Anti-CD25 antibody and application thereof

Abstract

The invention provides an anti-CD25 antibody and an application thereof. The antibody comprises the following complementarity determining region CDR-H1, wherein the complementarity determining region CDR-H1 contains an amino acid sequence shown in any one of SED ID NO: 1-9; the CDR-H2 contains an amino acid sequence as shown in any one of SEQ ID NO: 10 to SEQ ID NO: 23; the CDR-H3 contains an amino acid sequence as shown in any one of SEQ ID NO: 24 to 32 and SEQ ID NO: 54 to 55; the CDR-L1 contains an amino acid sequence as shown in any one of SEQ ID NO: 33 to 39; the CDR-L2 contains an amino acid sequence as shown in any one of SEQ ID NO: 40 to 45 and SEQ ID NO: 56 to 57; and the CDR-L3 contains an amino acid sequence as shown in any one of SEQ ID NO: 46-53. The antibody does not affect CD25 to bind to IL-2, and regulatory T cells Treg are specifically removed, so that the aim of treating tumors is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular to an anti-CD25 antibody and an application thereof. Background Art

[0002] Regulatory T cells (Treg) are a type of T cell subset with significant immunosuppressive effects, accounting for about 1%-2% of peripheral blood. They were discovered by Sakaguchi et al. in 1995, and are characterized by the expression of Foxp3, CD25, and CD4. Treg is a type of CD4+T cell subset with low proliferation capacity other than Th1, Th2, and Th17, and plays an important role in immune homeostasis, inducing immune tolerance, chronic inflammatory responses, and promoting tumor progression.

[0003] Treg is crucial for the regulation of immune homeostasis. There are a large number of Treg cells in the tumor microenvironment, which are currently believed to be one of the "culprits" that help tumor cells escape the body's immune surveillance. Treg cells have been found to be significantly increased in many malignant diseases such as lung, pancreatic and breast cancer. Treg is considered an important therapeutic target for tumor immunotherapy. Through the infiltration of Treg in established tumors, regulating the ratio of effector T cells (Teff) and Treg cells to escape immune surveillance is considered to be a key factor in tumor immunotherapy. Regulating the ratio of effector cells and Treg by regulating Teff to treat tumors has become a feasible option for tumor immunotherapy.

[0004] CD25 is one of the iconic proteins on the surface of Treg cells, also known as the alpha receptor of IL-2. IL-2 is a cytokine necessary for the survival of Treg and effector T cells. Compared with effector T cells, Treg mainly binds IL-2 through higher affinity receptors composed of α (CD25), β (CD122), and γ (CD132) subunits. In addition, Tregs have higher CD25 expression than effector T cells. This gives Tregs a competitive advantage in utilizing limited IL-2 in the tumor microenvironment (TME). At the same time, Tregs also secrete immunosuppressive molecules (including immunosuppressive cytokines) to suppress anti-tumor immunity. Tregs produce TGF-β, IL-10, and IL-35, leading to immunosuppression. Therefore, CD25 is an ideal target protein for clearing Tregs.

[0005] Currently on the market is the anti-CD25 monoclonal antibody daclizumab, which is used to treat multiple sclerosis (MS), an autoimmune disease. Basiliximab (Sule), approved for marketing in 2002, is a human / mouse chimeric monoclonal antibody that can antagonize the α chain of the IL-2 receptor (CD25), block the binding of T cells to IL-2, and inhibit the proliferation of Teff cells. The indication is to prevent early acute organ rejection after renal transplantation. Sansure's humanized anti-CD25 monoclonal antibody injection Jiannipai, which was launched in 2019, specifically acts on the activated T cell IL2 receptor α chain, and by binding to CD25, antagonizes the proliferation of T cells mediated by the binding of IL-2 to the receptor. The indication is to prevent acute rejection caused by renal transplantation. Traditional CD25 antibody drugs not only bind to CD25, but also block the binding of CD25 to IL-2. Therefore, the anti-tumor activity of traditional CD25 antibodies is very weak.

[0006] Recently, a study of tumor immunotherapy using CD25 as a target at the University of London in the UK found that the use of specially modified anti-mouse CD25 antibodies can specifically eliminate Treg cells without blocking the precursor of IL-2 / CD25 binding. The team concluded that anti-tumor CD25 antibody drugs need to selectively consume Treg but not affect Teff to better play a role in inhibiting tumors. The research results were transferred to Roche, and the anti-CD25 monoclonal antibody RG6292 is in the clinical evaluation stage (NCT04158583).

[0007] In addition to being expressed on the surface of T cells, CD25 is also expressed on activated B cells and some thymocytes. Due to the complexity of the immune system, anti-tumor drugs need to balance the activity of multiple immune cells. Therefore, the development of a new generation of CD25 antibody drugs has high clinical demand and commercial value. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present invention provides an anti-CD25 antibody and its application, which solves the problem of weak anti-tumor activity of CD25 antibody in the prior art.

[0009] In a first aspect, the present invention provides an anti-CD25 antibody comprising the following complementary determining regions:

[0010] CDR-H1, which contains the amino acid sequence shown in any one of SED ID NOs: 1-9;

[0011] CDR-H2, which contains the amino acid sequence shown in any one of SEQ ID NOs: 10-23;

[0012] CDR-H3, which contains the amino acid sequence shown in any one of SEQ ID NOs: 24-32 and SEQ ID NOs: 54-55;

[0013] CDR-L1, which contains the amino acid sequence shown in any one of SEQ ID NOs: 33-39;

[0014] CDR-L2, which contains the amino acid sequence shown in any one of SEQ ID NOs: 40-45 and SEQ ID NOs: 56-57;

[0015] CDR-L3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 46-53.

[0016] Further, the following complementarity determining regions are included:

[0017] (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46;

[0018] (2) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46;

[0019] (3) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 47;

[0020] (4) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 35, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 42, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48;

[0021] (5) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 28, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46;

[0022] (6) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 42, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48;

[0023] (7) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 15, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46;

[0024] (8) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 16, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 37, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 43, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 49;

[0025] (9) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 50;

[0026] (10) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46;

[0027] (11) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 514;

[0028] (12) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46;

[0029] (13) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46;

[0030] (14) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 22, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 32, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 52;

[0031] (15) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 53;

[0032] (16) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 47;

[0033] (17) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 15, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 57, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46;

[0034] (18) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 15, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 54, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46;

[0035] (19) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 15, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 55, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46.

