fcyriii binds with a higher affinity than fcyri, fcyriic, and/or fcyriib.

In certain embodiments, the anti-CD 25 antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising HCDR1-HCDR3 as set forth in SEQ ID NO 45, 43, 44, 48, 49, 59, 61, 63, 65, 67, 69, 71, 73 and a light chain variable region comprising LCDR1-LCDR3 as set forth in SEQ ID NO 47, 46, 50, 51, 52, 53, 60, 62, 64, 66, 68, 70, 72, 74 according to Kabat, IMGT, chothia or AbM numbering system.

In certain embodiments, the anti-CD 25 antibody, or antigen-binding fragment thereof, comprises 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 HCDR1 with 0, 1,2, 3, 4 or 5 amino acid differences from the amino acid sequence shown in 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,

2) The HCDR2 is selected from HCDR2 having a difference of 0, 1,2, 3, 4 or 5 amino acids from the amino acid sequence shown in 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

3) The HCDR3 is selected from HCDR3 with 0, 1,2, 3, 4 or 5 amino acid differences from the amino acid sequence shown in 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;

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

1) The LCDR1 is selected from LCDR1 with 0, 1,2, 3, 4 or 5 amino acid differences from the amino acid sequence shown in 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,

2) The LCDR2 is selected from LCDR2 having a difference of 0, 1, 2, 3, 4 or 5 amino acids from the amino acid sequence shown in 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

3) The LCDR3 is selected from LCDR3 with 0, 1,2, 3, 4 or 5 amino acid differences with the amino acid sequence shown in 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;

preferably, the CDRs are defined according to the Kabat numbering system.

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

1) The HCDR1 is selected from HCDR1 with 0,1, 2 or 3 amino acid differences from the amino acid sequence shown in 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,

2) The HCDR2 is selected from HCDR2 having 0, 1, 2 or 3 amino acid differences from the amino acid sequence shown in 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

3) The HCDR3 is selected from HCDR3 with 0, 1,2 or 3 amino acid differences with the amino acid sequence shown in 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;

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

1) The LCDR1 is selected from LCDR1 with 0, 1, 2 or 3 amino acid differences with the amino acid sequence shown in 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,

2) The LCDR2 is selected from LCDR2 having 0,1, 2 or 3 amino acid differences from the amino acid sequence shown in 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

3) The LCDR3 is selected from LCDR3 with 0, 1, 2 or 3 amino acid differences with the amino acid sequence shown in 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;

preferably, the CDRs are defined according to the Kabat numbering system.

In certain embodiments, the anti-CD 25 antibody or antigen-binding fragment thereof comprises 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 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 HCDR2 as 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

3) The HCDR3 is selected from 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 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 LCDR1 shown by replacing the 5 th amino acid in SEQ ID NO 33 with S and/or replacing the 7 th amino acid with G and/or replacing the 9 th amino acid with D,

2) The LCDR2 is selected from the group consisting of 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 LCDR2 shown by the following substitution of amino acid 1 in SEQ ID NO:34 with F and/or substitution of amino acid 4 with N, and

3) The LCDR3 is selected from 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.

In certain embodiments, the anti-CD 25 antibody or antigen-binding fragment thereof, wherein,

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 with the amino acid sequences shown in SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18 respectively,

The HCDR1, HCDR2 and HCDR3 have 0, 1,2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively,

The HCDR1, HCDR2 and HCDR3 have 0, 1,2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9 respectively,

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

The HCDR1, HCDR2 and HCDR3 have 0, 1, 2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24 respectively,

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

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

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

The LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21 respectively,

The LCDR1, LCDR2 and LCDR3 have 0, 1,2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively,

The LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15 respectively,

The LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO. 13, SEQ ID NO. 25 and SEQ ID NO. 26 respectively,

The LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO. 27, SEQ ID NO. 28 and SEQ ID NO. 29,

The LCDR1, LCDR2 and LCDR3 have 0, 1, 2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO. 33, SEQ ID NO. 34 and SEQ ID NO. 35 respectively,

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

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

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.

1) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 differ from the amino acid sequences set forth in SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18 by 0, 1,2 or 3 amino acids, respectively,

The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein 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;

2) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 differ from the amino acid sequences set forth in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 by 0, 1, 2 or 3 amino acids, respectively,

The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein 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;

3) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 differ from the amino acid sequences set forth in SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9 by 0, 1, 2 or 3 amino acids, respectively,

4) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 differ from the amino acid sequences set forth in SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12 by 0, 1,2 or 3 amino acids, respectively,

The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein 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;

5) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 differ from the amino acid sequences set forth in SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24 by 0, 1,2 or 3 amino acids, respectively,

The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein 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;

6) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 differ from the amino acid sequences set forth in SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24 by 0, 1,2 or 3 amino acids, respectively,

The light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 differ from the amino acid sequences shown in SEQ ID NO. 27, SEQ ID NO. 28, and SEQ ID NO. 29 by 0, 1, 2, or 3 amino acids, respectively;

7) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 differ from the amino acid sequences set forth in SEQ ID NO. 30, SEQ ID NO. 31 and SEQ ID NO. 32 by 0, 1,2 or 3 amino acids, respectively,

The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein 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;

8) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 differ from the amino acid sequences set forth in SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38 by 0, 1,2 or 3 amino acids, respectively,

The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein 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;

9) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 differ from the amino acid sequences set forth in SEQ ID NO. 30, SEQ ID NO. 31 and SEQ ID NO. 32 by 0, 1,2 or 3 amino acids, respectively,

The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1, LCDR2 and LCDR3 differ from the amino acid sequences shown in SEQ ID NO:34 and SEQ ID NO:41 by 0, 1, 2 or 3 amino acids, respectively, or

10 The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 differ from the amino acid sequences set forth in SEQ ID NO. 30, SEQ ID NO. 31 and SEQ ID NO. 32 by 0,1, 2 or 3 amino acids, respectively,

The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1, LCDR2 and LCDR3 differ from the amino acid sequences shown in SEQ ID NO. 42, SEQ ID NO. 34 and SEQ ID NO. 41 by 0, 1,2 or 3 amino acids, respectively.

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,

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 as SEQ ID NO. 39, LCDR2 shown as SEQ ID NO. 40 and LCDR3 shown as 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 as SEQ ID NO.30, HCDR2 shown as SEQ ID NO. 31 and HCDR3 shown as 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 as SEQ ID NO. 30, HCDR2 shown as SEQ ID NO. 31 and HCDR3 shown as SEQ ID NO. 32,

The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 is selected from the group consisting of SEQ ID NO. 33 or the 5 th amino acid substitution in SEQ ID NO. 33 is S, and/or the 7 th amino acid substitution is G, and/or the 9 th amino acid substitution is D;

the LCDR2 is selected from the sequence SEQ ID NO. 34 or the 1 st amino acid in the sequence SEQ ID NO. 34 is replaced by F and/or the 4 th amino acid is replaced by N;

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

2) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 differ from the amino acid sequences set forth in SEQ ID NO. 30, SEQ ID NO. 31 and SEQ ID NO. 32 by 0, 1,2 or 3 amino acids, respectively,

The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1, LCDR2 and LCDR3 differ from the amino acid sequences shown in SEQ ID NO:34 and SEQ ID NO:35 by 0, 1, 2 or 3 amino acids, respectively, or

3) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 differ from the amino acid sequences set forth in SEQ ID NO. 30, SEQ ID NO. 31 and SEQ ID NO. 32 by 0, 1,2 or 3 amino acids, respectively,

The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein 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;

preferably, the CDRs are defined according to the Kabat numbering system.

