Antibodies targeting CD25 and their production methods and applications

Abstract

The present invention discloses an antibody or variant thereof targeting CD25, the monoclonal antibody targeting CD25 is a naturally occurring antibody, has the activity of binding to human CD25 and cynomolgus monkey CD25, has a higher ADCC effect after Fc engineering, has the effect of eliminating Treg and lymphoma cells in in vitro experiments, and does not block the IL-2 signal pathway.

Description

[Technical field]

[0001] This application claims priority to Chinese patent application No. 2022100512339, filed on January 17, 2022. This application is incorporated herein by reference in its entirety. (Technical field)

[0002] The present application relates to the biomedicine field, specifically to an antibody or variant thereof that targets CD25, and its production method and application. [Background technology]

[0003] Regulatory T cells (Treg) are a subgroup of T cells that control autoimmune reactivity in the body and play an important role in maintaining immune tolerance, autoimmunity, and antitumor immunity in the body. Existing studies have found that an increase in tumor-infiltrating Treg cells is negatively correlated with the prognosis of various tumors. In addition, the ratio of effector T cells (Teff) to Treg in the tumor microenvironment affects tumor progression and therapeutic efficacy. Although these studies suggest that antitumor therapeutic schemes targeting Treg have potential value, there have been few successful cases of antitumor therapy targeting Treg in clinical practice, the main reason being that it is still unclear what can be used to design marker molecules that specifically target Treg. In mouse experiments, anti-CTLA-4 antibodies have shown the effect of eliminating tumor-infiltrating Treg cells, and this effect is achieved by activating Fc receptors (FcRs). However, in clinical experiments, the eliminating effect of anti-CTLA-4 antibodies on Treg is still unclear. Some studies have found that enhancing the affinity of anti-CTLA-4 antibodies with FcRs can improve the efficacy of antibody clinical trials, but not all CTLA-4 antibody treatments cause a decrease in Treg cells. Therefore, there is still a need to develop specific marker molecules that target Tregs in order to realize anti-tumor treatment that targets Tregs.

[0004] CD25 is the alpha chain of the interleukin-2 receptor (IL2Rα), which is constitutively highly expressed in Treg cells, but is less expressed in Teff cells. This property makes CD25 a potential target that can specifically target Treg cells. However, the lack of research on anti-CD25 antibodies in mice and clinical experiments has limited their development. Recent studies have found that in mice, reinforcing antibody-dependent cell-mediated cytotoxicity (ADCC) by increasing the affinity of anti-CD25 antibodies to FcRs can significantly improve the in vivo anti-tumor activity of the antibody. Studies have also found that anti-CD25 antibodies with enhanced ADCC function through Fc optimization can significantly improve the in vivo anti-tumor activity of anti-PD-1 antibodies. Meanwhile, CD25 is expressed in both Hodgkin's lymphoma and multiple non-Hodgkin's lymphomas, and the expression rate of CD25 is close to 100% in adult T-cell leukemia / lymphoma and hairy cell leukemia. Based on the high expression of CD25 in blood tumors, the research of antibody-drug conjugates (ADCs) targeting CD25 has become a hotspot. The conventional anti-CD25 ADC drug ADCT-301 has shown excellent therapeutic effects in clinical studies of Hodgkin's lymphoma. Based on the high expression of CD25 in Treg and lymphoma cells, the development of this targeted antibody drug can achieve anti-tumor effects by reinforcing ADCC function through Fc optimization, while it can also be developed by the ADC method.

[0005] The development of anti-CD25 antibody drugs has previously focused on blocking antibodies against the ligand interleukin-2, including monoclonal antibodies and ADCs. Recent studies have found that anti-CD25 antibodies that do not block interleukin-2 show better antitumor activity in mouse experiments, which may be because CD25 is also expressed on effector T cells (Teff), and blocking antibodies inhibit the activity of Teff by inhibiting the interleukin-2 signal pathway. Teff is the cell that plays a major role in the process of tumor immunity, and this property of blocking antibodies weakens its antitumor effect. Non-blocking anti-CD25 antibodies avoid this problem and have more prominent antitumor activity.

[0006] In the field of anti-CD25 monoclonal antibody development strategies, many companies are currently working on the development of anti-CD25 interleukin-2 non-blocking antibodies, the most advanced being Roche's RG6292, which is in phase 1 clinical studies and is indicated for solid tumors. In the field of ADC development strategies, many companies are also conducting research, but the most advanced is ADCT-301, which is currently in phase 2 clinical studies and is indicated for Hodgkin's lymphoma. However, clinically, there are no highly selective anti-CD25 interleukin-2 non-blocking antibodies yet. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides an anti-CD25 interleukin-2 non-blocking antibody or variant thereof with high affinity, high selectivity and high biological activity, and provides an antibody molecule that has the ability to be endocytosed by cells and is suitable for the development of ADC drugs. [Means for solving the problem]

[0008] In a first aspect of the invention there is provided an antibody or variant thereof targeting CD25, said antibody comprising a light chain variable region and a heavy chain variable region, wherein said antibody is selected from the group consisting of: the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 96, 111 and 133, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 20, 44 and 74, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 104, 111 and 127, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 22, 46 and 76, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 94, 110 and 134, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 23, 48 and 78, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 97, 113 and 129, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 16, 40 and 70, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 94, 116 and 135, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 23, 47 and 77, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 95, 111 and 127, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 14, 38 and 68, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 98, 114 and 130, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 17, 41 and 71, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 99, 115 and 131, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 18, 42 and 72, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 100, 113 and 132, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 14, 43 and 73, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 103, 110 and 134, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 21, 45 and 75, respectively; or The light chain variable region is characterized in that it comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 105, 111 and 136, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 18, 42 and 80, respectively.

[0009] As is well known to those skilled in the art, there are various methods for defining the CDRs of an antibody, including, for example, the Chothia method based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (World Wide Web imgt.cines.fr / ), and the North method based on affinity propagation clustering using a large number of crystal structures. CDR definitions In the present invention, the amino acid sequences of the recited CDRs are all shown according to the Chothia definition rules (sequences in the claims of the present invention are also shown according to the Chothia definition rules).

[0010] Here, Laa-Lbb may refer to the amino acid sequence from positions aa to bb from the N-terminus of the antibody light chain, and Haa-Hbb may refer to the amino acid sequence from positions aa to bb from the N-terminus of the antibody heavy chain. For example, L24-L34 may refer to the amino acid sequence from positions 24 to 34 according to the Chothia numbering rules from the N-terminus of the antibody light chain, and H26-H32 may refer to the amino acid sequence from positions 26 to 32 according to the Chothia numbering rules from the N-terminus of the antibody heavy chain.

[0011] As will be appreciated by those skilled in the art, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to cover the complementarity determining regions defined by any one of the above known schemes described in the present invention. Although the scope of what is claimed in the claims of the present invention is the sequence shown based on the Chothia definition rules, corresponding amino acid sequences based on other CDR definition rules also fall within the scope of protection of the present invention.

[0012] Therefore, when an antibody is defined by a specific CDR sequence defined in the present invention, the scope of said antibody further covers antibodies whose variable region sequences include said specific CDR sequences, but whose described CDR boundaries differ from the specific CDR boundaries defined in the present invention due to the application of a different scheme (e.g. a different CDR definition rule or combination).

[0013] In a specific embodiment, the mutant has one to three amino acid residues added, deleted or substituted in the amino acid sequence of the antibody targeting CD25.

[0014] Preferably, the mutant has an amino acid substitution at position 1 of HCDR1 and position 3 of HCDR2 of the heavy chain variable region of the antibody, and / or an amino acid substitution at positions 10 to 11 of LCDR1 and position 1 or 4 of LCDR3 of the light chain variable region of the antibody, Preferably, the variant has an amino acid sequence that has at least 85% sequence identity with the original amino acid sequence and maintains or improves binding of the antibody to an antigen of interest, said at least 85% sequence identity being preferably at least 90% sequence identity, more preferably at least 95%, 96%, 97% or 98% sequence identity, and most preferably at least 99% sequence identity.

[0015] More preferably, the variant is selected from the group consisting of: the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 101, 113 and 129, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 19, 40 and 70, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 101, 113 and 139, respectively; and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 19, 40 and 70, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 102, 113 and 129, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 19, 40 and 70, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 94, 116 and 137, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 23, 50 and 77, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 94, 116 and 138, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 23, 51 and 77, respectively; or The light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 94, 116 and 138, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 23, 50 and 77, respectively.