[0036] In one embodiment, the combination of the six CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) that may be included in the anti-CD25 antibody provided by the present invention is shown in Tables 1 and 2:

[0037] Table 1-1 CDR-H1, CDR-H2, CDR-H3 sequences

[0038]

[0039] Table 1-2 CDR-L1, CDR-L2, CDR-L3 sequences

[0040]

[0041] Table 2-1 CDR-H1, CDR-H2, CDR-H3 sequences

[0042]

[0043]

[0044] Table 2-2 CDR-L1, CDR-L2, CDR-L3 sequences

[0045] LCDR1 SEQ ID NO LCDR2 SEQ ID NO LCDR3 SEQ ID NO 133L2 KASQSVSNDVA 34 GASNRYT 56 QQDYNSPYT 47 133L3 KASQSVSNDVA 34 GASNRYT 56 QQDYNSPYT 47 134L2 RSSQSIVHSNGNTYLE 36 KVSNRFS 42 FQSHPYA 48 134L3 RSSQSIVHSNGDTYLE 35 KVSNRFS 42 FQSHPYA 48 163L1 RASENIYYSLA 33 NANSLED 40 QYNFPPT 46 163L2 RASENIYYSLA 33 NANSLAD 57 QYNFPPT 46 161L1 RASENIYYSLA 33 NANSLED 40 QYNFPPT 46 162L1 RSSQSIVHSNGNTYLE 36 KVSNRFS 42 FQSHPYA 48 167L1 RASESVDTYGKSFMH 37 RASNLQS 43 QQSNENPRT 49 174L1 RASENIYYSLA 33 NANSLED 40 QYNFPPT 46 177L1 RASENIYYSLA 33 NANSLED 40 KQAYKFPPT 50 189L1 RASENIYYSLA 33 NANSLED 40 QYNFPPT 46 191L1 RASENIYYSLA 33 NANSLED 40 QYNFPPT 46 195L1 QATQDIVKNLN 39 YATELAE 45 LQFYEFPLT 52 202L1 KASQSVSNDVA 34 YASNRYT 41 QQDYWSPWT 53

[0046] Further, comprising a heavy chain variable region and a light chain variable region,

[0047] The heavy chain variable region includes the amino acid sequence shown in SEQ ID NOs: 58-73; the light chain variable region includes the amino acid sequence shown in SEQ ID NOs: 74-86, 88-89;

[0048] Or the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 90-92, 94-102, 104, 106, 108-118; the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 119122, 125-133;

[0049] Preferably, the heavy chain variable region and light chain variable region include the following:

[0050] (1) the heavy chain variable region shown in SEQ ID NO:58, and the light chain variable region shown in SEQ ID NO:74;

[0051] (2) the heavy chain variable region shown in SEQ ID NO:59 and the light chain variable region shown in SEQ ID NO:75;

[0052] (3) the heavy chain variable region shown in SEQ ID NO:60, and the light chain variable region shown in SEQ ID NO:76;

[0053] (4) the heavy chain variable region shown in SEQ ID NO:61, and the light chain variable region shown in SEQ ID NO:77;

[0054] (5) the heavy chain variable region shown in SEQ ID NO:62, and the light chain variable region shown in SEQ ID NO:78;

[0055] (6) the heavy chain variable region shown in SEQ ID NO:63, and the light chain variable region shown in SEQ ID NO:79;

[0056] (7) the heavy chain variable region shown in SEQ ID NO:64, and the light chain variable region shown in SEQ ID NO:80;

[0057] (8) the heavy chain variable region shown in SEQ ID NO:65, and the light chain variable region shown in SEQ ID NO:81;

[0058] (9) the heavy chain variable region shown in SEQ ID NO:66, and the light chain variable region shown in SEQ ID NO:82;

[0059] (10) the heavy chain variable region shown in SEQ ID NO:67, and the light chain variable region shown in SEQ ID NO:83;

[0060] (11) the heavy chain variable region shown in SEQ ID NO:68, and the light chain variable region shown in SEQ ID NO:84;

[0061] (12) the heavy chain variable region shown in SEQ ID NO:69, and the light chain variable region shown in SEQ ID NO:85;

[0062] (13) the heavy chain variable region shown in SEQ ID NO:70, and the light chain variable region shown in SEQ ID NO:86;

[0063] (14) the heavy chain variable region shown in SEQ ID NO:71, and the light chain variable region shown in SEQ ID NO:78;

[0064] (15) the heavy chain variable region shown in SEQ ID NO:72, and the light chain variable region shown in SEQ ID NO:88;

[0065] (16) the heavy chain variable region shown in SEQ ID NO:73, and the light chain variable region shown in SEQ ID NO:89;

[0066] (17) the heavy chain variable region shown in SEQ ID NO:90, and the light chain variable region shown in SEQ ID NO:119;

[0067] (18) the heavy chain variable region shown in SEQ ID NO:91, and the light chain variable region shown in SEQ ID NO:120;

[0068] (19) the heavy chain variable region shown in SEQ ID NO:92, and the light chain variable region shown in SEQ ID NO:121;

[0069] (20) the heavy chain variable region shown in SEQ ID NO:92, and the light chain variable region shown in SEQ ID NO:122;

[0070] (21) the heavy chain variable region shown in SEQ ID NO:94, and the light chain variable region shown in SEQ ID NO:121;

[0071] (22) the heavy chain variable region shown in SEQ ID NO:95, and the light chain variable region shown in SEQ ID NO:121;

[0072] (23) the heavy chain variable region shown in SEQ ID NO:96, and the light chain variable region shown in SEQ ID NO:125;

[0073] (24) the heavy chain variable region shown in SEQ ID NO:97, and the light chain variable region shown in SEQ ID NO:126;

[0074] (25) the heavy chain variable region shown in SEQ ID NO:98, and the light chain variable region shown in SEQ ID NO:127;

[0075] (26) the heavy chain variable region shown in SEQ ID NO:99, and the light chain variable region shown in SEQ ID NO:128;

[0076] (27) the heavy chain variable region shown in SEQ ID NO: 100, and the light chain variable region shown in SEQ ID NO: 129;

[0077] (28) the heavy chain variable region shown in SEQ ID NO: 101, and the light chain variable region shown in SEQ ID NO: 130;

[0078] (29) the heavy chain variable region shown in SEQ ID NO: 102, and the light chain variable region shown in SEQ ID NO: 131;