In certain embodiments, the anti-CD 25 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 differ from the amino acid sequences set forth in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18 by 0,1, 2, or 3 amino acids, respectively,

The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1, LCDR2 and LCDR3 differ from the amino acid sequences shown in SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21 by 0, 1,2 or 3 amino acids, respectively.

In certain embodiments, the anti-CD 25 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 differ from the amino acid sequences set forth in SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32 by 0,1, 2, or 3 amino acids, respectively,

The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1, LCDR2 and LCDR3 differ from the amino acid sequences shown in SEQ ID NO. 33, SEQ ID NO. 34 and SEQ ID NO. 35 by 0, 1,2 or 3 amino acids, respectively.

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

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,

The LCDR3 is selected from a sequence SEQ ID NO. 35 or the 5 th amino acid in the SEQ ID NO. 35 is replaced by S;

preferably, the CDRs are defined according to the Kabat numbering system.

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

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,

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;

preferably, the CDRs are defined according to the Kabat numbering system.

In certain embodiments, the anti-CD 25 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,

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.

In certain embodiments, the anti-CD 25 antibody, or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1 shown as SEQ ID NO. 30, HCDR2 shown as SEQ ID NO. 31, and HCDR3 shown as 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.

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.

The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 is selected from the group consisting of SEQ ID NO. 33 or the 5 th amino acid substitution in SEQ ID NO. 33 is replaced with S, and/or the 7 th amino acid substitution is replaced with G, and/or the 9 th amino acid substitution is replaced with D;

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

As shown 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown 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

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 as shown in SEQ ID NO 73;

And 2) the amino acid sequence of the light chain variable region

As shown 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown in SEQ ID NO. 72 or 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 shown in SEQ ID NO. 74 or 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.

1) The heavy chain variable region has an amino acid sequence as shown in 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 to SEQ ID No. 45;

The light chain variable region has an amino acid sequence as shown in SEQ ID NO. 47 or 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 heavy chain variable region has an amino acid d sequence as shown in 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 to SEQ ID No. 49;

The light chain variable region has an amino acid sequence as shown in 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 to SEQ ID NO. 51, or

3) The heavy chain variable region has an amino acid sequence as shown in 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 to SEQ ID No. 49;

The light chain variable region has an amino acid sequence as shown in SEQ ID NO. 53 or 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.

In certain embodiments, the anti-CD 25 antibody or antigen-binding fragment thereof comprises a heavy chain constant region having an amino acid sequence as shown in SEQ ID No. 75 or 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 light chain constant region has an amino acid sequence as shown in SEQ ID NO. 76 or 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.

In certain embodiments, the anti-CD 25 antibody or antigen-binding fragment thereof comprises a heavy chain constant region having an amino acid sequence as set forth in SEQ ID No. 75 or having 1,2,3,4, or 5 amino acid differences from the amino acid sequence set forth in SEQ ID No. 75;

the amino acid sequence of the light chain constant region is shown as SEQ ID NO. 76 or has 1, 2, 3, 4 or 5 amino acid differences with the amino acid sequence shown as SEQ ID NO. 76.

In certain embodiments, the anti-CD 25 antibody or antigen-binding fragment thereof comprises a heavy chain constant region having an amino acid sequence as shown in SEQ ID NO. 75 and a light chain constant region having an amino acid sequence as shown in SEQ ID NO. 76.

In certain embodiments, the anti-CD 25 antibody or antigen-binding fragment thereof comprises an antibody heavy chain and an antibody light chain, wherein,

1) The amino acid sequence of the heavy chain of said antibody,

As shown 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

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 as shown in SEQ ID NO. 56;

and 2) the amino acid sequence of the light chain of said antibody,

As shown 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 shown 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 shown in SEQ ID NO. 58 or 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.

1) The amino acid sequence of the heavy chain of the antibody is 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 therewith;

The amino acid sequence of the antibody light chain is 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 therewith;

2) The amino acid sequence of the heavy chain of the antibody is 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 therewith;

the antibody light chain has an amino acid sequence as shown in SEQ ID NO 57 or 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 of the antibody is 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 therewith;

the amino acid sequence of the antibody light chain is 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 therewith.

The amino acid sequence of the heavy chain of the antibody is 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 therewith;

The amino acid sequence of the antibody light chain is 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 therewith.

The amino acid sequence of the heavy chain of the antibody is 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 therewith;

the amino acid sequence of the antibody light chain is 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 therewith.

1) The amino acid sequence of the heavy chain of said antibody,

As shown in SEQ ID NO. 54 or with 1,2,3,4 or 5 amino acid differences, or

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

and 2) the amino acid sequence of the light chain of said antibody,

As shown in SEQ ID NO. 55 or having a1, 2, 3, 4 or 5 amino acid difference therefrom,

As shown in SEQ ID NO 57 or with 1,2,3,4 or 5 amino acid differences, or

As shown in SEQ ID NO 58 or with 1, 2, 3, 4 or 5 amino acid differences thereto.

1) The amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO. 54 or has 1,2, 3, 4 or 5 amino acid differences with the heavy chain;

The amino acid sequence of the antibody light chain is shown as SEQ ID NO. 55 or has 1,2, 3, 4 or 5 amino acid differences with the antibody light chain;

2) The amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO. 56 or has 1,2, 3, 4 or 5 amino acid differences with the heavy chain;

the amino acid sequence of the antibody light chain is shown as SEQ ID NO 57 or has 1,2, 3, 4 or 5 amino acid differences with the antibody light chain, or

3) The amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO. 56 or has 1,2, 3, 4 or 5 amino acid differences with the heavy chain;

The amino acid sequence of the antibody light chain is shown as SEQ ID NO. 58 or has 1, 2,3,4 or 5 amino acid differences with the antibody light chain.

The amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO. 54 or has 1,2, 3, 4 or 5 amino acid differences with the heavy chain;

the amino acid sequence of the antibody light chain is shown as SEQ ID NO. 55 or has 1,2, 3, 4 or 5 amino acid differences with the antibody light chain.

The amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO. 56 or has 1,2, 3, 4 or 5 amino acid differences with the heavy chain;

the amino acid sequence of the antibody light chain is shown in SEQ ID NO. 57 or has 1,2, 3, 4 or 5 amino acid differences with the antibody light chain.

1) The amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO. 54, and the amino acid sequence of the light chain of the antibody is shown as SEQ ID NO. 55;

2) The amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO. 56, the amino acid sequence of the light chain of the antibody is shown as SEQ ID NO. 57, or

3) The amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO. 56, and the amino acid sequence of the light chain of the antibody is shown as SEQ ID NO. 58.

In certain embodiments, the anti-CD 25 antibody is selected from a murine, chimeric, humanized or fully human antibody, the antigen binding fragment is selected from Fab, fab ', F (ab') 2, fv, scFv or sdAb, and the anti-CD 25 antibody is selected from an IgG antibody selected from an IgG1 type, an IgG2 type or an IgG4 type.