[0016] In a specific embodiment, the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 171 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 171; and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 153 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 153; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 173 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 173; and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 155 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 155; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 175 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 175; and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 157 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 157; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 165 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 165; and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 148 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 148; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 174 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 174; and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 156 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 156; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 163 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 163; and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 146 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 146; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 166 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 166; and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 149 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 149; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 167 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 167; and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 150 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 150; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 168 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 168; and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 151 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 151; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 172 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 172, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 154 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 154; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 177 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 177; and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 159 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 159; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 169 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 169; and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 152 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 152; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 180 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 180; and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 152 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 152; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 170 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 170; and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 152 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 152; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 178 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 178; and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 160 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 160; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 179 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 179, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 161 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 161; or The light chain variable region comprises a VL whose amino acid sequence is set forth in SEQ ID NO:179 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:179, and the heavy chain variable region comprises a VH whose amino acid sequence is set forth in SEQ ID NO:160 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:160.

[0017] In certain specific embodiments, the antibody or variant thereof comprises a chimeric antibody, a humanized antibody, or a fully human antibody.

[0018] Preferably, the structure of the antibody or variant thereof is Fab, Fab', F(ab')2, Fv, IgG, Fd, dAb, or a bispecific or multispecific antibody comprising the antibody or variant thereof, or a monoclonal or polyclonal antibody produced from the antibody, and the Fv is preferably an scFv, and preferably, the antibody or variant thereof having the IgG structure comprises a light chain and a heavy chain, the light chain comprises an amino acid sequence as set forth in SEQ ID NO:207 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:207, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:189 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:189; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:209 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:209, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:191 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:191; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:211 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:211, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:193 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:193; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:201 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:201, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:184 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:184; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:210 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:210, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:192 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:192; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:199 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:199, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:182 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:182; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:202 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:202, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:185 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:185; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:203 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:203, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:186 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:186; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:204 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:204, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:187 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:187; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:208 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:208, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:190 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:190; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:213 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:213, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:195 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:195; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:205 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:205, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:188 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:188; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:216 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:216, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:188 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:188; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:206 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:206, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:188 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:188; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:214 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:214, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:196 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:196; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:215 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:215, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:197 or which has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:197; or The light chain comprises the amino acid sequence set forth in SEQ ID NO:215, and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:196.

[0019] In the present invention, a "Fab fragment" consists of one light chain and one heavy chain CH1 and variable region. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fc" region contains two heavy chain fragments including the CH2 and CH3 domains of an antibody. The two heavy chain fragments consist of two or more disulfide bonds and are held together by the hydrophobic effect of the CH3 domain. A "Fab' fragment" contains one light chain and a portion of one heavy chain including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, which allows the two heavy chains of the two Fab' fragments to form an interchain disulfide bond to form a F(ab')2 molecule. A "F(ab')2 fragment" contains two light chains and two heavy chains including a portion of the constant region between the CH1 and CH2 domains, which allows the two heavy chains to form an interchain disulfide bond to form a F(ab')2 molecule. Thus, an F(ab')2 fragment consists of two Fab' fragments held together via disulfide bonds between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody, but lacks the constant region.

[0020] In the present invention, said scFv (single chain antibody fragment) may be a conventional single chain antibody in the art, which comprises a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids, in which the VL and VH domains are paired to form a monovalent molecule via a linker that allows them to be produced as a single polypeptide chain [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules may have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeated G4S amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (G4S)4 or (G4S)3 may be used, although variants thereof may also be used.

[0021] In the present invention, the Fd may be a conventional antibody fragment in the art, which includes the portion of Fab other than VL and CL, contains approximately 225 amino acid residues, and includes VH, CH1 and a part of the hinge region.

[0022] In the present invention, said dAb may be any conventional in the art, being some minimal functional antibody fragment consisting of a VH or VL domain, retaining full antigen-binding specificity, and having a molecular weight approximately one-tenth the molecular weight of a normal antibody.

[0023] The term "multispecific antibodies" is used in the broadest sense to cover antibodies with specificity for multiple epitopes, including, but not limited to, antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), where the VH-VL unit has specificity for multiple epitopes, antibodies with two or more VL and VH regions, where each VH-VL unit binds to a different target or a different epitope on the same target, antibodies with two or more single variable regions, where each single variable region binds to a different target or a different epitope on the same target, full length antibodies, antibody fragments, diabodies, and triabodies, antibody fragments linked together covalently or non-covalently, etc.

[0024] The antibody or variant thereof of the present invention includes a monoclonal antibody. A monoclonal antibody or mAb or Ab according to the present invention refers to an antibody obtained from a single clonal cell line, which is not limited to a eukaryotic, prokaryotic or phage clonal cell line.

[0025] In a second aspect of the invention, there is provided a chimeric antigen receptor, said chimeric antigen receptor comprising an antibody or variant thereof according to the first aspect of the invention.

[0026] In a third aspect of the invention there is provided an isolated nucleic acid, said nucleic acid encoding an antibody or variant thereof according to the first aspect of the invention, or a chimeric antigen receptor according to the second aspect of the invention.

[0027] In a fourth aspect of the present invention there is provided a recombinant expression vector comprising the isolated nucleic acid according to the third aspect of the present invention, preferably the recombinant expression vector is a plasmid, cosmid, phage or viral vector, preferably the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

[0028] In a fifth aspect of the present invention, there is provided a transformant, the transformant comprising a recombinant expression vector according to the fourth aspect of the present invention in a host cell, preferably the host cell is a prokaryotic or eukaryotic cell, more preferably the host cell is selected from a yeast cell, a mammalian cell or other cell suitable for producing an antibody or an antigen-binding fragment thereof, wherein the mammalian cell is, for example, a 293 cell or a CHO cell.

[0029] In a sixth aspect of the invention there is provided an immune cell, said immune cell comprising a chimeric antigen receptor according to the second aspect of the invention, preferably said immune cell is a T cell or a NK cell, more preferably said NK cell is a peripheral blood NK cell, a cord blood derived NK cell, a stem cell differentiated NK cell, or a NK cell line such as the NK-92 cell line.

[0030] In a seventh aspect of the present invention, there is provided a method for producing an antibody or variant thereof that targets CD25, the method comprising culturing a transformant described in the fifth aspect of the present invention, and obtaining an antibody or variant thereof that targets CD25 from the culture.

[0031] In an eighth aspect of the present invention there is provided an antibody drug conjugate comprising a cytotoxic agent and an antibody or variant thereof targeting CD25 according to the first aspect of the present invention.

[0032] In a ninth aspect of the present invention, there is provided a pharmaceutical composition, the pharmaceutical composition comprising an antibody or variant thereof targeting CD25 according to the first aspect of the present invention, an isolated nucleic acid according to the third aspect of the present invention, a recombinant expression vector according to the fourth aspect of the present invention, a transformant according to the fifth aspect of the present invention, an immune cell according to the sixth aspect of the present invention and / or an antibody-drug conjugate according to the eighth aspect of the present invention, and a pharma- ceutical acceptable carrier; Preferably, the pharmaceutical composition further comprises one or more of the group consisting of hormonal agents, targeted small molecule agents, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0033] In some embodiments, the pharmaceutical composition or pharmaceutical formulation of the present invention comprises a suitable pharmaceutically acceptable carrier, e.g., pharmaceutical carriers, pharmaceutical excipients known in the art, including pharmaceutical additives, e.g., buffering agents. As used herein, "pharmaceutical acceptable carriers" or "pharmaceutical carriers" include any and all physiologically compatible solvents, dispersion media, isotonicity agents, and absorption delaying agents, etc. Pharmaceutical carriers suitable for the present invention may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a suitable carrier when the pharmaceutical composition is administered intravenously. Saline, aqueous dextrose and glycerin solutions may also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, diol, water, ethanol, and the like. The use of excipients and their applications are also described in "Handbook of Pharmaceutical Excipients", 5th Edition, RC Rowe, PJ Eskey and SCOwen, Pharmaceutical Press, London, Chicago. If desired, the compositions may further contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral formulations may contain standard pharmaceutical carriers and / or excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin, and the like. The pharmaceutical formulations or compositions according to the present invention may be prepared by mixing the antibody or antigen-binding fragment thereof of the present invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Edition, edited by Osol, A. (1980)), preferably in the form of a lyophilized formulation or aqueous solution.The pharmaceutical composition or formulation of the present invention may further comprise more than one active ingredient as required for the particular indication being treated, preferably with those active ingredients having complementary activities that do not adversely affect each other. For example, it may be desirable to further provide other anti-infective active ingredients, such as other antibodies, anti-infective active agents, small molecule drugs or immunomodulators. The active ingredients are present in suitable combinations in amounts effective for the intended use. Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody or antigen-binding fragment thereof of the present invention, the matrices being in the form of shaped articles, such as films or microcapsules.