[0079] (30) the heavy chain variable region shown in SEQ ID NO: 102, and the light chain variable region shown in SEQ ID NO: 132;

[0080] (31) the heavy chain variable region shown in SEQ ID NO: 104, and the light chain variable region shown in SEQ ID NO: 131;

[0081] (32) the heavy chain variable region shown in SEQ ID NO: 104, and the light chain variable region shown in SEQ ID NO: 132;

[0082] (33) the heavy chain variable region shown in SEQ ID NO: 106, and the light chain variable region shown in SEQ ID NO: 131;

[0083] (34) the heavy chain variable region shown in SEQ ID NO: 106, and the light chain variable region shown in SEQ ID NO: 132;

[0084] (35) the heavy chain variable region shown in SEQ ID NO: 108, and the light chain variable region shown in SEQ ID NO: 133;

[0085] (36) the heavy chain variable region shown in SEQ ID NO: 109, and the light chain variable region shown in SEQ ID NO: 133;

[0086] (37) the heavy chain variable region shown in SEQ ID NO: 110, and the light chain variable region shown in SEQ ID NO: 133;

[0087] (38) the heavy chain variable region shown in SEQ ID NO: 111, and the light chain variable region shown in SEQ ID NO: 133;

[0088] (39) the heavy chain variable region shown in SEQ ID NO: 112, and the light chain variable region shown in SEQ ID NO: 133;

[0089] (40) the heavy chain variable region shown in SEQ ID NO: 113, and the light chain variable region shown in SEQ ID NO: 133;

[0090] (41) the heavy chain variable region shown in SEQ ID NO: 114, and the light chain variable region shown in SEQ ID NO: 133;

[0091] (42) the heavy chain variable region shown in SEQ ID NO: 115, and the light chain variable region shown in SEQ ID NO: 133;

[0092] (43) the heavy chain variable region shown in SEQ ID NO: 116, and the light chain variable region shown in SEQ ID NO: 133;

[0093] (44) the heavy chain variable region shown in SEQ ID NO: 117, and the light chain variable region shown in SEQ ID NO: 133;

[0094] (45) The heavy chain variable region shown in SEQ ID NO:118 and the light chain variable region shown in SEQ ID NO:133.

[0095] In one embodiment, the combinations of heavy chain variable regions and light chain variable regions that may be included in the anti-CD25 antibodies provided by the present invention are shown in Tables 3 and 4:

[0096] Table 3-1 Heavy chain variable region

[0097]

[0098]

[0099] Table 3-2 Light chain variable region

[0100]

[0101] Table 4-1 Heavy chain variable region

[0102]

[0103]

[0104]

[0105] Table 4-2 Light chain variable region

[0106]

[0107]

[0108]

[0109] Further, the antibody is selected from a monoclonal antibody, an antibody fragment that specifically binds to human CD25, and a fusion protein that specifically binds to an antibody fragment that specifically binds to human CD25;

[0110] Preferably, the antibody is a scFv;

[0111] Preferably, the antibody is a murine antibody or a humanized antibody;

[0112] Preferably, the antibody does not affect the binding of CD25 to IL-2.

[0113] In the present invention, the antibody may refer to a fragment derived from an anti-CD25 antibody and retaining the antigen (CD25) binding affinity of the antibody. In one embodiment, the antibody fragment may be a polypeptide comprising the six CDRs of the above-mentioned anti-CD25 antibody, for example, scFv, scFv-Fc, scFv-Cκ (κ constant region), scFv-Cλ (λ constant region), Fab, but not limited thereto.

[0114] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned anti-CD25 antibody.

[0115] The third aspect of the present invention provides a recombinant vector, which comprises the above-mentioned nucleic acid molecule.

[0116] In one embodiment of the present invention, the recombinant vector may include a nucleic acid molecule encoding a light chain variable region or a light chain and a nucleic acid molecule encoding a heavy chain variable region or a heavy chain, respectively (e.g., in two separate vectors) or all together (e.g., in one vector). The recombinant vector may be used as an expression vector.

[0117] In a fourth aspect, the present invention provides a recombinant cell, wherein the recombinant cell comprises the above-mentioned nucleic acid molecule or recombinant vector.

[0118] In a fifth aspect, the present invention provides a pharmaceutical composition, comprising the above-mentioned anti-CD25 antibody, a nucleic acid molecule, and a recombinant vector;

[0119] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0120] In the present invention, the pharmaceutically acceptable carrier can be selected from any carrier commonly used in antibody preparations. For example, the pharmaceutically acceptable carrier can be selected from lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. One or more, but not limited to this. The pharmaceutical composition may also include one or more selected from diluents, excipients, lubricants, wetting agents, sweeteners, flavor enhancers, emulsifiers, suspending agents, preservatives, etc. commonly used in the manufacture of pharmaceutical compositions.

[0121] The pharmaceutical composition or the antibody can be administered orally or parenterally in a pharmaceutically effective amount. Parenteral administration can be intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, intranasal administration, intrapulmonary administration, rectal administration or topical administration in a lesion. Since proteins or peptides are digested when administered orally, the active ingredient in the composition for oral administration can be coated or formulated to prevent digestion in the stomach. In addition, the antibody or the composition can be administered using an optional device that can deliver the active ingredient to the target cell.

[0122] The pharmaceutical composition may be formulated in the form of a solution, suspension, syrup, emulsified solution, extract, powder, granule, tablet or capsule in oil or aqueous medium, and may further contain a dispersant or stabilizer for formulation.

[0123] In a sixth aspect, the present invention provides the use of the anti-CD25 antibody, the nucleic acid molecule, and the recombinant vector in the preparation of a drug, wherein the drug has any one of the following effects (1) to (6):

[0124] (1) Treat or prevent cancer;

[0125] (2) Specific elimination of Treg cells;

[0126] (3) Activate anti-tumor immunity;

[0127] (4) Eliminate immunosuppressive factors;

[0128] (5) regulating the ratio of effector T cells and Treg cells;

[0129] (6) It does not block IL-2 signal transduction and can induce ADCC effect.

[0130] In a seventh aspect, the present invention provides a method for preparing an anti-CD25 antibody, the method comprising culturing the above-mentioned recombinant cell.

[0131] The technical principle of the present invention is:

[0132] The present invention obtains anti-human CD25 antibodies by immunizing mice with human CD25 protein, and obtains antibodies that do not affect the binding of CD25 to IL-2 through screening, and then specifically eliminates regulatory T cells Treg through the ADCC effect of the Fc part to achieve the purpose of treating tumors.