In certain embodiments, the anti-CD 25 antibody is selected from a murine, chimeric, humanized or fully human antibody, the antigen binding fragment is selected from Fab, fab ', F (ab') 2, fv, scFv or sdAb, and the anti-CD 25 antibody is selected as IgG1 type or IgG4 type.

In certain embodiments, the anti-CD 25 antibody is selected from a humanized antibody or a fully human antibody.

In certain embodiments, the anti-CD 25 antibody is selected from an IgG 1-type or IgG 4-type antibody.

In certain embodiments, the anti-CD 25 antibody is selected to be an IgG1 type antibody.

In certain embodiments, the anti-CD 25 antigen binding fragment is selected from Fab, fab ', F (ab') 2, fv, scFv, or sdAb.

In certain embodiments, the anti-CD 25 antibody has a defucosylation modification in the Fc region to enhance binding to fcyriiia, and/or to reduce binding to fcyriib.

The present invention provides a polynucleotide molecule encoding an anti-CD 25 antibody or antigen-binding fragment thereof according to any one of the above aspects.

The invention also provides an expression vector comprising a polynucleotide molecule according to any one of the above schemes.

In certain embodiments, the expression vector is a eukaryotic expression vector.

The invention also provides a host cell or an engineered cell comprising a polynucleotide molecule according to any one of the above schemes or an expression vector according to any one of the above schemes.

In certain embodiments, the host cell or engineered cell is selected from bacterial, yeast or mammalian cells.

In certain embodiments, the host cell or engineered cell is selected from the group consisting of escherichia coli, pichia pastoris, chinese hamster ovary cells, or human embryonic kidney cells 293.

The invention also provides a pharmaceutical formulation comprising an anti-CD 25 antibody or antigen-binding fragment thereof according to any of the above, a polynucleotide molecule according to any of the above, and one or more pharmaceutically acceptable carriers or excipients.

The present invention provides a pharmaceutical formulation comprising an expression vector as described in any one of the above schemes or a host cell or engineered cell as described in any one of the above schemes, and one or more pharmaceutically acceptable carriers or excipients.

In certain embodiments, the pharmaceutical formulation further comprises a second active ingredient selected from a chemical anticancer agent, an anti-tumor antigen or marker antibody, an immune checkpoint inhibitor, or an immune modulating antibody.

In certain embodiments, the second active ingredient is selected from an immune checkpoint inhibitor or an immune modulating antibody.

In certain embodiments, the second active ingredient is selected from the group consisting of PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, TIGIT antibodies, TIM-3 antibodies, B7-H3 antibodies, CD73 antibodies, LAG3 antibodies, CD27 antibodies, CD70 antibodies, 4-1BB antibodies, GITR antibodies, OX40 antibodies, CD47 antibodies, CD39 antibodies, ILDR antibodies, VISTA antibodies, BTLA antibodies, or VTCN-1 antibodies.

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

The invention also provides a pharmaceutical composition comprising an anti-CD 25 antibody or antigen-binding fragment thereof according to any of the above, a polynucleotide molecule according to any of the above, an expression vector according to any of the above, or a host cell or engineered cell according to any of the above, and one or more second active ingredients.

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 immune modulating antibody.

The invention also provides the use of an anti-CD 25 antibody or antigen-binding fragment thereof according to any one of the above, a polynucleotide molecule according to any one of the above, a pharmaceutical formulation according to any one of the above, in the manufacture of a medicament for the treatment and/or prophylaxis of a CD25 mediated disease or condition.

The invention also provides the use of an expression vector according to any one of the above schemes, a host cell or an engineering cell according to any one of the above schemes, and a pharmaceutical composition according to any one of the above schemes in the preparation of a medicament for treating and/or preventing a CD25 mediated disease or disorder.

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

In certain embodiments, the cancer is selected from squamous cell carcinoma, myeloma, small-cell lung carcinoma, non-small cell lung carcinoma, glioma, liver cancer, hodgkin's lymphoma, non-hodgkin's lymphoma, leukemia, colorectal cancer, gastrointestinal cancer, renal cancer, ovarian cancer, endometrial cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, gastric cancer, bladder cancer, breast cancer, and head and neck cancer.

The invention also provides a kit comprising an anti-CD 25 antibody or antigen-binding fragment thereof according to any of the above, a polynucleotide molecule according to any of the above, an expression vector according to any of the above, a host cell or engineered cell according to any of the above, a pharmaceutical formulation according to any of the above, or a pharmaceutical composition according to any of the above.

The partial relevant sequences of the anti-CD 25 antibodies of the invention are shown in table 1:

TABLE 1 heavy chain/light chain CDR region sequences

Sequence numbering	Amino acid sequence	Sequence numbering	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

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, however, for a better understanding of the present invention, the following definitions of some terms are provided. When the definition and interpretation of terms provided by the present invention are not identical to the meanings commonly understood by those skilled in the art, the definition and interpretation of terms provided by the present invention is in control. The abbreviations for amino acids (including residues) are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

"Antibody" refers to an immunoglobulin that is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains joined by interchain disulfide bonds. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins, igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α and ε chains, respectively. The same class of Ig can be further classified into different subclasses according to the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chain, e.g., igG can be classified into IgG1, igG2, igG3, and IgG4. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each of the five classes of Ig may have either a kappa chain or a lambda chain. The sequences of the heavy and light chains of the antibody near the N-terminus vary widely, being the variable region (V region), and the remaining amino acid sequences near the C-terminus are relatively stable, being the constant region (C region). The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs).

"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.

"Binds to CD25" or "binds to CD25 protein" means capable of interacting with CD25 or an epitope thereof, which CD25 or epitope thereof may be of human origin.

"CD 25 binding antibody" or "anti-CD 25 antibody" refers to an antibody that is capable of binding to the CD25 subunit of the IL-2 receptor. This subunit is also known as the alpha subunit of the IL-2 receptor.

"Affinity" or "binding affinity" refers to the inherent 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 binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art.

An "epitope" refers to a site on an antigen molecule that is capable of specifically binding to an immunoglobulin or antibody (including antibodies or antigen binding fragments of the present disclosure) binding site and determining the specificity of an antigen. Epitopes can be formed by contiguous amino acids, or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically maintained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation detection assays, among others. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography, two-dimensional nuclear magnetic resonance, and the like.

For the determination or definition of "CDRs," deterministic delineation of CDRs and identification of residues comprising the binding site of an antibody can be accomplished by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. This may be accomplished by any of a variety of 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, kabat numbering system, chothia numbering system, abM numbering system, IMGT numbering system, contact definition, conformational definition. The CDRs of an antibody or antibody binding fragment thereof according to the invention can be defined according to any of the methods described above. The CDRs are defined using the kabat numbering convention in the embodiments of the present disclosure, but those skilled in the art will appreciate that CDRs may also be redefined according to any of Chothia, extended, abM, IMGT, contact, and/or conformational definitions.

Sequence "homology" or "identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both comparison sequences is occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, in the optimal alignment of sequences, if there are 6 matches or homologies at 10 positions in the two sequences, then the two sequences are 60% homologous. In general, a comparison is made when two sequences are aligned to give the greatest percent homology.