[0034] The present invention relates to an application of an antibody or variant thereof targeting CD25 according to any one of the first aspect of the present invention, a chimeric antigen receptor according to the second aspect of the present invention, an isolated nucleic acid according to the third aspect of the present invention, a recombinant expression vector according to the fourth aspect of the present invention, a transformant according to the fifth aspect of the present invention, an immune cell according to the sixth aspect of the present invention, an antibody-drug conjugate according to the eighth aspect of the present invention and / or a pharmaceutical composition according to the ninth aspect of the present invention in the manufacture of a drug for diagnosing, preventing and / or treating a tumor.

[0035] In a tenth aspect of the present invention, there is provided a kit, the kit comprising an antibody targeting CD25 or a variant thereof according to the first aspect of the present invention, a chimeric antigen receptor according to the second aspect of the present invention, an isolated nucleic acid according to the third aspect of the present invention, a recombinant expression vector according to the fourth aspect of the present invention, a transformant according to the fifth aspect of the present invention, an immune cell according to the sixth aspect of the present invention, an antibody-drug conjugate according to the eighth aspect of the present invention, or a pharmaceutical composition according to the ninth aspect of the present invention, Preferably, the kit further comprises (i) a device for administering the antibody or variant thereof, or chimeric antigen receptor, or immune cells, or antibody drug conjugate, or pharmaceutical composition, and / or (ii) instructions for use.

[0036] In an eleventh aspect of the present invention there is provided a kit of parts comprising kit A and kit B, wherein The kit A contains an antibody or an antigen-binding fragment thereof targeting CD25 according to the first aspect of the present invention, a chimeric antigen receptor according to the second aspect of the present invention, an isolated nucleic acid according to the third aspect of the present invention, a recombinant expression vector according to the fourth aspect of the present invention, a transformant according to the fifth aspect of the present invention, an immune cell according to the sixth aspect of the present invention, an antibody-drug conjugate according to the eighth aspect of the present invention, and / or a pharmaceutical composition according to the ninth aspect of the present invention; The kit B contains another anti-tumor antibody or a pharmaceutical composition comprising the other anti-tumor antibody, and / or one or more of the group consisting of a hormonal preparation, a targeted small molecule preparation, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic agent, a cytotoxic agent, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.

[0037] In a twelfth aspect of the present invention there is provided a method of detecting CD25, said method comprising using an antibody or variant thereof targeting CD25 according to the first aspect of the invention, a chimeric antigen receptor according to the second aspect of the invention, an immune cell according to the sixth aspect of the invention, an antibody drug conjugate according to the eighth aspect of the invention and / or a pharmaceutical composition according to the ninth aspect of the invention.

[0038] Preferably, said detection is not for diagnostic and / or therapeutic purposes.

[0039] In a thirteenth aspect of the present invention, there is provided a method for preventing, treating or alleviating a disease in a subject, said method comprising administering to said subject a therapeutically effective amount of an antibody or variant thereof targeting CD25 as described in any one of the first aspect of the present invention, an isolated nucleic acid as described in the third aspect of the present invention, a recombinant expression vector as described in the fourth aspect of the present invention, a transformant as described in the fifth aspect of the present invention, an immune cell as described in the sixth aspect of the present invention, an antibody drug conjugate as described in the eighth aspect of the present invention and / or a pharmaceutical composition as described in the ninth aspect of the present invention.

[0040] In some specific embodiments, the subject's condition is a proliferative disease, e.g., a tumor or cancer. In some embodiments, the subject is afflicted with a formed tumor, e.g., a solid tumor.

[0041] In some embodiments, there is provided a method for reducing intratumoral or tumor-infiltrating Treg cell number in a subject, and in some embodiments, there is provided a method for eliminating or inhibiting intratumoral or tumor-infiltrating Treg cell activity in a subject, each comprising administering to the subject an effective amount of an antibody or variant thereof targeting CD25 according to any one of the first aspect of the invention, a chimeric antigen receptor according to the second aspect of the invention, an isolated nucleic acid according to the third aspect of the invention, a recombinant expression vector according to the fourth aspect of the invention, a transformant according to the fifth aspect of the invention, an immune cell according to the sixth aspect of the invention, an antibody drug conjugate according to the eighth aspect of the invention and / or a pharmaceutical composition according to the ninth aspect of the invention.

[0042] In some embodiments, there is provided a method of increasing intratumoral Teff / Treg ratio in a subject, comprising administering to said subject an antibody or variant thereof targeting CD25 according to any one of the first aspect of the invention, a chimeric antigen receptor according to the second aspect of the invention, an isolated nucleic acid according to the third aspect of the invention, a recombinant expression vector according to the fourth aspect of the invention, a transformant according to the fifth aspect of the invention, an immune cell according to the sixth aspect of the invention, an antibody drug conjugate according to the eighth aspect of the invention and / or a pharmaceutical composition according to the ninth aspect of the invention.

[0043] In some embodiments, there is provided a method of enhancing ADCC against tumor cells in a subject, comprising administering to said subject an antibody or variant thereof targeting CD25 according to any one of the first aspect of the invention, a chimeric antigen receptor according to the second aspect of the invention, an isolated nucleic acid according to the third aspect of the invention, a recombinant expression vector according to the fourth aspect of the invention, a transformant according to the fifth aspect of the invention, an immune cell according to the sixth aspect of the invention, an antibody drug conjugate according to the eighth aspect of the invention and / or a pharmaceutical composition according to the ninth aspect of the invention.

[0044] In some specific embodiments, the ADCC effect against tumor cells in the subject is potentiated.

[0045] In some embodiments, there is provided use of the antibody or variant thereof targeting CD25 according to any one of the first aspect of the invention, the chimeric antigen receptor according to the second aspect of the invention, the isolated nucleic acid according to the third aspect of the invention, the recombinant expression vector according to the fourth aspect of the invention, the transformant according to the fifth aspect of the invention, the immune cell according to the sixth aspect of the invention, the antibody drug conjugate according to the eighth aspect of the invention and / or the pharmaceutical composition according to the ninth aspect of the invention for the manufacture of a medicament for preventing, treating or alleviating a disease in a subject, for the manufacture of a medicament for reducing the number of intratumoral or tumor-infiltrating Treg cells in a subject, for the manufacture of a medicament for eliminating or inhibiting intratumoral or tumor-infiltrating Treg cell activity in a subject, for the manufacture of a medicament for increasing the intratumoral Teff / Treg ratio in a subject, for the manufacture of a medicament for enhancing ADCC against tumor cells in a subject.

[0046] In some specific embodiments, the subject is afflicted with or has a proliferative disease (e.g., cancer or tumor). The tumor includes, but is not limited to, carcinoma, lymphoma, leukemia, germ cell mass, and sarcoma. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (HCC), Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), various myelomas, gastrointestinal (ductal) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, renal cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer.

[0047] In some specific embodiments, the cancer or tumor may be a solid tumor, including, but not limited to, sarcoma (including cancers arising from transformed cells of mesenchymal origin in tissues (e.g., cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells, or fibrous connective tissues)), carcinoma (including tumors arising from epithelial cells), mesothelioma, neuroblastoma, retinoblastoma, and the like. Cancers related to solid tumors include, but are not limited to, brain cancer, lung cancer, gastric cancer, duodenal cancer, esophageal cancer, breast cancer, colon and rectal cancer, renal cancer, bladder cancer, kidney cancer, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, oral cancer, sarcoma, eye cancer, thyroid cancer, urethral cancer, vaginal cancer, cervical cancer, lymphoma, and the like.

[0048] In some specific embodiments, the cancer is related to tumors that express CD25, including, but not limited to, lymphomas, such as Hodgkin's lymphoma, and lymphocytic leukemias, such as chronic lymphocytic leukemia (CLL).

[0049] Each of the above preferred conditions can be arbitrarily combined in accordance with common knowledge in the art to obtain each of the preferred embodiments of the present invention.

[0050] All of the reagents and materials used in the present invention are commercially available.