[0133] Compared with the prior art, the present invention has the following beneficial effects:

[0134] The present invention uses a method of immunizing mice with human CD25 protein and obtains anti-CD25 antibodies that do not affect the binding of CD25 to IL-2 through multiple screenings. In later efficacy experiments, some anti-CD25 antibodies show stronger efficacy than RG6292. BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figure 1 The figure is a diagram showing the effect of Phage on the binding of anti-CD25 antibodies to CD25 in ELISA in Example 1 of the present invention, wherein the anti-CD25 antibodies are numbered 105, 133, 134, 161, 162, 163, 167, 174, 177, 181, 183, 189, 191, 195, 202;

[0136] Figure 2 This is a graph showing the mean fluorescence intensity of 10 μg / mL anti-CD25 antibody binding to KARPASS and SUDHL-1 cells in Example 5 of the present invention, wherein the anti-CD25 antibodies are numbered 105, 131, 133, 134, 161, 162, 163, 167, 174, 177, 181, 183, 189, 191, 195, 202;

[0137] Figure 3 is the average fluorescence intensity of the anti-CD25 antibody binding to KARPASS and SU-DHL-1 cells in Example 5 of the present invention, wherein the anti-CD25 antibodies are numbered 133nH6L2, 133nH6L3, 133nH7L2, 133nH7L3, 133nH9L2, 133nH9L3, and RG6292;

[0138] Figure 4 is the average fluorescence intensity of the anti-CD25 antibodies binding to KARPASS and SU-DHL-1 cells in Example 5 of the present invention, wherein the anti-CD25 antibodies are numbered 163nH1L2, 163H4L1, 163H5L1, 134H3L2, 134H4L2, 134H9L2, 134H12L2, 134H13L2, 134H14L2, 134H16L2, 134H19L2, 134H21L2, 134H24L2, 134H29L2, and RG6292;

[0139] Figure 5 is the average fluorescence intensity of the binding of the anti-CD25 antibody to KARPASS and SU-DHL-1 cells in Example 5 of the present invention, wherein the anti-CD25 antibodies are numbered 161H1L1, 162H1L1, 163nH1L1, 167H1L1, 174H1L1, 177H1L1, 189H1L1, 195H1L1, 202H1L1, and RG6292;

[0140] Figure 6 This is a graph showing changes in the percentage of Tregs in human peripheral blood mononuclear cells (hPBMCs) removed by CD25 antibodies in Example 6 of the present invention;

[0141] Figure 7 The absolute count of Treg cells in the experiment of CD25 antibody depleting Treg in human peripheral blood mononuclear cells (hPBMC) in Example 6 of the present invention;

[0142] Figure 8 This is a diagram showing the percentage of Treg in the spleen and blood after the efficacy experiment of the three antibodies 133nH9L, 134H24L2 and RG6292 in Example 7 of the present invention on the Karpas299 / NCG-hIL15 mouse animal model reconstructed with human PBMCs;

[0143] Fig. 9 This is the Treg% in the spleen and blood after the efficacy experiment of the three antibodies 133nH9L, 134H24L2, RG6292 in Example 8 of the present invention on the MC38 / B-hIL2RA humanized mouse model was completed. DETAILED DESCRIPTION

[0144] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0145] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0146] Technical terms

[0147] The term "antibody" may refer to a protein that specifically binds to a specific antigen, and may be a protein produced by antigen stimulation in the immune system, or a protein produced by chemical synthesis or recombinant production, without specific limitations. The antibody may be non-naturally occurring, for example, produced by recombinant or synthetic production. The antibody may be an animal antibody (e.g., a mouse antibody, etc.), a chimeric antibody, a humanized antibody, or a human antibody. The antibody may be a monoclonal or polyclonal antibody.

[0148] The term "heavy chain" may refer to a full-length heavy chain and fragments thereof, wherein the full-length heavy chain may include a variable region VH (including an amino acid sequence sufficient to provide specificity for an antigen), three constant regions CH1, CH2 and CH3, and a hinge.

[0149] The term "light chain" may refer to a full-length light chain and fragments thereof, wherein the full-length light chain may include a variable region VL (including an amino acid sequence sufficient to provide specificity for an antigen), and a constant region CL.

[0150] The term "complementarity determining region (CDR)" may refer to a portion of the variable region of an antibody that confers antigen binding specificity, and may refer to an amino acid sequence found in the hypervariable region of a heavy or light chain of an immunoglobulin. The heavy and light chains may include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3), respectively. The CDRs may provide contact residues that play an important role in the binding of an antibody to its antigen or antigenic epitope.

[0151] The term "scFv" refers to an antibody heavy chain variable region and a light chain variable region connected by a short peptide of 15-20 amino acids, without an Fc fragment.

[0152] The anti-CD25 antibody can be a monoclonal antibody or a polyclonal antibody, and is, for example, a monoclonal antibody. Monoclonal antibodies can be prepared using methods well known in the art, such as using phage display technology. Alternatively, the anti-CD25 antibody can be constructed in the form of a mouse-derived monoclonal antibody by conventional methods.

[0153] Example 1 Immune library construction and screening

[0154] 1. Animal immunization

[0155] The present invention purchases commercial CD25 (ACRO Lot #ILA-H52H9) to immunize Balb / C mice, and performs booster immunization every 2 weeks. After 4-5 immunizations, tail vein blood is collected to detect the serum titer of anti-CD25 antibodies. According to the titer detection results, mice with the best immune effect are selected to collect spleen cells.

[0156] 2. Database construction

[0157] Splenocytes were collected from immunized mice, and spleen B cells were isolated therefrom. RNA was extracted using Trizol (Thermofisher, Cat. No. 15596018), and RT-PCR was performed using a reverse transcription kit (Thermofisher, Cat. No. 11756500) according to the instructions to obtain total cDNA.

[0158] In the first round of PCR, cDNA was used as a template, and the degenerate primers of mouse antibodies were used to amplify the heavy chain fragment and light chain fragment of the antibody respectively. In the second round, overlapping PCR was performed, the heavy chain fragment mixture and the light chain fragment mixture were mixed, forward primers and reverse primers were added, and the heavy and light chain fragments were spliced ​​by PCR to obtain a single-chain antibody ScFv fragment with restriction sites at both ends. The phage display vector pcomb3X and the ScFv fragment were digested with SfiI (NEB), and the DNA fragments were recovered, and the recovered products were mixed in an appropriate proportion and connected with T4 ligase. TG1 electroporation competent state was prepared, and the ligation product was electroporated into the TG1 competent state to obtain the original library of the antibody library.