"Back mutation" refers to the complete or partial restoration of the mutant (mutant) to its original genotype or phenotype by a second mutation. The complete recovery is also called true back mutation because the base sequence of the mutation is changed to the original base sequence after the second mutation, and the partial recovery is because the second mutation occurs at another position, resulting in partial recovery of the original phenotype. "back mutation" as described in this disclosure includes complete and/or partial recovery.

Antibodies of the present disclosure may be polyclonal, monoclonal, xenogenic, allogeneic, syngeneic, or modified versions thereof, with monoclonal antibodies being particularly useful in a variety of embodiments. Generally, the antibodies of the present disclosure are recombinant antibodies. "recombinant" as used herein refers broadly to products such as cells or nucleic acids, proteins or vectors, which means that the cells, nucleic acids, proteins or vectors have been modified by the introduction of heterologous nucleic acids or proteins or alteration of native 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 native (non-recombinant) cell forms or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all.

"Monoclonal antibody" or "mab" refers to an antibody obtained from a single clonal cell line, which is not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen binding fragments can be obtained by recombinant techniques such as hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques (e.g., CDR-grafting), or other prior art techniques.

"Murine antibodies" or "hybridoma antibodies" are in the present disclosure murine monoclonal antibodies directed against human CD25 or an epitope thereof, prepared according to the knowledge and skill in the art. The preparation is performed by injecting a test subject with a CD25 antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties. In a specific embodiment of the present disclosure, the murine anti-human CD25 antibody or antigen binding fragment thereof may further comprise a light chain constant region of murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of murine IgG1, igG2, igG3 or IgG4 or variant thereof.

The "chimeric antibody" is an antibody in which a variable region of a murine antibody and a constant region of a human antibody are fused, and can reduce an immune response induced by the murine antibody. The method comprises the steps of establishing chimeric antibody, selecting hybridoma secreting murine specific monoclonal antibody, cloning variable region genes from mouse hybridoma cells, cloning constant region genes of human antibody according to requirements, connecting the mouse variable region genes and the human constant region genes into chimeric genes, inserting the chimeric genes into an expression vector, and finally expressing chimeric antibody molecules in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of a human antibody may be selected from the heavy chain constant region of human IgG1, igG2, igG3 or IgG4 or variants thereof.

"Humanized antibody", also known as a CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting the CDR sequences of a mouse into the variable region framework of a human antibody. The strong immune response induced by chimeric antibodies due to the large number of mouse protein components can be overcome. To avoid a decrease in activity while reducing immunogenicity, the human antibody variable region may be subjected to minimal reverse mutation to maintain activity. The antibodies of the present disclosure may be affinity matured humanized antibodies, with the CDRs of the affinity matured antibody parent sequences (including all sequences) being at least 80% identical, e.g., 90% identical. Affinity matured antibodies are antibodies having one or more altered amino acids in one or more CDRs, which results in an improved affinity of the antibody for CD25 as compared to the parent antibody without the altered amino acids.

"Human antibodies" include antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., 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").

By "isolated antibody" is meant a purified state of the bound compound, and in this case it is meant 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 media. The term "isolated" does not mean that such materials are completely absent or that water, buffer or salt are absent unless they are present in amounts that would significantly interfere with the experimental or therapeutic use of the binding compounds described herein.

An "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of the antibody, typically including at least one fragment of an 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 ', 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, e.g., sc-Fv, nanobodies (nanobodies) formed from antibody fragments, and multispecific antibodies. When the binding activity of an 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 an antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as "conservative variants" or "functional conservative variants" of the antibody). In some embodiments, the anti-CD 25 antibodies of the present disclosure encompass bispecific antibodies.

"Single chain Fv" or "scFv" antibodies refer to antibody fragments comprising the VH and VL domains of the antibody, wherein these domains are present in a single polypeptide chain. Fv polypeptides generally also comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

An "isotype" antibody refers to the class of antibodies provided by the heavy chain constant region gene (e.g., igM, igE, igG such as IgGl, igG2, or IgG 4). Isoforms also include modified forms of one of these species, wherein modifications have been made to alter Fc function, e.g., to enhance or attenuate effector function or binding to Fc receptors.

The "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise indicated herein, 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, 5 th edition, public HEALTH SERVICE, national Institutes of Health, bethesda, MD (1991).

"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, to which cell membrane surface antigens bind.

"Complement dependent cytotoxicity" or "CDC" refers to the effector functions of IgG and IgM antibodies, which when bound to surface antigens elicit typical complement pathways, including the formation of membrane attack complexes and target cell lysis. The antibodies of the invention, when bound to CD25, elicit CDC against cancer cells.

The "defragmentation" of the antibody can be performed (1) by producing the defragmentation antibody in a host cell having a fucosylation defect, such as a knockout of the FUT8 gene (Yamane-Ohnuki N et., biotechnol bioeng.87 (5): 614-22, 2004), or (2) by having a S239D, I332E, A L substitution (Kabat numbering) or a combination of any or all of these variations in the Fc domain of the antibody (Lazar GAet al., proc NATL ACAD SCI U S a.103 (11): 4005-4010, 2006).

The Fc region of IgG antibodies interacts with several cellular fcγ receptors (fcγr) to stimulate and regulate downstream effector mechanisms. There are five activating receptors, fcyri (CD 64), fcyriia (CD 32 a), fcyriic (CD 32 c), fcyriiia (CD 16 a) and fcyriiib (CD 16 b), and one inhibitory receptor fcyriib (CD 32 b). Communication of IgG antibodies to the immune system is controlled and mediated by fcγr, which conveys information on antibody perception and collection to the immune system, thereby providing a link between the innate and adaptive immune systems, especially in the context of biological therapy (Hayes J et al, 2016.JInflamm Res 9:209-219). The IgG subclasses differ in their ability to bind fcγr, and this differential binding determines their ability to elicit a range of functional responses. For example, in humans fcγriiia is the primary receptor involved in antibody-dependent cell-mediated cytotoxicity (ADCC) activation, igG1 followed by IgG3 show the highest affinity for this receptor, reflecting their ability to effectively induce ADCC, igG2 has weak binding to this receptor, but it has been found that anti-CD 25 antibodies with the human IgG2 isotype also deplete tregs effectively.

"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 specifically limited, the term includes nucleic acids containing known analogues of natural nucleotides that have similar binding properties to the reference nucleic acid 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 with uridine replaced by pseudouridine, methods of synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases 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))

An "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected or transduced into a host cell. The expression vector contains one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to provide amplification in the host if desired. One type of expression vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of expression vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Upon introduction into a host cell, other vectors (e.g., non-episomal mammalian vectors) integrate into the genome of the host cell and thereby replicate together with the host genome. In addition, certain vectors are capable of directing the expression of genes that are operably linked.