[0051] The positive effects of the present invention are as follows: The monoclonal antibody targeting CD25 of the present invention is a naturally occurring antibody, and has the activity of binding to human CD25 and cynomolgus monkey CD25. After Fc engineering, it has a higher ADCC effect, and has a good effect of eliminating Treg and lymphoma cells in in vitro experiments, while not blocking the signal pathway of IL-2, not affecting the activity of Teff cells, and has good internalization activity, making it a potential antibody molecule suitable for the development of ADC drugs. [Brief description of the drawings]

[0052] [Figure 1] FIG. 1 shows binding of anti-CD25 H2L2 antibody to CHO-K1 cells overexpressing human CD25. [Diagram 2] FIG. 1 shows binding of anti-CD25 H2L2 antibody to HEK293 cells overexpressing monkey CD25. [Diagram 3] FIG. 1 shows binding of anti-CD25 H2L2 antibody to the tumor cell line SU-DHL-1, which endogenously expresses human CD25. [Figure 4] FIG. 1 shows binding of anti-CD25 H2L2 antibody to the tumor cell line Karpas299, which endogenously expresses human CD25. [Diagram 5] FIG. 1 shows binding of anti-CD25 H2L2 antibody to human Treg and Teff cells. [Figure 6] FIG. 1 shows that anti-CD25 H2L2 antibody does not block the binding of human interleukin-2 to CHOK1-huCD25 cells. [Figure 7] FIG. 1 shows that anti-CD25 H2L2 antibody does not block the binding of human interleukin-2 to SU-DHL-1 cells. [Figure 8] AlphsLisa detected an anti-CD25 H2L2 antibody that does not inhibit the phosphorylation level of STAT5, a downstream protein in the human interleukin-2 signaling pathway. [Figure 9]This is FACS that detected anti-CD25 H2L2 antibody, which does not inhibit the phosphorylation level of STAT5, a downstream protein in the human interleukin-2 signal pathway. [Figure 10] FIG. 1 shows detection of the ADCC effect of anti-CD25 H2L2 antibody against SU-DHL-1 cell line using a reporter gene cell line. [Figure 11] FIG. 1 shows detection of the ADCC effect of anti-CD25 H2L2 antibody on human Treg cells using a reporter gene cell line. [Figure 12] FIG. 1 shows detection of the ADCC effect of anti-CD25 H2L2 antibody against human Teff cells using a reporter gene cell line. [Figure 13] FIG. 1 shows the endocytosis of Karpas 299 cells toward anti-CD25 H2L2 antibody. [Figure 14] FIG. 1 shows the endocytosis of SU-DHL-1 cells toward anti-CD25 H2L2 antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the above examples. The following examples do not specify the specific conditions of the experimental methods, but they can be selected according to conventional methods and conditions or according to the product instructions.

[0054] Example 1. Obtaining anti-CD25 antibody molecules The CD25 recombinant protein is used to immunize laboratory animals to obtain antibody molecules that specifically bind to CD25, and the laboratory animals may be mice, rats, rabbits, sheep, camels, etc. Usually, the obtained antibody molecules are of non-human origin. After obtaining antibodies of non-human origin, it is necessary to carry out humanization operations on these molecules using antibody engineering techniques to reduce immunogenicity and increase drug discovery potential. However, the humanization process of antibodies is technically complex, and humanized molecules often have reduced affinity for antigens. Meanwhile, advances in transgenic technology have made it possible to create genetically engineered mice that have a human immunoglobulin immune repertoire and delete their endogenous mouse immune repertoire. The Harbour H2L2 mouse (Harbour Antibodies BV) is a transgenic mouse with a human immunoglobulin immune repertoire, and the antibodies produced from such transgenic mice have a fully human-derived sequence, so no further humanization operations are required, greatly improving the efficiency of developing therapeutic antibodies.

[0055] 1.1. Mouse immunization Harbour H2L2 mice were immunized multiple times with soluble recombinant human CD25-his fusion protein (Sino Biological, #10165-H08H) as the antigen. The antigen protein and the immune adjuvant were mixed to form an immunogen reagent, which was then injected subcutaneously into the groin or intraperitoneally. In each round of immunization, the total injection volume received by each mouse was 100 microliters. In the first immunization, each mouse was immunized with an immunogen reagent prepared by mixing 50 micrograms of the antigen protein with complete Freund's adjuvant (Sigma, #F5881) in a 1:1 volume ratio. In each subsequent round of boosting, each mouse was immunized with an immunogen reagent prepared by mixing 25 micrograms of the antigen protein with Sigma Adjuvant System adjuvant (Sigma, #S6322). The interval between each round of boosting was at least 2 weeks, and there were usually 6 to 7 rounds of boosting. The immunization times were days 0, 14, 28, 42, 56, 70, 84, and 98, and antibody titers in mouse serum were detected on days 49 and 77. Five days before splenic B cell isolation of H2L2 mice, the final booster was administered at a dose of 25 micrograms of antigen protein per mouse.

[0056] 1.2. Serum titer detection At specific time points, mouse serum was collected and the titers of CD25-binding antibodies in the serum were detected by FACS method.

[0057] A cDNA encoding human CD25 (whose Genebank number is NM000417) was obtained by gene synthesis, cloned into an expression vector pcDNA3.1, and the expression vector was transfected into CHO cells to construct CHOK1-huCD25 cells that highly express human CD25.

[0058] CHOK1-huCD25 cells were incubated with serially diluted mouse serum at 4°C for 1 hour, and the cells were washed twice. The secondary antibody anti-rat IgG (H+L) (Life technologies, A11006) was added and incubated at 4°C for 1 hour. After washing twice, the cells were resuspended and detected by flow cytometry (BD, CantoII). CHOK1 cells were used as background control.

[0059] 1.3. Screening for anti-CD25 antibodies using hybridoma technology A cDNA encoding cynomolgus monkey CD25 (whose Genebank number is NM001283704) was obtained by gene synthesis, cloned into the expression vector pcDNA3.1, and the expression vector was transfected into HEK293 cells to construct HEK293-cynoCD25 cells that highly express cynomolgus monkey CD25.

[0060] Mice with high serum titers were selected, and sacrificed after one final immunization. Splenocytes and SP2 / 0 myeloma cells (ATCC, CRL-1581) were taken for electrofusion. The cell ratio was 4:1, and the electrofusion parameters were V1:50V, t1:15s, V2:600V, t2:20μs, t3: 0.5s, n:1, t4: 7s, V+ / -: +, fade: on. The cells were resuspended in a culture medium of DMEM containing 20% ​​FBS and HT, and 1 × 10 5After 24 hours, 100μL / well of DMEM containing 20% ​​FBS and 2× HT was added and the culture was continued. The supernatant was then taken to detect the antibody titer. Generally, 9 to 15 days after fusion, the supernatant was taken from the mouse immunized with recombinant human CD25-his protein and pre-screened by ELISA to detect the binding with recombinant human CD25-his protein, and the positive clones were further confirmed by FACS to detect the binding ability with CHOK1 cell line overexpressing human CD25 (CHOK1-huCD25) and HEK293 cell line overexpressing cynomolgus monkey CD25 (HEK293-cynoCD25). In addition, the blocking effect of the positive clones on the binding of interleukin-2 to CHOK1-huCD25 was detected by FACS method. The wells that showed no blocking effect and were positive were further subcloned by finite dilution method, and then further cloned by ELISA and FACS method. Clones that bind well to human and monkey CD25 were selected and sequenced. In this example, the sequences of the variable domains of the anti-CD25 monoclonal antibody molecules obtained from immunized Harbour H2L2 mice are human-derived antibody sequences.

[0061] 1.4. Production of fully human recombinant antibodies After obtaining the sequences of the light and heavy chain variable domains encoding the antibody molecule, the sequences of the light and heavy chain variable domains and the sequences of the corresponding light and heavy chain constant domains of a human antibody can be fused and expressed using conventional recombinant DNA technology to obtain a recombinant antibody molecule. In this example, an antibody heavy chain variable domain sequence (VH) was genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding a human IgG1 antibody heavy chain constant domain sequence to encode a full-length heavy chain that produces an IgG1 antibody. An antibody light chain variable domain sequence (VL) was genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding a human antibody Igκ light chain constant domain sequence to encode a full-length light chain that produces an antibody. In this example, the sequences of the anti-CD25 monoclonal antibody molecule variable domains obtained from the immunized Harbour H2L2 mice are human-derived antibody sequences, so that a fully human-derived anti-CD25 recombinant IgG1 antibody was also obtained in this example.

[0062] Both the plasmid encoding the antibody heavy chain and the plasmid encoding the antibody light chain can be transfected into mammalian host cells (e.g., human embryonic kidney cells HEK293) and purified CD25 recombinant antibodies in which the light and heavy chains are correctly paired and assembled can be obtained using conventional recombinant protein expression and purification techniques. Specifically, HEK293 cells can be transfected with FreeStyle TM The cells were cultured in F17 Expression Medium (Thermo, #A1383504). Before the start of transient transfection, the cell concentration was 6–8 × 10 5 The cells were adjusted to 1.2 × 10 cells / ml and cultured for 24 hours in a shaker at 37°C with 8% CO2 until the cell concentration reached 1.2 × 10 6cells / ml. 30 ml of cultured cells were prepared. The above plasmids encoding the antibody heavy chain and the above plasmids encoding the antibody light chain were mixed in a ratio of 2:3, and a total of 30 μg of plasmid was dissolved in 1.5 ml of Opti-MEM serum-reduced medium (Thermo, 31985088), filtered through a 0.22 μm filter membrane, and sterilized. Next, 1.5 ml of Opti-MEM was taken, dissolved in 120 μl of 1 mg / ml PEI (Polysciences, Inc #23966-2), and left to stand for 5 minutes. PEI was slowly added to the plasmid, incubated at room temperature for 10 minutes, and the plasmid-PEI mixed solution was slowly added dropwise while shaking the culture flask slowly, and cultured for 5 days in a shaker at 37 °C and 8% CO2. After 5 days, the cell viability was observed. The culture was collected, centrifuged at a rotation speed of 3300 g for 10 minutes, and the supernatant was taken, and the supernatant was centrifuged at high speed to remove impurities. MabSelect TM A gravity column (Bio-Rad, #7311550) containing CD25 antibody (GE Healthcare Life Science, #71-5020-91 AE) was equilibrated with PBS (pH 7.4) and washed with 2-5 column volumes. The supernatant sample was passed through the column and the column was washed with 5-10 column volumes of PBS. The target protein was then eluted with 0.1 M glycine at pH 3.5, then neutralized to pH 8.0 Tris-HCl, and finally concentrated and exchanged into PBS buffer with an ultrafiltration tube (Millipore, UFC901024) to obtain the purified CD25 antibody solution. Finally, the column was filtered with a NanoDrop (Thermo Scientific TM NanoDrop TM The concentration was measured using a 300 mL aliquot dispenser, which was then stored and put on hold.