[0159] The original antibody library was cultured in 2YT medium containing 100ug / ml ampicillin. When the OD value reached 0.8, M13K07 was added and allowed to stand at 37℃ for 30 minutes. Then centrifuged, the precipitate was resuspended in 2YT-AK (100μg / mL Amp, 20μg / mL Kana) medium, and cultured overnight at 30℃ and 220 rpm. The overnight culture was centrifuged, the supernatant was collected and 1 / 4 volume of PEG / NaCl was added for precipitation. Centrifuged at 12000rpm for 20 minutes, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended in PBS and sterilized by filtration with a 0.22 micron filter to obtain the phage display antibody library.

[0160] 3. Screening

[0161] The direct coating method was used for well plate screening. 10 μg / mL of CD25 protein was coated in a 96-well plate with 0.1M sodium bicarbonate buffer at pH 9.6, and the phage display antibody library was added to bind to CD25, and eluted with 0.1M Glycine buffer at pH 2.2. After 4 rounds of screening, phage display clones that bind to CD25 were enriched.

[0162] After the screening, 400 monoclones were randomly selected from the 4th round of screening products for phage display. Coat with CD25 antigen 1 μg / mL at 37 degrees for 1 hour. After washing 3 times with PBS, add 100uL of the phage to be tested and bind at 37 degrees for 1 hour. Wash 3 times with 0.1% PBST, add HRP-labeled mouse anti-M13 antibody and bind at 37 degrees for 1 hour. After washing 3 times with 0.1% PBST, add tetramethylbenzidine (TMB, Biolegend, 421101) for color development, stop the color development with 1M HCl, and read the absorbance at 450nm with an enzyme reader. Sequence the positive clones to obtain the antibody sequence. Figure 1The ELISA effect diagram of monoclonal antibodies 105, 133, 134, 161, 162, 163, 167, 174, 177, 181, 183, 189, 191, 195, and 202 binding to CD25 is shown for Phage.

[0163] Example 2 Synthesis, expression and purification of antibody genes

[0164] 1. Synthesis of antibody genes

[0165] The present invention entrusts Suzhou Jinweizhi Biotechnology Co., Ltd. to perform gene synthesis of positive antibodies to obtain heavy chain and light chain antibody genes with complete constant regions. According to the operating method mentioned in the Molecular Cloning Experiment Guide, DH10B is transformed and the bacterial solution is cultured for transient expression plasmid preparation. According to the operating methods mentioned in the Qiagen Mini-prep Kit and the Qiagen Endofree Maxi-prep Kit, the plasmid used for expression is prepared. Express according to the standard operating procedure for transient expression. Specifically, prepare the required volume and adjust the cell density to 2.0-2.5E6 / ml Expi293 cell suspension, mix the plasmid and transfection reagent PEI in a certain proportion and place at room temperature for 10 minutes, then add the mixed solution to the adjusted cell suspension, and culture it in a shaker at 115rpm, 37°C, and 5% CO2. After 24 hours, add 50×KT-Feed to a final concentration of 1×, continue to culture in the shaker for 5-7 days, centrifuge at 8500rpm for 15min to collect the cell supernatant, and then purify it.

[0166] 2. Purification

[0167] The purification process includes two steps of purification: Protein A affinity chromatography and ultrafiltration concentration. Specifically, add 4M sodium chloride solution to the centrifuged cell culture supernatant to a final concentration of 0.2M sodium chloride, and adjust the pH to 7.5 with 1M tris(hydroxymethyl)aminomethane; use 2×PBS pH 7.4 to balance the AT Protein A Diamond (Borgron) filler, and rinse with equilibrium buffer until the baseline is stable after loading to remove most non-specifically bound impurities (such as host proteins, host DNA, etc.); then add 1M tris(hydroxymethyl)aminomethane pH8.0 to the tube where the sample is to be collected, and add 1mL 1M tris(hydroxymethyl)aminomethane pH8.0 to 10mL of sample; finally, elute with 100mM glycine, 25mM L-arginine, and 10mM sodium chloride pH3.2, and collect the eluted protein. The final sample is exchanged with 1×PBS, pH7.4 buffer using a 30kDa ultrafiltration concentration tube.

[0168] Example 3 Affinity Detection

[0169] Antibody affinity detection was performed using Biacore 8K. PROAsensor was selected to capture the antibody to be tested, and CD25 purified protein was used as the analyte for affinity test. The antibody to be tested was diluted to 1-2 μg / mL, the buffer was 0.02% PBST, the flow rate was 10 μL / min, and the capture time was 60S. The purified CD25 recombinant protein was gradiented from 200nm and set to zero concentration. The binding time of the antibody to be tested and CD25 was 150-200S, the dissociation time was 400-1000S, and the flow rate was 30uL / min. The test results are shown in Table 5.

[0170] Table 5 Affinity of CD25 antibody binding to Human CD25

[0171]

[0172]

[0173] It can be seen that the affinity of the antibody is 10 -7 ~10 -10 range. This indicates that the antibody has a high affinity.

[0174] Example 4 Antibody Competition Experiment

[0175] Fortebio Red 96E was used to detect whether the binding epitope of the antibody to CD25 was in competition with IL-2. The experimental steps are as follows: Step 1: Activate the PROA chip; Step 2: Dilute the antibody to be tested to 10μg / mL with PBST 0.02% Tween20|0.1% BSA, and use PROA Sensor to capture the CD25 antibody to be tested, with a binding time of 120S-180S; Step 3: Bind to 200nM CD25, and the binding time is generally set to 120-180S; Step 4: Bind to a mixture of 200nM CD25 and 200nM IL-2, with a binding time of 120-180S. Each clone needs to set a control, so there will be 3 control groups, which are simply represented as follows:

[0176] The first test group: PROAsensor captures the antibody to be tested + binds CD25 + binds CD25 / IL-2mix

[0177] The second control group 1: PROAsensor captures the antibody to be tested + binds CD25 + binds CD25

[0178] Group 3 Control Group 2: PROAsensor binding buffer + binding CD25 + binding CD25

[0179] The fourth group, control group 3: PROAsensor binding buffer + binding CD25 + binding CD25 / IL-2mix.

[0180] The test results are shown in Table 6.