"Host cell" refers to any type of cellular system that can be engineered to produce antibodies disclosed herein. The host cell is selected from a prokaryotic cell, a yeast or a mammalian cell. Prokaryotes include eubacteria such as gram-negative and gram-positive organisms, for example, enterobacteriaceae (Enterobacteriaceae) such as Escherichia (E.coli), enterobacter (Enterobacter), erwinia (Erwinia), klebsiella (Klebsiella), proteus (Proteus), salmonella (Salmonella) such as Salmonella typhimurium (S.tyrmium), serratia (Serratia) such as Serratia marcescens (S.marcescens) and Shigella (Shigella), and Bacillus (Bacillus) such as Bacillus subtilis (B.subsuitis) and Bacillus licheniformis (B.licformis), pseudomonas (Pseudomonas) such as Pseudomonas aeruginosa (P.aegerita) and Streptomyces sp. 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 and not limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody encoding vectors. Saccharomyces cerevisiae (Saccharomyces cerevisiae) is a common baker's yeast, 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 (Schizosaccharomyces pombe), kluyveromyces hosts such as, for example, kluyveromyces lactis (k.lactis), kluyveromyces fragilis (k.fragilis) (ATCC 12,424), kluyveromyces bulgaricus (k.bulgarisus) (ATCC 16,045), k.wintermi (ATCC 24,178), k.walti (ATCC 56,500), kluyveromyces drosophila (ATCC 36,906), kluyveromyces thermotolerans (k.thermoderans) and Kluyveromyces marxianus (k.marxianis), yarrowia (EP 402,226), pichia pastoris (EP 183,070), candida (Candida reesei (Trichoderma reesia) (EP 244,234), candida rugosa (Neurospora crassa), aspergillus, such as, for example, aspergillus kawachii (Aspergillus kawachii), aspergillus, and Aspergillus such as, the Candida (Candida) and Aspergillus sp.

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 subclone 293 cells for growth in suspension culture), baby hamster kidney cells (BHK, ATCC CCL 10), chinese hamster ovary cells/-DHFR (CHO), mouse support cells (servoli cells) (TM 4), monkey kidney cells (CV 1 ATCC CCL 70), african green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cancer cells (Hep G2, HB 8065), mouse mammary tumors (MMT 060562,ATCC CCL51, TRI FS4 cells.

"1, 2, 3, 4 Or 5 amino acid differences" refers to substitutions, additions or deletions of 1,2, 3, 4 or 5 amino acids based on the original sequence.

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

"Administration," "administering," and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administration," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a reagent with the cell, and contacting the reagent with a fluid, wherein the fluid is in contact with the cell. "administration," "administering," and "treatment" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treatment" is meant administration of an internally or externally used therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments thereof of the invention, or conjugates thereof, to a subject already suffering from, suspected of suffering from, predisposed to suffering from, or having symptoms of, one or more diseases or symptoms of which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the subject or population being treated in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. Whether a disease symptom has been reduced can be assessed by any clinical test method that a physician or other healthcare professional typically uses to assess the severity or progression of the symptom. Although embodiments of the invention (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating symptoms of a target disease in a subject, they should alleviate symptoms of the target disease in a statistically significant number of subjects, as determined by any statistical test methods 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.

An "effective amount" as used herein refers to a dosage of a drug that is capable of preventing, alleviating, delaying, inhibiting or curing a condition in a subject. The size of the dose administered is related to the mode of administration of the drug, the pharmacokinetics of the agent, the severity of the disease, the individual sign (sex, weight, height, age) of the subject, etc.

"Preventing" herein refers to delaying or preventing the onset of symptoms of cancer. Prevention may be absolute (and thus no disease occurs) or only effective in some individuals or for a limited period of time.

"Cell", "cell line" and "cell culture" are used interchangeably and all such designations include progeny thereof. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to deliberate or unintentional mutations. Including mutant progeny having the same function or biological activity as screened in the original transformed cell.

Advantageous effects of the invention

1. The anti-CD 25 antibody has higher affinity with human CD25 protein, and the K _D value is less than 5 multiplied by 10 ^-7 M.

2. The anti-CD 25 antibodies of the invention do not inhibit or substantially inhibit IL-2 binding to CD25, and do not affect or substantially affect the function of the Teff cells.

3. The compounds of the present invention have good tumor inhibiting activity and can trigger enhanced ADCC, CDC and/or ADCP reactions.

4. The anti-CD 25 antibodies of the invention bind fcyriiia with higher affinity than fcyri, fcyriic, and/or fcyriib.

Drawings

FIG. 1 inhibitory Activity of antibodies against the cellular interleukin 2 (IL-2) signaling pathway. KA-1292 is a control antibody RG6292 analogue, the constant region sequence of which is derived from human wild type IgG1, the inventors prepared by themselves according to the information of the patent (US 10745485B 2), KA-1329 is a control antibody Daclizumab analogue (Daclizumab, trade name ZINBRYTA, a marketed drug, the sequence of which is obtained from IMGT website), and the sample was prepared by the inventors themselves.

FIGS. 2A-2C data on binding of humanized anti-CD 25 antibodies to Treg cells and Teff cells. FIG. 2A shows the binding data of humanized antibody hu-KD-143-v3, FIG. 2B shows the binding data of humanized antibody hu-KD-161-v2, and FIG. 2C shows the binding data of humanized antibody hu-KD-161-v 4.

FIG. 3 ADCC activity of humanized anti-CD 25 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.

FIG. 4 tumor inhibitory activity of antibodies hu-KD-0143-v3, hu-KD-0161-v4 and hu-KD-0161-v2 in MC38 mouse models.

FIG. 5 percent change in body weight of tumor-bearing mice following dosing in the MC38 in vivo model.

FIG. 6 evaluation results of antitumor activity of antibody hu-KD-0143-v3 alone and in combination with PD-1 antibody in MC38 in vivo model.

Fig. 7 in vivo model of mc38, mice were tested for body weight changes during dosing.

FIG. 8 effect of anti-CD 25 antibodies on immune cell infiltration in tumor tissue.

Detailed Description

The technical scheme of the present invention will be described in detail below with reference to specific embodiments, but the scope of the subject matter of the present invention should not be construed as being limited to the following examples. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

Example 1 production of anti-CD 25 monoclonal antibodies

1.1 Immunization of mice

First, a fusion protein CD25-mFc of human CD25 extracellular domain and mouse IgG2a-Fc (Genbank accession number: AAH 31470.1) was prepared, and the activity of these protein samples was verified by positive CD25 antibody binding. Then, following the common hybridoma mouse immunization scheme, the CD25-mFc protein and Freund's adjuvant are emulsified and then used for immunizing BALB/C healthy mice with the age of 6-8 weeks, and the total number of the BALB/C healthy mice is 6. After immunization for a certain number of times, the mice were collected from the orbits, and serum was collected by centrifugation after standing overnight at 4 ℃, and serum titers (titers) were detected by ELISA and FACS methods. Two mice with the highest serum titers (mice #1 and #2, see table 2) were boosted, and after 3 days, the mice were sacrificed and the spleen was removed to prepare spleen cells.

TABLE 2.6 detection of titers of mice after secondary immunization

1.2 Preparation of hybridomas

Taking myeloma cells SP2/0 with good growth state (the number of living cells is more than 95%), mixing myeloma cells with spleen cells prepared in the step 1.1 according to the proportion of 1:2, re-suspending and cleaning the cell mixture by using an electrotransfer buffer solution, adding a proper amount of electrotransfer buffer solution to re-suspend cells, and adding the cells into an electrotransfer fusion tank for fusion. The cell suspension was added to a complete medium containing HAT medium (SIGMA, cat#h0262) in a pre-prepared volume, 96-well plates were plated, 150-200 μl/well, 37 ℃ and CO ₂ incubator for incubation and observation. Culturing for 7-10 days, changing HAT culture solution into HT culture solution (SIGMA, cat#H20137) for culturing, culturing for 3-4 days, taking supernatant of each hole, performing ELISA and FACS detection, and performing the specific operation in the steps (1) - (3) of the step 1.3.