[0063] 1.5 Enhancement of ADCC function by Fc region mutation The antibody heavy chain variable domain sequence (VH) was genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the human IgG1 antibody heavy chain constant domain sequence, and the S239D and I332E mutations (according to EU numbering, the serine at position 239 is replaced with aspartic acid, and the isoleucine at position 332 is replaced with glutamic acid) were introduced into the CH2 region of the IgG1 heavy chain constant region to increase the ADCC effect function. Then, the plasmids encoding the antibody heavy chain and the antibody light chain were transfected into mammalian host cells using the method of Example 1.4 to produce recombinant antibody protein.

[0064] 1.6 Enhancement of ADCC function by removing fucose In this example, the fucose moiety in the antibody sugar chain (N297) was removed to change the Fc conformation and further enhance the ADCC effect function. Then, using the method described in Example 1.4, both the plasmid encoding the IgG1 antibody heavy chain and the plasmid encoding the antibody light chain were transfected into mammalian host cells, and 2-fluoro peracetylated fucose (2FF) (Sigma-Aldrich, #344827) was added, and the steps of cell culture, collection, purification, etc. were carried out according to the method described in Example 1.4. The addition of compound 2FF inhibited glycosidase activity and inhibited the formation of the fucose moiety in the sugar chain.

[0065] The recombinant antibody produced in this example has the same amino acid sequence as the recombinant antibody produced in Example 1.4, but has a different glycosylation. To distinguish between the different antibody protein samples produced by the two methods that have the same amino acid sequence, in this example, "AF" was used as the suffix in the antibody sample name to indicate that the sample was a "low fucose" or "no fucose" sample.

[0066] For example, PR004639 is a recombinant antibody containing a normal IgG1 constant region, and the corresponding antibody protein sample with normal glycan structure was produced using the method of Example 1.4. PR004639AF is an antibody protein sample with the same amino acid sequence as PR004639 and with the fucose modification removed, obtained by the method of this example.

[0067] In some examples, the same antibody amino acid sequence can be used to produce an antibody sample (numbered PRxxxxxx, where xxxxxx is a number) using the method of Example 1.4, and an antibody sample (numbered PRxxxxxxAF, where xxxxxx is a number) can be produced using the method of this example, and these samples have different glycan structures.

[0068] 1.7 Anti-CD25 fully human recombinant antibody sequence Table 2-1 lists the amino acid sequences of the light and heavy chain variable domains of the CD25 antibody in the present application, the full-length light chain amino acid sequence, the full-length heavy chain amino acid sequence, and the amino acid sequences of the CDRs defined according to the Chothia definition rules.

[0069] The control antibody used in several examples of the present application is RG6292, whose sequence is from patent application US20190284287A1, and its corresponding recombinant IgG1 antibody number is PR004639. Using the method described above in Example 1.6, the fucose-depleted antibody protein sample PR004639AF, an analog of RG6292, was prepared. The sequence of PR004639 (PR004639AF, RG6292) is shown in Table 2-5.

[0070] The control antibody used in the examples of the present application is daclizumab (trade name Zenapax, Roche), the corresponding recombinant IgG1 antibody number is PR003689, its heavy chain sequence is as shown in SEQ ID NO.217, and its light chain sequence is as shown in SEQ ID NO.218.

[0071] The control antibody used in the examples of the present application is HuMax-Tac (Genmab), the sequence of which is taken from patent application WO 2014057119A1 and the corresponding recombinant IgG1 antibody number PR007722.

[0072] Example 2 Antibody sequence analysis and sequence optimization The heavy chain variable domain sequence of an antibody is derived from events such as gene rearrangement and somatic hypermutation of germline gene V, D, and J gene segments of a heavy chain gene group in a chromosome, and the light chain variable domain sequence is derived from events such as gene rearrangement and somatic hypermutation of germline gene V and J gene segments of a light chain gene group. Gene rearrangement and somatic hypermutation are the main factors that increase the diversity of antibodies. Although antibodies from the same germline V gene segment can produce different sequences, they are generally highly similar. Using several algorithms, such as IMGT / DomainGapAlign (http: / / imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi) or NCBI / IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ), the possible germline gene segments when gene rearrangement occurs can be predicted from the variable domain sequence of an antibody. The antibody sequences in Example 1 and Table 2-1 were analyzed, and the germline V gene segments of their heavy chain variable domains (VH) and light chain variable domains (VL) are shown in Table 2-2.

[0073] After translational synthesis in cells, the amino acid chain of a protein or polypeptide may introduce chemical modifications called post-translational modifications (PTMs). In the case of antibodies, some PTM sites are very conserved; for example, the conserved amino acid asparagine Asn at position 297 (EU numbering) of the constant domain of human IgG1 antibodies is usually glycosylated to form a glycan, and the glycan structure is quite important for antibody structure and related effector functions. However, if PTMs are present in the variable domain of an antibody, particularly in the antigen-binding region (e.g., CDR), the presence of these PTMs may have a significant effect on antigen binding and may also change the physicochemical properties of the antibody. For example, glycosylation, deamidation, isomerization, oxidation, etc., may all increase the instability or heterogeneity of the antibody molecule, thereby increasing the difficulty and risk of antibody development. Therefore, avoiding some potential PTMs is very important for the development of therapeutic antibodies. With the accumulation of experience, it has been found that some PTMs are highly correlated with the composition of amino acid sequences, especially the "pattern" of the composition of adjacent amino acids, which allows the prediction of potential PTMs from the primary amino acid sequence of a protein. For example, the sequence pattern of NxS / T (position 1 is asparagine, position 2 is any amino acid other than non-proline, and position 3 is serine or threonine) predicts an N-linked glycosylation site. The amino acid sequence pattern that causes PTMs may be derived from germline gene sequences, for example, human germline gene fragment IGHV3-33, which naturally has a glycosylation pattern NST in the FR3 region, or may be derived from somatic hypermutation. Table 2-2 lists the predicted PTMs of the variable domains VH and VL of the antibody of Example 1. Specifically, NSS or NLT may be glycosylation sites, and NS or NT or NN may be deamidation sites.

[0074] The formation of a particular PTM can be reduced or eliminated by disrupting the amino acid sequence pattern of the PTM by amino acid mutation. The method of designing the mutation varies depending on the antibody sequence and the PTM sequence pattern. One method is to replace the "hot spot" amino acid (e.g., N or S in NS pattern) with an amino acid with similar physicochemical properties (e.g., N to Q mutation), or to replace it with any other amino acid by saturation mutation method. If the PTM sequence pattern is derived from somatic hypermutation but does not exist in the germline gene sequence, another method is to replace the sequence pattern with the corresponding germline gene sequence. In practical operation, various mutation design methods can be used for the same PTM sequence pattern.

[0075] Table 2-3 lists new antibody molecules (called PTM mutants) obtained by performing amino acid mutations on the antibody sequences with potential PTM sites from Example 1. Table 2-4 lists the light and heavy chain variable domain amino acid sequences, full-length light chain amino acid sequences, full-length heavy chain (human IgG1) amino acid sequences, and amino acid sequences of CDRs defined according to the Chothia definition rules of these PTM mutants in this example. All designed PTM mutants were further verified in subsequent functional experiments by obtaining purified recombinant antibodies according to the method described in Example 1.4.

[0076] [Table 1]

[0077] [Table 2]

[0078] [Table 3]

[0079] [Table 4]

[0080] [Table 5]

[0081] [Table 6-1] [Table 6-2]

[0082] Example 3. Production of pre-expanded human Treg cells and pre-activated human Teff cells To prepare pre-expanded human Treg cells, Treg cells (EasySep TM Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit, #18063) were isolated, and Treg expansion beads (Miltenyi Biotec Treg Expansion Kit, human #130-095-345) and IL-2 (peprotech, 200-02) were added, and the cells were incubated at 37°C in a 5% CO2 incubator for 10 days to obtain pre-expanded human Treg cells.