[0181] Table 6 Antibody competition experiment results

[0182]

[0183]

[0184] The above results indicate that the binding epitope of the antibody to CD25 is a non-competitive epitope and does not compete with IL-2.

[0185] Example 5 Binding experiment of anti-CD25 antibody and cells with high expression of CD25

[0186] Flow cytometry was used to detect the binding of anti-CD25 antibodies to CD25-expressing lymphoma cell lines SU-DHL-1 and KARPAS-299. Cells were collected during the logarithmic growth phase, resuspended in PBS containing 2% FBS, and 10 μg / mL was added to each well of a 96-well plate. 5 Cells (50 μL), then add different concentrations of anti-CD25 antibodies to make the final concentration range from 0 to 10 μg / mL, and set up a blank control. Incubate at room temperature for 1 hour, add 1 μg / mL FITC-labeled goat anti-mouse Fc II (Invetrogen, 406001), and incubate at room temperature for 30 minutes. Use a flow cytometer (Cytek, NL-3000) to detect the binding of antibodies. The tested anti-CD25 antibodies can bind to the surface of cells SU-DHL-1 and KARPAS-299. The results are as follows Figure 2 , Figure 3 , Figure 4 , Figure 5 shown.

[0187] Example 6 CD25 antibody eliminates Treg cells in human peripheral blood mononuclear cells (hPBMC)

[0188] Resuscitate hPBMC, wash with PBS, and adjust the cell number to 2 × 10 6 cell / mL, 1.5mL / well was plated in a 6-well plate. The sample to be tested was diluted to a starting concentration of 8mg / mL, and then a 2-fold serial dilution was performed for a total of 4 concentrations, 1.5mL / well was added to the plated cells, and hPBMC and the sample to be tested were co-incubated in a 37°C, 5% CO2 incubator for 7 days.

[0189] For each sample, 1.5×10 6The cells were centrifuged at 300g for 5 min, the supernatant was discarded, 1mL staining buffer (BD, catalog number: 554656) was added, and the supernatant was discarded after centrifugation at 300g for 5 min. The cells were resuspended in 100μL staining buffer, and APC-H7 mouse Anti-human CD3 (BD, catalog number: 560176), BV650 mouse Anti-human CD4 (BD, catalog number: 563875), BV605 mouse Anti-human CD8 (BD, catalog number: 564116), BB515 mouse Anti-human CD56 (BD, catalog number: 564488), PE Mouse anti-human CD127 (BD, catalog number: 557938), and APC mouse Anti-human CD25 (BD, catalog number: 555434) antibodies were added to each tube. Mix well, incubate at 4℃ in the dark for 30min, add 1mL staining buffer, centrifuge at 300g for 5min, and the supernatant was discarded. Add 400 μL staining buffer to each tube to resuspend the cells, detect using flow cytometry, use Flowjo [v10.8] for data analysis, and Graphpad Prism [v9.0] for graphing. Figure 6 , Figure 7 shown.

[0190] The results show that, compared with NTC, the percentage and absolute count of Treg in all tested samples were downregulated to varying degrees.

[0191] Example 7 Anti-CD25 Antibody Animal Efficacy Experiment

[0192] In order to further verify the anti-tumor effect of anti-CD25 antibodies in animal models, the present invention selected two anti-CD25 antibodies obtained by screening for animal experiments. In this experiment, PBMC (human peripheral blood mononuclear cells) from healthy adult donors were inoculated into experimental mice one day before tumor inoculation, and the cell inoculation amount was 6.5×10 6 On the day of inoculation, KARPAS-299 tumor cells, which belong to human anaplastic large cell lymphoma, were inoculated subcutaneously on the right back of NCG-hIL15 female mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) at a cell inoculation volume of 5×10 6 / mouse. When the tumor grows to 78mm 3 The animals were divided into groups and administered at about 1:00 pm, with a total of 4 groups, including 1 negative control group and 2 groups of RG6292 treatment group and antibody treatment group, with 6 animals in each group. The dosage was 20 mg / kg, twice a week for two weeks, and the administration was intraperitoneal injection.

[0193] 1. Inspect animal weight and tumor volume

[0194] The tumor volume was measured twice a week using a vernier caliper and the mice were weighed using an electronic balance. The long and short diameters of the tumor were measured and the volume was calculated using the following formula: tumor volume TV (mm 3 )=0.5×long diameter(mm)×short diameter(mm) 2 Tumor growth inhibition rate TGI TV % = (1 - T / C) × 100%, T / C = RTV of treatment group / RTV of control group.

[0195] At the end of the experiment, the animals were euthanized and the tumor growth inhibition rate was calculated. Specific data are shown in Table 7.

[0196] Table 7 Tumor inhibition rate on day 15 (%)

[0197] Antibody Name Tumor inhibition rate on day 15 (%) RG6292-human IgG1 35.90 133nH9L2-human IgG1 45.73 13424L2-human IgG1 20.76

[0198] In this animal efficacy experiment, all antibodies showed good anti-human anaplastic large cell lymphoma activity, among which the 133-human IgG1 antibody had a tumor growth inhibition rate of 45.73% relative to the control group on day 15. In this experiment, the animals gained normal weight and no drug-related toxic side effects were observed.

[0199] 2. Detection of spleen and peripheral blood of experimental mice

[0200] After the experiment, the spleen and peripheral blood of the mice were taken separately, and the spleen and peripheral blood of 6 mice in each group were mixed for subsequent experiments. The spleen was ground into a single cell suspension, and 600 μL was taken from each group to a 15 mL centrifuge tube, centrifuged at 300g for 5 minutes, and the supernatant was discarded. 600 μL of peripheral blood was taken from each group to a 15 mL centrifuge tube. Then 1800 μL RBC lysis buffer (CWBIO, catalog number: CW0613S) was added to each tube, incubated at 4°C for 15 minutes, centrifuged at 300g for 5 minutes, and the supernatant was discarded. 1200 μL RBC lysis buffer was added to each tube, incubated at 4°C for 15 minutes, centrifuged at 300g for 5 minutes, and the supernatant was discarded. 2 mL PBS was added to each tube, centrifuged at 300g for 5 minutes, and the supernatant was discarded. Repeat once, and finally add 2 mL PBS to resuspend.