1.3 Selection of hybridoma antibodies

(1) ELISA detection of binding of hybridoma antibodies to human CD25-HIS protein

Human CD25-HIS protein was diluted in PBS, coated on plates at 50 ng/well and left overnight at 4 ℃. The next day, the plate was blocked with PBS/1% BSA for 2h, hybridoma cell supernatants were added, 50. Mu.L/well, and incubated for 1 hour at room temperature. PBST was washed 5 times. Adding secondary antibody, selecting dilution multiple according to the titer of the enzyme-labeled secondary antibody, incubating for 40min at 37 ℃ with 50 mu L/hole, washing for 5 times, and drying. TMB developing solution 50. Mu.L/well was added and developed at 37℃for 15min to 30min. Stop solution 50. Mu.L/well was added. The OD value of each well was measured at a single wavelength of 450nm (multifunctional microplate reader, molecular Devices, spectraMax M). ELISA is combined with rescreening, positive clones are selected according to the result, and the next screening is carried out.

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

A number of well-grown 293T-hu-CD25 cells (Kyinno, KC-1472) were collected, washed by centrifugation with FACS buffer (1 XPBS+2% FBS), and added to a deep well plate at 2X 10-5 cells/50 ul/well. Adding the supernatant of the hybridoma positive clone obtained in the step (1), placing in a refrigerator at a temperature of 4 ℃ for incubation for 1h, centrifuging, re-suspending cells by using FACS buffer, centrifuging and washing three times. Fluorescence-labeled secondary antibody was added and incubated for 40min at 4 ℃. After the cells were resuspended by FACS buffer centrifugation three times, 200ulFACS buffer, the fluorescence signal was detected by an upper cell flow machine.

(3) ELISA detection of binding of hybridoma antibodies to monkey CD25 protein

ELISA plates were coated with CD25/IL2RAProtein, cynomomolgus, recombinant (His Tag) (available from Yiqiao China/90265-C08H) protein, 50 ng/100. Mu.L/well. The rest steps are the same as the step (1).

(4) Hybridoma cell subcloning

According to the screening results of the 1.3 steps (1) - (3), selecting positive clones which are cross-combined with human and monkey CD25, carrying out cell subcloning culture, culturing in HT culture medium for 7-10 days, selecting monoclonal cell supernatant, screening positive clones by ELISA and FACS method, and screening the parent clones of the 1.3 steps (1) - (3).

(5) Preparation and screening of subcloned antibodies

Positive clones were selected by screening the subcloned cell supernatants, cultured in 50ml, and IgG antibodies in the supernatant were purified for EC ₅₀ assays in ELISA and FACS. At the same time, it was examined whether these antibodies would affect the binding of interleukin 2 (IL-2) to CD25 and thus the downstream signaling pathway. The specific operation is shown in the steps (6) - (8).

(6) ELISA assay for determining EC binding to CD25-HIS protein ₅₀

Human CD25-HIS protein was diluted in PBS, coated on plates at 50 ng/well and left overnight at 4 ℃. The next day, the plates were blocked with PBS/1% BSA for 2h, the hybridoma antibodies to be tested were diluted 3.16-fold, 10 gradients, starting at 20ug/ml, 100. Mu.L/well, and incubated for 1h at room temperature. PBST was washed 5 times. Adding secondary antibody, selecting dilution multiple according to the titer of the enzyme-labeled secondary antibody, incubating for 40min at 37 ℃ with 50 mu L/hole, washing for 5 times, and drying. TMB developing solution 50. Mu.L/well was added and developed at 37℃for 15min to 30min. Stop solution 50. Mu.L/well was added. The OD value of each well was measured at a single wavelength of 450nm (multifunctional microplate reader, molecular Devices, spectraMax M). The data were fitted with the dose-response model in GRAPHPAD PRISM software to give the values for EC ₅₀. The results are shown in Table 3.

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

A number of well-grown 293T-hu-CD25 cells were collected, washed by centrifugation with FACS buffer (1 XPBS+2% FBS), and added to a deep well plate at 2 XPBS 5 cells/50 ul/well. The hybridoma antibodies to be tested were diluted 3.16-fold, 10 gradients, starting at 20ug/ml,100 μl/well, and incubated for 1h at 4 ℃. The cells were resuspended with FACS buffer, centrifuged and washed three times. Fluorescence-labeled secondary antibody was added and incubated for 40min at 4 ℃. After three times washing by FACS buffer centrifugation, 200ulFACS buffer re-suspending the cells, the fluorescence signal was detected by an upflow machine and the MFI read to fit the data in GRAPHPAD PRISM software to obtain the EC ₅₀ values. The results are shown in Table 3.

TABLE 3 binding data for murine antibodies to CD25-HIS protein and 293T-hu-CD25 cells

(8) Inhibitory Activity of antibodies against the cellular interleukin 2 (IL-2) Signal pathway

NK92 cells were starved overnight in serum-free medium, on day 2, a small amount of cells were taken and assayed for CD25 expression on the cell surface with PE-anti-human CD25 antibody, hybridoma antibodies of different concentrations were prepared with serum-free RPMI-1640, the antibodies and starved NK92 cells were mixed in a proportion, the incubator was incubated for 30 min, IL-2 was diluted with whole medium and added to each cell/antibody well, incubator was incubated for 20 min to stimulate p-STAT5 expression, 4x cell lysate was prepared, 10. Mu.L cell lysate was added per well, after mixing, room temperature was increased by 30 min, 4xp-STAT5 antibody mix was prepared, 10. Mu.L Phospho-STAT5 Eu Cryptate antibody was added to 190. Mu. L detection buffer, 10. Mu.L Phospho-STAT 5d 2 antibody was added to 190. Mu. L detection buffer, two antibodies 200AL were mixed together, a total of 400. Mu.L was added to each well in a small volume white plate, the above 16. Mu.L cell lysate and 4. Mu.L of the above-STAT antibody were added to each well, and the Signal-HTp-STAT 5 mix was read out at room temperature (RF-Signal-radio Signal-radio frequency Signal-Signal radio frequency (RF-radio frequency Signal) was measured at room temperature of 665 nm). The results are shown in FIG. 1.

CD25 is a subunit of the cytokine IL2 receptor, and when IL2 binds to its receptor, it activates its downstream signaling pathway, resulting in an increase in STAT5 phosphorylation levels. Experimental results show that Daclizumab analogs that compete for binding to IL2 can cause a significant decrease in STAT5 phosphorylation levels compared to isotype control antibody mIgG, whereas RG6292 analogs that do not compete for binding to IL2 and most of the CD25 antibody clones of the application do not reduce the increase in STAT5 phosphorylation levels caused by IL2, indicating that these antibodies do not compete for binding to CD25 with IL2 and thus do not inhibit IL2 activation signals to T cells.