[0083] In the method for producing preactivated Teffs, T cells (Miltenyi, #130096535) were isolated from fresh human PBMCs and added to 6-well plates coated with OKT3 (Invitrogen, #16-0037-85, 2 μg / ml) at a density of 1 × 10 6 The cells were cultured at 100x the concentration of Teff cells per ml, and then anti-CD28 antibody (Invitrogen, #16-0289-85, 1 μg / ml) was added. After culturing for 72 hours in a cell incubator, IL-2 (peprotech 200-02, 10 ng / ml) was added and cultured for 72 hours to obtain pre-activated Teff cells.

[0084] Example 4. Detection of the binding ability of anti-CD25 antibodies to CD25 by FACS This example is to study the in vitro binding activity of anti-human CD25 H2L2 monoclonal antibody to human / cynomolgus monkey CD25. Human CD25 overexpressing CHOK1 cell line (CHOK1-huCD25, Harbour), cynomolgus monkey CD25 overexpressing HEK293 cell line (HEK293-cynoCD25, Harbour), and human CD25 highly expressing cell lines SU-DHL-1 (ATCC® CRL-2955), Karpas299 (Nanjingke BaicBP60271), pre-expanded human Treg cells, and pre-activated human Teff cells were used to carry out antibody binding experiments at the cellular level. Briefly, CHOK1-huCD25 cells, HEK293-cynoCD25 cells were digested, and SU-DHL-1 cells, Karpas299 cells, Treg cells, and Teff cells were collected and resuspended in PBS containing 2% BSA at a cell density of 1 × 10 6 The cells were adjusted to 100 μL / mL. 100 μL cells / well were seeded into a 96-well V-bottom plate (Corning, #3894), and then 100 μL / well of the detection antibody and positive control antibody RG6292, which were serially diluted at 5-fold concentrations, which is twice the final concentration, were added. The cells were incubated at 4°C for 1 hour in the dark. Then, 100 μL / well of pre-cooled 2% BSA in PBS was added, the cells were rinsed twice, centrifuged at 500g at 4°C for 5 minutes, and the supernatant was discarded. Next, fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #109-545-098, diluted 1:1000) was added at 100 μL / well and incubated at 4°C for 1 hour in the dark. The cells were washed twice with 100 μL / well of pre-chilled PBS containing 2% BSA, centrifuged at 500 g for 5 min at 4° C., and the supernatant was discarded. Finally, the cells were resuspended with 200 μL / well of pre-chilled PBS containing 2% BSA, and the fluorescence signal was read using a BD CantoII flow cytometer.

[0085] A summary of antibody binding to cell surface human CD25, cynomolgus monkey CD25, and CD25 on the tumor cells SU-DHL-1 and Karpas is shown below (Table 4-1, Table 4-2, Table 4-3, Table 4-4, Table 4-5, Table 4-6, Table 4-7, Table 4-8, Table 4-9).

[0086] [Table 7]

[0087] [Table 8]

[0088] [Table 9]

[0089] [Table 10]

[0090] [Table 11]

[0091] [Table 12]

[0092] [Table 13]

[0093] [Table 14]

[0094] [Table 15]

[0095] The results of antibody binding to CHO-K1 cells overexpressing human CD25 are shown in Figure 1 (Figure 1A and Figure 1B), Table 4-1 and Table 4-2, and PR005433, PR005435, PR005436 and PR005497 have higher binding activity than the control antibody. The results of antibody binding to HEK293 cells overexpressing monkey CD25 are shown in Figure 2 (Figure 2A to Figure 2F). Referring to Table 4-1, Table 4-2 and Table 4-3, it was found that the initial antibodies PR005433, PR005435, PR005436, PR005497, PR006122, PR006927, PR006930, PR006931 and PR006932 have good cross-binding activity with cynomolgus monkey CD25, and Table 4 -5 and Tables 4-7, it was found that mutants PR006769, PR006771 and PR008197 obtained from PR005436 by PTM mutation have good cross-binding activity with cynomolgus monkey CD25, and with reference to Tables 4-6, it was found that mutants PR008014 and PR008016 obtained from PR006931 by PTM mutation have good cross-binding activity with cynomolgus monkey CD25.

[0096] The results of antibody binding to the tumor cell line SU-DHL-1, which endogenously expresses human CD25, are shown in Figure 3 (Figure 3A to Figure 3I). With reference to Table 4-1, Table 4-2, Table 4-3, Table 4-4 and Table 4-9, it was found that the initial antibodies PR005433, PR005436, PR005497, PR006110, PR006122, PR006927, PR006928, PR006930, PR006931, PR006932 and PR007181 have high binding activity to SU-DHL-1, and with reference to Table 4-1 and Table 4-4, it was found that PR005435 and PR006984 have weak binding activity. Referring to Tables 4-5 and 4-7, it was found that mutants PR006769, PR006771 and PR008197 obtained from PR005436 by PTM mutation have high binding activity to SU-DHL-1, and referring to Tables 4-6 and 4-8, it was found that mutants PR008014, PR008015 and PR008016 obtained from PR006931 by PTM mutation have high binding activity to SU-DHL-1.

[0097] The results of antibody binding to the tumor cell line Karpas299, which endogenously expresses human CD25, are shown in Figure 4 (Figure 4A to Figure 4I). With reference to Table 4-1, Table 4-2, Table 4-3, Table 4-4 and Table 4-9, it was found that the initial antibodies PR005433, PR005497, PR006110, PR006122, PR006927, PR006928, PR006930, PR006932 and PR007181 have high binding activity to Karpas299, and with reference to Table 4-1, Table 4-3 and Table 4-4, it was found that PR005435, PR005436, PR006931 and PR006984 have weak binding activity. Referring to Table 4-5, it was found that the mutants PR006769 and PR006771 obtained from PR005436 by PTM mutation have binding activity to Karpas299 equivalent to that of the control antibody RG6292, and according to Table 4-7, it was found that the binding activity of PR008197 was relatively weak. Referring to Tables 4-6 and 4-8, it was found that the mutant PR008015 obtained from PR006931 by PTM mutation has high binding activity to Karpas299, and PR008014 and PR008016 have weak binding activity.

[0098] The results of antibody binding to human Treg cells and Teff cells are shown in Figure 5 (Figure 5A to Figure 5F), and PR005436, PR008014, PR008016, PR008197, and PR006927 were able to bind to human Treg, but had weak binding strength to human Teff.

[0099] Example 5. Detection of blocking effect of anti-CD25 antibody on binding of human interleukin-2 to CD25 by FACS This example is to study the blocking effect of anti-CD25 antibodies on the binding of human interleukin-2 to CD25. Using CHOK1 cell line overexpressing human CD25 (CHOK1-huCD25, Harbour) or cell line SU-DHL-1 (ATCC® CRL-2955) highly expressing human CD25 and biotin-labeled human interleukin-2 (Acro, IL2-H82F3), the blocking effect of the antibodies was detected by FACS method. Briefly, CHOK1-huCD25 cells were digested or SU-DHL-1 (ATCC® CRL-2955) cells were collected and resuspended in PBS containing 2% BSA. The cell density was 1×10 6 The cells were adjusted to 100 μL / well. 90 μL of cells / well were seeded into a 96-well V-bottom plate (Corning, #3894), and then 100 μL / well of the detection and control antibodies RG6292 or PR003689, which were serially diluted at 5-fold concentrations, which was twice the final concentration, was added, followed by 10 μL / well of PBS containing a biotin label and a human interleukin-2 concentration of 10 μg / ml. The cells were incubated at 4°C for 1 hour, protected from light. Then, 100 μL / well of pre-cooled 2% BSA in PBS was added, the cells were rinsed twice, centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was discarded. 100 μL / well of fluorescent secondary antibody (eBioscience #12-4317-87, diluted 1:200) was added, and incubated at 4°C for 1 hour, protected from light. The cells were washed twice with 100 μL / well of pre-chilled PBS containing 2% BSA, centrifuged at 500 g for 5 min at 4°C, and the supernatant was discarded. Finally, the cells were resuspended in 200 μL / well of pre-chilled PBS containing 2% BSA, and the fluorescence signal was read using a BD CantoII flow cytometer.

[0100] The results of detecting the blocking effect of antibodies on the binding of human interleukin-2 to CHOK1-huCD25 cells by the FACS method are shown in Figure 6 (Figures 6A to 6C). PR005433, PR005435, PR005436, PR005497 and PR006122 were unable to block the binding of interleukin-2 to CD25 on the surface of CHOK1-huCD25 cells.