[0201] Take 2×10 6 The cells were transferred to the flow cytometry tubes and centrifuged at 300 g for 5 min to discard the supernatant. The cells were resuspended in 100 μL staining buffer and FITC Anti-Human CD45 (Tonbo, Catalog No.: 35-0459-T100), violetFluor TM450Anti-Human CD3 (Tonbo, item number: 75-0038-T100), redFluor TM 710Anti-Human CD4(Tonbo, item number: 80-0048-T100), Brilliant Violet650 TM Anti-human CD127 (Biolegend, Catalog No.: 351326), PE Anti-Human CD25 (Tonbo, Catalog No.: 50-0259-T100) antibodies. Mix well, incubate at room temperature in the dark for 30 minutes, add 2mL staining buffer to each tube, centrifuge at 300g for 5 minutes, and discard the supernatant. Add 400μL staining buffer to each tube to resuspend the cells, detect using flow cytometer, use Flowjo [v10.8] for data analysis, and Graphpad Prism [v9.0] for graphing. The results are shown in Figure 8 shown.

[0202] The results show that after the efficacy experiment of the three antibodies, 133nH9L, 134H24L2, and RG6292, on the Karpas299 / NCG-hIL15 mouse model reconstructed with human PBMC, the Treg% in the spleen and blood were downregulated to varying degrees.

[0203] Example 8 Anti-CD25 Antibody Animal Efficacy Experiment

[0204] The present invention selected two anti-CD25 antibodies obtained by screening and one control for animal efficacy experiments. In this experiment, MC38 tumor cells belonging to mouse colon cancer were inoculated subcutaneously on the right back of B-hIL2RA female mice (Biocytogenetics Jiangsu Gene Biotechnology Co., Ltd.), and the cell inoculation amount was 1×10 6 / mouse. When the tumor grows to 81mm 3The mice were divided into groups and administered at about 1:10 pm, with a total of 5 groups, including 1 negative control group, 3 RG6292 treatment groups and antibody treatment groups, with 6 animals in each group. The dosage was 5 mg / kg, administered twice a week for two weeks, and intraperitoneal injection. After 2 weeks, the spleen and peripheral blood of the mice were taken respectively, and the spleen and peripheral blood of 6 mice in each group were mixed for subsequent experiments. The spleen was ground into a single cell suspension, and 600 μL was taken from each group to a 15 mL centrifuge tube, centrifuged at 300g for 5 minutes, and the supernatant was discarded. 600 μL of peripheral blood was taken from each group to a 15 mL centrifuge tube. Subsequently, 1800 μL RBC lysis buffer (CWBIO, catalog number: CW0613S) was added to each tube, incubated at 4°C for 15 minutes, and centrifuged at 300g for 5 minutes to discard the supernatant. 1200 μL RBC lysis buffer was added to each tube, incubated at 4°C for 15 minutes, and centrifuged at 300g for 5 minutes to discard the supernatant. Add 2 mL of PBS to each tube, centrifuge at 300 g for 5 min, discard the supernatant, repeat once, and finally add 2 mL of PBS to resuspend.

[0205] Take 2×10 6 The cells were transferred to the flow cytometry tube and centrifuged at 300 g for 5 min to discard the supernatant. The cells were resuspended in 100 μL staining buffer and FITC Anti-Mouse CD45 (Tonbo, Catalog No.: 35-0451-U100), violetFluor TM 500 Anti-Mouse CD3 (Tonbo, Catalog No.: 85-0032-U100), APC-Cyanine7 Anti-Mouse CD4 (Tonbo, Catalog No.: 25-0042-U100), PerCP-Cyanine5.5 Anti-Mouse CD127 (Tonbo, Catalog No.: 65-1271-U100), PE Anti-Mouse CD25 (Tonbo, Catalog No.: 50-0251-U100) antibodies. Mix well, incubate at room temperature in the dark for 30 minutes, add 2mL staining buffer to each tube, centrifuge at 300g for 5 minutes, and discard the supernatant. Add 400μL staining buffer to each tube to resuspend the cells, detect using a flow cytometer, use Flowjo [v10.8] for data analysis, and Graphpad Prism [v9.0] for graphing. The results are shown in Fig. 9 shown.

[0206] The results show that after the efficacy experiments on the MC38 / B-hIL2RA humanized mouse model, the Treg% in the spleen and blood of the three antibodies, 133nH9L, 134H24L2, and RG6292, were downregulated to varying degrees.

[0207] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. An anti-CD25 antibody, characterized in that: Contains the following complementarity determining regions: CDR-H1, which contains the amino acid sequence shown in any one of SED ID NOs: 1-9; CDR-H2, which contains the amino acid sequence shown in any one of SEQ ID NOs: 10-23; CDR-H3, which contains the amino acid sequence shown in any one of SEQ ID NOs: 24-32 and SEQ ID NOs: 54-55; CDR-L1, which contains the amino acid sequence shown in any one of SEQ ID NOs: 33-39; CDR-L2, which contains the amino acid sequence shown in any one of SEQ ID NOs: 40-45 and SEQ ID NOs: 56-57; CDR-L3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 46-53.

2. An anti-CD25 antibody according to claim 1, characterized in that: Contains the following complementarity determining regions: (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46; (2) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46; (3) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 47; (4) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 35, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 42, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; (5) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 28, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46; (6) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 42, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; (7) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 15, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46; (8) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 16, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 37, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 43, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 49; (9) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 50; (10) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46; (11) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 514; (12) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46; (13) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46; (14) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 22, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 32, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 52; (15) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 53; (16) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 47; (17) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 15, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 57, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46; (18) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 15, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 54, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46; (19) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 15, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 55, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

46.