Example 2 sequencing, production and identification of candidate hybridoma antibodies

(1) According to the screening result of subcloned antibody, selecting m-KD-0140, m-KD-0142, m-KD-0143, m-KD-0150, m-KD-0151, m-KD-0161 and m-KD-0165, and carrying out antibody V gene sequencing on 7 cells, wherein the method is as follows:

Culturing selected monoclonal hybridoma cells, collecting cell sediment, extracting RNA, reversing and recording to prepare ccDNA, adopting a set of mouse V gene primers to respectively amplify light chain and heavy chain V genes of an antibody by PCR, connecting PCR fragments to a T carrier for sequencing analysis, and providing a basis for the next experiment through analysis and comparison after sequence information is obtained. The specific sequences obtained are shown in tables 4-1 and 4-2.

TABLE 4-1 amino acid sequences of the sequence-specific cloned light chain variable region and heavy chain variable region

TABLE 4-2 SEQ ID NO. s corresponding to CDRs of each clone in TABLE 4-1

(CDR regions are defined by Kabat numbering; KD-0140_V1 and KD-0140_V2 have identical VL, different VH sequences)

(2) According to the screening data of the hybridoma mouse antibody, m-KD-0140_V2, m-KD-0143, m-KD-0151, m-KD-0161, m-KD-0165,5 antibody clones were selected to construct plasmids of chimeric antibodies by amplifying the signal peptide + variable region gene fragment using conventional molecular biology techniques, cloning the gene fragment encoding VH into vector pComB-VH carrying the human IgG1 heavy chain constant region (SEQ ID NO:75, see Table 4-3 below) gene fragment, and inserting the gene fragment encoding VL into vector pComB-VL carrying the human antibody Kappa light chain constant region gene fragment (Ck) (SEQ ID NO:76, see Table 4-3 below). HEK293 cells were transfected with the heavy and light chain plasmids of each antibody simultaneously, cultured in a shaker at 37℃for 6-7 days, and the culture supernatants were collected and purified for each cloned IgG antibody by Protein A affinity chromatography.

TABLE 4-3 heavy chain/light chain constant region sequences

(3) ELISA assay for determining EC binding to CD25-HIS protein ₅₀

The EC ₅₀ of these several chimeric antibodies was determined in the same manner as in example 1, and the results are shown in table 5.

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

Table 5 determination of chimeric antibody binding to CD25

The assay results demonstrate that these antibody sequences encode the hybridoma antibodies that are screened for specific binding to CD 25. Clones KD-0143, KD-0151, KD-0161 and KD-0163 have stronger binding force (smaller EC ₅₀ value) than the positive control antibody RG6292 analogue, and KD-0143 and KD-0161 are selected as the lead sequences for humanization.

EXAMPLE 3 humanized engineering of candidate hybridoma antibodies

(1) Clones KD-0143 and KD-0161 were engineered to reduce the risk of immunogenicity of the antibodies in clinical applications.

The humanized design includes the main steps of finding out the sequence similar to that of female parent antibody, or the sequence of fully human antibody with excellent development, as acceptor sequence, CDR grafting, three-dimensional structure modeling, and back mutation with reference to the structure model to avoid the degradation of affinity caused by humanized. The heavy and light chains of the designed humanized sequences are shown in tables 6-1 and 6-2 below, and after preparing these humanized antibodies, the optimal humanized sequences were determined experimentally.

TABLE 6-1 heavy chain/light chain variable region sequences of humanized antibodies

TABLE 6-2 SEQ ID NO: 6-1 corresponding to CDRs of each heavy chain/light chain variable region

(2) Production of humanized antibodies

DNA fragments encoding the above antibody sequences were synthesized and cloned into human IgG1 transient expression vectors using conventional molecular biology techniques (the procedure was the same as the construction of chimeric antibody expression plasmids). Different versions of the heavy chain sequence and the light chain sequence were recombined, HEK293 cells were transfected simultaneously with the combined plasmids, cultured in a 37℃shaker for 6-7 days, and after the culture supernatants were collected, igG antibodies were purified by Protein A affinity chromatography.

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

The corresponding sequences of hu-KD-0143-v3, hu-KD-161-v2 and hu-KD-161-v4 are shown in the following tables 7-1 and 7-2.

TABLE 7-1 correspondence between humanized antibodies and VH and VL

Cloning	Corresponding heavy chain variable region codes	Corresponding light chain variable region codes
			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

TABLE 7-2 heavy chain/light chain sequences corresponding to humanized antibodies

The 3 humanized antibodies were expressed by CHO-K1-D57 cells to give 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 genetically engineered to knock out the Fut8 gene (ZL 201610194325.7) responsible for fucosylation modification. Antibodies prepared using this platform are free of fucosylation and have enhanced ADCC activity.

(3) Characterization of selected humanized antibodies

Determination of affinity the binding constants (K _D) of the humanized antibodies and CD25-ECD-His protein were determined using a molecular interaction analyzer (Fortebio 96 e). 5ug/ml of antibody to be tested is prepared, 4x gradient diluted CD25-ECD-His protein samples (HIS-tagged CD25 extracellular region proteins of human and monkey are all produced by the company) are prepared, and the highest concentration is 20ug/ml. Firstly, a Protein A probe is used for adsorbing an antibody to be detected, then the probe is transferred into a buffer solution (running buffer), then transferred into a CD25-ECD-His Protein sample hole for antigen-antibody combination, and after 100 seconds, the probe is transferred into the buffer solution for dissociation. The instrument analyzes the data with its own software and calculates the binding constant (K _D). The results are shown in Table 8.

TABLE 8 affinity assay results for humanized antibodies

Regulatory T cells (tregs) have high levels of CD25 expression, and effector T cells (teffs) have significantly elevated CD25 levels in activated Teff cells, although they are low. To avoid killing of Teff cells, the KD of CD25 antibodies should not be too high, so that both Treg cells can be efficiently bound by affinity interaction (avidity) and binding to Teff cells with low expression of CD25 is reduced.

The data in Table 8 shows that humanized antibodies hu-KD-143-v3 and hu-KD-161-v2 have similar affinities to the positive control antibody, but hu-KD-161-v4 have approximately 3-fold weaker affinities to both human CD25-His and monkey CD 25-His.

The selected humanized antibodies all have slower dissociation rates (Koff) than the positive control antibodies, and may exhibit higher Fc-mediated ADCC activity, thereby eliminating Treg cells more effectively.

(4) Differential binding to activated primary T cells

Human PBMC cells were incubated with CD3/CD28 antibody magnetic beads for 7-10 days at 37 ℃. Antibody samples of different concentrations were formulated and incubated with a quantity of activated T cells. The cells were washed, and fluorescence-labeled anti-human Fc secondary antibody was added and incubated. Cells were washed, fixed, and perforated with amphoteric agents. And fluorescent-labeled Foxp3 antibody and CD3 antibody. Foxp3 served as a marker for Treg cells. Cells were washed and data was analyzed by a flow cytometer. The results are shown in FIGS. 2A-2C, which show that humanized antibodies hu-KD-143-v3 (FIG. 2A), hu-KD-161-v2 (FIG. 2B) and hu-KD-161-v4 (FIG. 2C) bind significantly more strongly to Treg cells than to Teff cells at the same concentrations.