[0101] The results of detecting the blocking effect of antibodies on the binding of human interleukin-2 to SU-DHL-1 (ATCC® CRL-2955) cells by FACS method are shown in Figure 7 (Figure 7A to Figure 7D). Initial antibodies PR006927, PR006930, PR006931 and PR006932 were unable to block the binding of interleukin-2 to CD25 on the surface of SU-DHL-1 cells, mutants PR006769, PR006771 and PR008197 derived from PR005436 by PTM mutation were unable to block the binding of interleukin-2 to CD25 on the surface of SU-DHL-1 cells, and mutants PR008014 and PR008016 derived from PR006931 by PTM mutation were unable to block the binding of interleukin-2 to CD25 on the surface of SU-DHL-1 cells.

[0102] Example 6. Detection of the effect of anti-CD25 antibody on the phosphorylation level of the downstream signal molecule STAT5 induced by human interleukin-2 in Teff using AlphaLisa (PerkinElimer, #ALSU-PST5-B500) Preactivated Teff cells were cultured in medium without IL-2 for 4 h, after which the cells were harvested and the cell density was adjusted to 2.5 × 10 7The cells were adjusted to cells / ml and plated at 4 μL / well in a 384-well plate. Then, 4x the final concentration of antibodies and the control antibody PR003689 were added at 2 μL / well and incubated in the incubator for 1 hour. Then, 5x the final concentration of IL-2 was added at 2 μL / well and incubated in the incubator for 10 minutes. 5x lysis solution was added immediately at 2 μL / well and incubated at room temperature for 15 minutes. Then, the acceptor mixture was added at 5 μL / well and incubated at room temperature for 1 hour, and then the donor mixture was added at 5 μL / well and incubated at room temperature for 1 hour. Signal values ​​were read using a PerkinElimer Envision.

[0103] The effect of the antibody on the phosphorylation level of the downstream protein STAT5 of the human interleukin-2 signal pathway was detected by the AlphaLisa method as shown in FIG. 8. PR006122 had no inhibitory effect on the phosphorylation level of the downstream protein STAT5 of the human interleukin-2 signal pathway in Teff.

[0104] Example 7. Detection by FACS of the effect of anti-CD25 antibody on the phosphorylation level of the downstream signal molecule STAT5 induced by human interleukin-2 in Teff Preactivated Teff cells were cultured in medium without IL-2 for 4 h, after which the cells were harvested and the cell density was adjusted to 2 × 10 6The cells were adjusted to 100 μL / well. 50 μL cells / well were seeded into a 96-well V-bottom plate (Corning, #3894), and then 50 μL / well of the detection and control antibodies PR003689 or RG6292, serially diluted at 5-fold concentrations, which is twice the final concentration, were added and incubated in an incubator for 1 hour. 1 μL / well of IL-2 at 1 μg / ml was then added and incubated in an incubator for 10 minutes. Fixative (Biolegend, #420801) was immediately added at 100 μL / well and incubated at 37°C for 15 minutes, followed by centrifugation at 500g at 4°C for 5 minutes and discarding the supernatant. 100 μL / well of pre-chilled 2% BSA in PBS was added to rinse the cells, then centrifuged at 500g at 4°C for 5 minutes and discarding the supernatant. Then transmembrane buffer was added at 100 μL / well, cells (Biolegend, #425401) were resuspended and incubated overnight in a -20°C refrigerator. The next day, cells were removed at 1000g, centrifuged at 4°C for 5 minutes, the supernatant was discarded, pre-chilled 2% BSA in PBS was added at 100 μL / well, cells were rinsed twice, then centrifuged at 500g at 4°C for 5 minutes, and the supernatant was discarded. Anti-phosphorylated STAT5 antibody (Biolegend, #936904) was added at 100 μL / well, and incubated at 4°C for 1 hour. Pre-chilled 2% BSA in PBS was added at 100 μL / well, cells were rinsed twice, then centrifuged at 500g at 4°C for 5 minutes, and the supernatant was discarded. Finally, cells were resuspended in 200 μL / well pre-chilled 2% BSA in PBS, and fluorescence signal values ​​were read using a BD CantoII flow cytometer.

[0105] The results are shown in Figure 9. PR008014, PR008016, PR008197 and PR006927 had no inhibitory effect on the phosphorylation level of STAT5, a downstream protein of the human interleukin-2 signal pathway, in Teff.

[0106] Example 8. Detection of ADCC effect of anti-CD25 antibody against tumor cell line SU-DHL-1 using a reporter gene cell line SU-DHL-1 (ATCC® CRL-2955) cells were harvested and diluted to 1.2 × 10 6 The cells / mL were adjusted and seeded at 25 μL / well into a 96-well plate (PerkinElmer, 6005225). cDNAs encoding human CD16a (whose Genebank number is NM000569.8) and human NFAT (whose Genebank number is NM_145912.8) were obtained by gene synthesis, cloned into expression vector pcDNA3.1, and the expression vector was transfected into Jurkat cells to construct Jurkat / FCγRIII-NFAT stable cell line.

[0107] Jurkat / FCγRIII-NFAT cells (Harbour) were collected and 1.2 × 10 6 The cells were adjusted to 100 cells / mL and plated at 25 μL / well in the same 96-well plate. Antibodies and control antibody RG6292 at twice the final concentration were then added at 50 μL / well and incubated at 37°C in a 5% CO2 incubator for 6 hours. One-Glo (Promega, #E6120) was added at 60 μL / well and incubated at room temperature for 10 minutes in the dark. Signals were read using a PerkinElmer Envision.

[0108] The ADCC results of the antibodies against SU-DHL-1 are shown below (Tables 8-1 and 8-2).

[0109] [Table 16]

[0110] [Table 17]

[0111] The results are shown in FIG. 10 (FIG. 10A and FIG. 10B), and PR006927AF, PR006931AF, PR006769AF, and PR006771AF have high ADCC activity against SU-DHL-1.

[0112] Example 9. Detection of ADCC effect of anti-CD25 antibodies on pre-expanded human Treg cells and pre-activated human Teff cells using reporter gene cell lines Pre-expanded human Treg cells and pre-activated human Teff cells were collected and diluted to 1.2 × 10 6 Jurkat / FCγRIII-NFAT cells (Harbour) were harvested and 1.2 × 10 6 The cells were adjusted to 100 cells / mL and plated at 25 μL / well in the same 96-well plate. Antibodies were then added at 2x the final concentration at 50 μL / well and incubated at 37°C in a 5% CO2 incubator for 6 hours. One-Glo (Promega, #E6120) was added at 60 μL / well and incubated at room temperature for 10 minutes protected from light. Signals were read using a PerkinElmer Envision.

[0113] The ADCC results of the antibodies against Treg and Teff are shown below (Tables 9-1, 9-2, and 9-3).

[0114] [Table 18]

[0115] [Table 19]

[0116] [Table 20]

[0117] The results are shown in Figure 11 (Figure 11A to Figure 11C) and Figure 12 (Figure 12A to Figure 12C). PR005433, PR005436, PR005497 and PR006122 had high ADCC activity against human Treg cells, but had no clear activity against human Teff. PR005435 had a weak ADCC activity against human Treg.

[0118] Example 10. Antibody internalization experiments This example is to study the internalization of antibodies. The internalization of antibodies was detected by FACS using tumor cell lines SU-DHL-1 (ATCC® CRL-2955) and Karpas299 (Nanjing Ke BaicBP60271) that highly express human CD25. The experimental method is as follows: SU-DHL-1 cells or Karpas299 cells were harvested and resuspended in PBS containing 2% BSA. The cell density was 1×10 6The cells were adjusted to 100 μL / mL. 100 μL cells / well were seeded into a 96-well V-bottom plate (Corning, #3894), and then 100 μL / well of the detection antibody and control antibody PR007722, which were serially diluted at 5-fold concentration, which is twice the final concentration, were added. The cells were incubated at 4°C, protected from light, for 1 hour. Then, 100 μL / well of pre-cooled 2% BSA in PBS was added, the cells were rinsed twice, centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was discarded. The cells were resuspended in 100 μL of 2% BSA in PBS, and incubated at 37°C in a 5% CO2 incubator for 4 hours. Then, 100 μL / well of pre-cooled 2% BSA in PBS was added, the cells were rinsed twice, centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was discarded. Then, 100 μL / well of fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #109-545-098, 1:1000 dilution) was added and incubated for 1 h at 4°C, protected from light. The cells were washed twice with 100 μL / well of pre-chilled PBS containing 2% BSA, centrifuged at 500g for 5 min at 4°C, and the supernatant was discarded. Finally, the cells were resuspended with 200 μL / well of pre-chilled PBS containing 2% BSA, and the fluorescence signal was read using a BD CantoII flow cytometer.