3. An anti-CD25 antibody according to claim 2, characterized in that: comprising a heavy chain variable region and a light chain variable region, The heavy chain variable region includes the amino acid sequence shown in SEQ ID NOs: 58-73; the light chain variable region includes the amino acid sequence shown in SEQ ID NOs: 74-86, 88-89; Or the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 90-92, 94-102, 104, 106, 108-118; the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 119-122, 125-133; Preferably, the heavy chain variable region and light chain variable region include the following: (1) the heavy chain variable region shown in SEQ ID NO:58, and the light chain variable region shown in SEQ ID NO:74; (2) the heavy chain variable region shown in SEQ ID NO:59 and the light chain variable region shown in SEQ ID NO:75; (3) the heavy chain variable region shown in SEQ ID NO:60, and the light chain variable region shown in SEQ ID NO:76; (4) the heavy chain variable region shown in SEQ ID NO:61, and the light chain variable region shown in SEQ ID NO:77; (5) the heavy chain variable region shown in SEQ ID NO:62, and the light chain variable region shown in SEQ ID NO:78; (6) the heavy chain variable region shown in SEQ ID NO:63, and the light chain variable region shown in SEQ ID NO:79; (7) the heavy chain variable region shown in SEQ ID NO:64, and the light chain variable region shown in SEQ ID NO:80; (8) the heavy chain variable region shown in SEQ ID NO:65, and the light chain variable region shown in SEQ ID NO:81; (9) the heavy chain variable region shown in SEQ ID NO:66, and the light chain variable region shown in SEQ ID NO:82; (10) the heavy chain variable region shown in SEQ ID NO:67, and the light chain variable region shown in SEQ ID NO:83; (11) the heavy chain variable region shown in SEQ ID NO:68, and the light chain variable region shown in SEQ ID NO:84; (12) the heavy chain variable region shown in SEQ ID NO:69, and the light chain variable region shown in SEQ ID NO:85; (13) the heavy chain variable region shown in SEQ ID NO:70, and the light chain variable region shown in SEQ ID NO:86; (14) the heavy chain variable region shown in SEQ ID NO:71, and the light chain variable region shown in SEQ ID NO:78; (15) the heavy chain variable region shown in SEQ ID NO:72, and the light chain variable region shown in SEQ ID NO:88; (16) the heavy chain variable region shown in SEQ ID NO:73, and the light chain variable region shown in SEQ ID NO:89; (17) the heavy chain variable region shown in SEQ ID NO:90, and the light chain variable region shown in SEQ ID NO:119; (18) the heavy chain variable region shown in SEQ ID NO:91, and the light chain variable region shown in SEQ ID NO:120; (19) the heavy chain variable region shown in SEQ ID NO:92, and the light chain variable region shown in SEQ ID NO:121; (20) the heavy chain variable region shown in SEQ ID NO:92, and the light chain variable region shown in SEQ ID NO:122; (21) the heavy chain variable region shown in SEQ ID NO:94, and the light chain variable region shown in SEQ ID NO:121; (22) the heavy chain variable region shown in SEQ ID NO:95, and the light chain variable region shown in SEQ ID NO:121; (23) the heavy chain variable region shown in SEQ ID NO:96, and the light chain variable region shown in SEQ ID NO:125; (24) the heavy chain variable region shown in SEQ ID NO:97, and the light chain variable region shown in SEQ ID NO:126; (25) the heavy chain variable region shown in SEQ ID NO:98, and the light chain variable region shown in SEQ ID NO:127; (26) the heavy chain variable region shown in SEQ ID NO:99, and the light chain variable region shown in SEQ ID NO:128; (27) the heavy chain variable region shown in SEQ ID NO: 100, and the light chain variable region shown in SEQ ID NO: 129; (28) the heavy chain variable region shown in SEQ ID NO: 101, and the light chain variable region shown in SEQ ID NO: 130; (29) the heavy chain variable region shown in SEQ ID NO: 102, and the light chain variable region shown in SEQ ID NO: 131; (30) the heavy chain variable region shown in SEQ ID NO: 102, and the light chain variable region shown in SEQ ID NO: 132; (31) the heavy chain variable region shown in SEQ ID NO: 104, and the light chain variable region shown in SEQ ID NO: 131; (32) the heavy chain variable region shown in SEQ ID NO: 104, and the light chain variable region shown in SEQ ID NO: 132; (33) the heavy chain variable region shown in SEQ ID NO: 106, and the light chain variable region shown in SEQ ID NO: 131; (34) the heavy chain variable region shown in SEQ ID NO: 106, and the light chain variable region shown in SEQ ID NO: 132; (35) the heavy chain variable region shown in SEQ ID NO: 108, and the light chain variable region shown in SEQ ID NO: 133; (36) the heavy chain variable region shown in SEQ ID NO: 109, and the light chain variable region shown in SEQ ID NO: 133; (37) the heavy chain variable region shown in SEQ ID NO: 110, and the light chain variable region shown in SEQ ID NO: 133; (38) the heavy chain variable region shown in SEQ ID NO: 111, and the light chain variable region shown in SEQ ID NO: 133; (39) the heavy chain variable region shown in SEQ ID NO: 112, and the light chain variable region shown in SEQ ID NO: 133; (40) the heavy chain variable region shown in SEQ ID NO: 113, and the light chain variable region shown in SEQ ID NO: 133; (41) the heavy chain variable region shown in SEQ ID NO: 114, and the light chain variable region shown in SEQ ID NO: 133; (42) the heavy chain variable region shown in SEQ ID NO: 115, and the light chain variable region shown in SEQ ID NO: 133; (43) the heavy chain variable region shown in SEQ ID NO: 116, and the light chain variable region shown in SEQ ID NO: 133; (44) the heavy chain variable region shown in SEQ ID NO: 117, and the light chain variable region shown in SEQ ID NO: 133; (45) The heavy chain variable region shown in SEQ ID NO:118 and the light chain variable region shown in SEQ ID NO:

133.

4. An anti-CD25 antibody according to claim 1, characterized in that: The antibody is selected from a monoclonal antibody, an antibody fragment that specifically binds to human CD25, and a fusion protein that specifically binds to an antibody fragment that specifically binds to human CD25; Preferably, the antibody is a scFv; Preferably, the antibody is a murine antibody or a humanized antibody; Preferably, the antibody does not affect the binding of CD25 to IL-2.

5. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the anti-CD25 antibody according to any one of claims 1 to 4.

6. A recombinant vector, characterized in that: Comprising the nucleic acid molecule of claim 5.

7. A recombinant cell, characterized in that: Comprising the nucleic acid molecule according to claim 5 or the recombinant vector according to claim 6.

8. A pharmaceutical composition, characterized in that: Comprising the anti-CD25 antibody according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, and the recombinant vector according to claim 6; Preferably, a pharmaceutically acceptable carrier is also included.

9. Use of the anti-CD25 antibody according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, and the recombinant vector according to claim 6 in the preparation of a drug, wherein the drug has any one of the following effects (1) to (6): (1) Treat or prevent cancer; (2) Specific elimination of Treg cells; (3) Activate anti-tumor immunity; (4) Eliminate immunosuppressive factors; (5) regulating the ratio of effector T cells and Treg cells; (6) It does not block IL-2 signal transduction and can induce ADCC effect.

10. A method for preparing an anti-CD25 antibody, the method comprising culturing the recombinant cell according to claim 7.

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