(5) ADCC Activity of antibodies

KARPAS299 cells (the pseudoplastic JY 803) are human anaplastic large cell lymphoma cells, moderately expressing CD25. KARPAS299 cells in logarithmic growth phase were added to a U-bottom/V-bottom 96-well cell culture plate at a cell concentration of 2X 10 ⁵ cells/mL, 50. Mu.L/well. 50 μl of diluted antibody solution was added to each well of a 96-well plate inoculated with cells, and the highest concentration of antibody in the final system was 50000ng/mL, 5-fold dilution, 9 concentrations, 2 multiplex wells were set for each concentration. The 96-well plate was incubated at 4℃for 30min. Effector cells in the logarithmic growth phase (Jurkat-NFAT-Luc 2-CD16a-V158 reporter cell line) (Kang Yuanbo, cat# KC-1507) were taken and the cell concentration was adjusted to 1X 10 ⁵ cells/mL for use. After incubating the antibody and target cells in 96-well plates twice with PBS, effector cells were added at 100. Mu.L/well. 96-well plates were incubated at 37℃under 5% CO ₂ for 6h. 100 μl of detection reagent (Stable-Lite ^TM Luciferase ASSAY SYSTEM) was added to each well of the 96-well plate, the cells were thoroughly lysed by shaking at room temperature (at least 5 min), 100 μl of lysate was transferred to the full white 96-well plate per well, and the luminescence value was detected using a multifunctional microplate reader. The results are shown in FIG. 3 and Table 9.

TABLE 9 humanized defucose antibodies and non-defucose antibody ADCC Activity

Sample (wild type)	IC₅₀(ng/mL)	Sample (defucose)	IC₅₀(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

Example 4 tumor inhibitory Activity in mouse model

In vivo pharmacodynamics evaluation of CD25 transgenic mouse colon cancer MC38 model

Human CD25 transgenic mice (hIL 2RA, C57B6, baiocin) were used. MC38 cells were inoculated subcutaneously into the anterior costal region of 86 female B-hIL2RA mice and were dosed in groups of 6 mice each at an average tumor volume of 59mm ³. Each group was administered 2 times per week by intraperitoneal injection. The body weight and tumor volume of the mice were measured 3 times a week, and the changes in body weight and tumor volume of the tumor-bearing mice were recorded as a function of the time of administration. Tumor growth inhibition rates TGI _TV (%) and tumor weight inhibition rates TGI _TW (%) were calculated and statistically analyzed.

The results showed that hu-KD-0143-v3 had a remarkable effect of inhibiting tumor growth at a dose of 10mg/kg, hu-KD-0161-v4 and hu-KD-0161-v2 had a certain effect of inhibiting tumor growth at a dose of 10mg/kg (see FIG. 4), while the tumor-bearing mice of each group had good tolerability, stably increased body weight, no other abnormal manifestations and a good general state (see FIG. 5).

EXAMPLE 5 tumor inhibitory Activity in mouse model

(1) In vivo pharmacodynamics evaluation of CD25 transgenic mouse colon cancer MC38 model

Since the humanized antibodies did not bind to murine CD25, human CD25 transgenic mice (hIL 2RA, C57B6, baiocigram) were used to establish tumor models. MC38 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, 100U/ml penicillin and 100. Mu.g/ml streptomycin. MC38 cells in an exponential growth phase are collected, serum is removed, and then tumor cells are resuspended by serum-free basal medium to prepare cell suspension, wherein the cell suspension is prepared by 2X 10 ⁶/mL, and matrigel is added in a ratio of 1:1. Female hCD25 humanized mice (C57 BL/6N) were subcutaneously inoculated with 0.1 mM MC38 cells subcutaneously on the right flank, periodically observed for tumor growth, and dosed until tumor volume had grown to about 97mm ³. The experiments were performed in 4 groups, namely group 1 (normal saline control group), group 2 (hu-KD-0143-v 3,10mg/kg group), group 3 (anti-mPD-1, 1mg/kg group) (antibody source: bioxcell, # BP 0146), group 4 (hu-KD-0143-v 310 mg/kg+anti-mPD-1 mg/kg group), 6 animals each, and were given by intraperitoneal administration (IP) twice per week (BIW) 6 times. Efficacy was evaluated according to tumor inhibition (TGI) and safety was evaluated according to weight change and death of animals. And statistically analyzed.

The results showed that hu-KD-0143-v3 had a remarkable tumor growth inhibition effect at a dose of 10mg/kg, and showed further synergistic effect when combined with anti-mPD1 (see FIG. 6, table 10), whereas tumor-bearing mice of each group had good tolerability, stable body weight elevation, no other abnormal manifestation, and good general status (see FIG. 7).

Table 10 tumor volumes and TGI for each group on day 21 after initiation of MC38 tumor model dosing

Group of	Day 1 (mm) ³)^a	Day 21 (mm ³)	TGI(%)	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

A. mean ± standard error, b. comparison with the veccle group.

(2) Effect of CD25 antibodies on immune cell infiltration in tumor tissue

Following test step (1) above, 5 mice were taken in the combination administration group (hu-KD-0143-v3+ anti-mPD-1) 4 days after the 6 th administration (22 days after the grouping) of the test mice, and 6 mice were taken in each of the remaining groups. Removing fat, fiber, membrane and necrotic tissue from the tumor, cutting the tumor into small pieces, adding tumor tissue digestive enzyme, shaking at 37 ℃ for 20-30 min at low speed, filtering, washing, and re-suspending to PBS to prepare tumor single cell suspension of each mouse. Cell blocking was performed in a 1.5mL EP tube, cells were homogenized, incubated at 4℃for 15-20 min with a suitable amount of fluorescent-labeled T-cell marker antibody, staining was stopped with 1mL of wash solution, cells were washed centrifugally, 1mL of Foxp3 fixation/membrane disruption working solution was added, thoroughly mixed, incubated at 4℃for 60 min with 1 Xmembrane disruption working solution centrifugally washed, recommended amounts of Foxp3 antibody were added, thoroughly mixed, incubated at 4℃for 45 min with 2mL of 1 Xmembrane disruption working solution centrifugally washed, and the cell suspension was transferred into a flow tube after resuspension with 300uLPBS, whereupon the abundance of several immune cells was examined, total immune cells (CD45+, corresponding to FIG. 8A), CD8+ T cells (corresponding to FIG. 8C), tcon cells (CD4+ Foxp3-) (corresponding to FIG. 8B) and Treg cells (CD4+ Foxp3+) (corresponding to FIG. 8D), as a result, see FIG. 8. Compared with a control group, the hu-KD-0143-v3 single drug and the hu-KD-0143-v3 combined anti-mPD-1 can reduce the proportion of Treg cells in mouse MC38 tumor, increase tumor infiltration of CD8+ T cells, tcon cells (CD4+ Foxp 3-) and total immune cells (CD45+), improve tumor immune microenvironment and improve 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) binds to CD25 antigen with a _KD of 100 nM or less;

4) The EC ₅₀ value of binding to CD25 is ≤5ug/mL as determined by ELISA; preferably, the EC ₅₀ value of binding to CD25 is ≤1ug/mL;

5) inducing an enhanced CDC, ADCC and/or ADCP response; preferably, inducing an increased ADCC response;

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

7) Has no or little effect 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,

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 the group consisting of 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.