[0119] The results are shown in Figures 13 and 14. The human tumor cell line Karpas299 has obvious endocytosis effects on PR005433, PR005497, PR006122, PR006930, and PR006932.

Claims

1. 1. An antibody or variant thereof targeting CD25, said antibody comprising a light chain variable region and a heavy chain variable region, said antibody being selected from the group consisting of: the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 96, 111 and 133, respectively; and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 20, 44 and 74, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 104, 111 and 127, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 22, 46 and 76, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 94, 110 and 134, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 23, 48 and 78, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 97, 113 and 129, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 16, 40 and 70, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 94, 116 and 135, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 23, 47 and 77, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 95, 111 and 127, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 14, 38 and 68, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 98, 114 and 130, respectively; and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 17, 41 and 71, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 99, 115 and 131, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 18, 42 and 72, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 100, 113 and 132, respectively; and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 14, 43 and 73, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 103, 110 and 134, respectively; and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 21, 45 and 75, respectively; or The light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 105, 111 and 136, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 18, 42 and 80, respectively.

2. The variant is one in which 1 to 3 amino acid residues are added, deleted or substituted in the amino acid sequence of an antibody targeting CD25, and preferably, the variant is one in which an amino acid residue is substituted at position 1 of HCDR1 and position 3 of HCDR2 in the heavy chain variable region of the antibody, and / or, an amino acid residue is substituted at positions 10 to 11 of LCDR1 and position 1 or 4 of LCDR3 in the light chain variable region of the antibody, and more preferably, the variant is selected from the group consisting of: the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 101, 113 and 129, respectively; and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 19, 40 and 70, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 101, 113 and 139, respectively; and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 19, 40 and 70, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 102, 113 and 129, respectively; and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 19, 40 and 70, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 94, 116 and 137, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 23, 50 and 77, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 94, 116 and 138, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 23, 51 and 77, respectively; or The antibody or variant thereof targeting CD25 according to claim 1, characterized in that the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 94, 116 and 138, respectively, and / or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 23, 50 and 77, respectively.

3. the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 171 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 171, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 153 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 153; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 173 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 173, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 155 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 155; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 175 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 175, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 157 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 157; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 165 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 165, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 148 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 148; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 174 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 174, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 156 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 156; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 163 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 163, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 146 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 146; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 166 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 166, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 149 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 149; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 167 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 167, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 150 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 150; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 168 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 168, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 151 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 151; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 172 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 172, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 154 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 154; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 177 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 177, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 159 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 159; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 169 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 169, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 152 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 152; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 180 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 180, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 152 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 152; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 170 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 170, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 152 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 152; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 178 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 178, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 160 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 160; or the light chain variable region comprises a VL having an amino acid sequence set forth in SEQ ID NO: 179 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 179, and the heavy chain variable region comprises a VH having an amino acid sequence set forth in SEQ ID NO: 161 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 161; or 3. The antibody or variant thereof according to claim 1 or 2, characterized in that the light chain variable region comprises a VL whose amino acid sequence is set forth in SEQ ID NO: 179 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity with SEQ ID NO: 179, and the heavy chain variable region comprises a VH whose amino acid sequence is set forth in SEQ ID NO: 160 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity with SEQ ID NO:

160.

4. The antibody is selected from the group of a chimeric antibody, a humanized antibody and a fully human antibody, preferably The structure of the antibody or variant thereof is Fab, Fab', F(ab')2, Fv, IgG, Fd, dAb, or a bispecific or multispecific antibody comprising the antibody or variant thereof, or a monoclonal or polyclonal antibody produced from the antibody, and the Fv is preferably an scFv; Preferably, the antibody or variant thereof having an IgG structure comprises a light chain and a heavy chain, the light chain comprises an amino acid sequence as set forth in SEQ ID NO:207 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:207, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:189 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:189; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:209 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity thereto, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:191 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity thereto; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:211 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:211, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:193 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:193; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:201 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity thereto, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:184 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity thereto; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:210 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity thereto, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:192 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity thereto; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:199 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity thereto, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:182 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity thereto; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:202 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity thereto, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:185 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity thereto; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:203 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:203, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:186 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:186; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:204 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:204, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:187 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:187; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:208 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:208, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:190 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:190; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:213 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:213, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:195 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:195; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:205 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:205, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:188 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:188; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:216 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:216, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:188 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:188; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:206 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:206, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:188 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:188; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:214 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:214, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:196 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:196; or the light chain comprises an amino acid sequence as set forth in SEQ ID NO:215 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:215, and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:197 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:197; or The antibody or variant thereof according to any one of claims 1 to 3, characterized in that the light chain comprises an amino acid sequence shown in SEQ ID NO: 215 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 215, and the heavy chain comprises an amino acid sequence shown in SEQ ID NO: 196 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO:

196.

5. A chimeric antigen receptor comprising the antibody or variant thereof according to any one of claims 1 to 4.

6. An isolated nucleic acid encoding the antibody or variant thereof according to any one of claims 1 to 4, or the chimeric antigen receptor according to claim 5.

7. 7. A recombinant expression vector comprising the isolated nucleic acid of claim 6, preferably said recombinant expression vector being a plasmid, cosmid, phage or viral vector, said viral vector being preferably a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

8. 10. A transformant comprising a host cell comprising the recombinant expression vector according to claim 7, wherein the host cell is preferably a prokaryotic or eukaryotic cell, more preferably the host cell is selected from a yeast cell, a mammalian cell or other cell suitable for producing an antibody or an antigen-binding fragment thereof, wherein the mammalian cell is, for example, a 293 cell or a CHO cell.

9. 6. An immune cell comprising the chimeric antigen receptor of claim 5, preferably the immune cell is a T cell or a NK cell, more preferably the NK cell is a peripheral blood NK cell, a cord blood derived NK cell, a stem cell differentiated NK cell, or a NK cell line, e.g. the NK-92 cell line.

10. A method for producing an antibody or a variant thereof that targets CD25, comprising culturing the transformant described in claim 8 and obtaining an antibody or a variant thereof that targets CD25 from the culture.

11. An antibody-drug conjugate comprising a cytotoxic agent and an antibody or variant thereof targeting CD25 according to any one of claims 1 to 4.

12. A pharmaceutical composition comprising an antibody or variant thereof targeting CD25 according to any one of claims 1 to 4, an isolated nucleic acid according to claim 6, a recombinant expression vector according to claim 7, a transformant according to claim 8, an immune cell according to claim 9, and / or an antibody-drug conjugate according to claim 11, and a pharma- ceutical acceptable carrier; Preferably, the pharmaceutical composition further comprises one or more of the group consisting of hormonal agents, targeted small molecule agents, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

13. 13. Use of an antibody or variant thereof targeting CD25 according to any one of claims 1 to 4, a chimeric antigen receptor according to claim 5, an isolated nucleic acid according to claim 6, a recombinant expression vector according to claim 7, a transformant according to claim 8, an immune cell according to claim 9, an antibody-drug conjugate according to claim 11, and / or a pharmaceutical composition according to claim 12 in the manufacture of a drug for diagnosing, preventing and / or treating a tumor.

14. A kit comprising an antibody or variant thereof targeting CD25 according to any one of claims 1 to 4, a chimeric antigen receptor according to claim 5, an isolated nucleic acid according to claim 6, a recombinant expression vector according to claim 7, a transformant according to claim 8, an immune cell according to claim 9, an antibody-drug conjugate according to claim 11, or a pharmaceutical composition according to claim 12, Preferably, the kit further comprises (i) a device for administering the antibody or variant thereof, or chimeric antigen receptor, or immune cell, or antibody drug conjugate, or pharmaceutical composition, and / or (ii) an instruction manual.

15. A parts kit including kit A and kit B, The kit A comprises an antibody or antigen-binding fragment thereof targeting CD25 according to any one of claims 1 to 4, a chimeric antigen receptor according to claim 5, an isolated nucleic acid according to claim 6, a recombinant expression vector according to claim 7, a transformant according to claim 8, an immune cell according to claim 9, an antibody-drug conjugate according to claim 11, and / or a pharmaceutical composition according to claim 12; Kit B is a kit of parts containing another anti-tumor antibody or a pharmaceutical composition comprising the other anti-tumor antibody, and / or one or more of the group consisting of a hormonal preparation, a targeted small molecule preparation, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic agent, a cytotoxic agent, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.

16. 13. A method for detecting CD25, comprising using an antibody or variant thereof according to any one of claims 1 to 4, a chimeric antigen receptor according to claim 5, an immune cell according to claim 9, an antibody drug conjugate according to claim 11 and / or a pharmaceutical composition according to claim 12, preferably wherein said detection is not for diagnostic and / or therapeutic purposes.

17. A method for preventing, treating or alleviating a disease symptom in a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of an antibody or variant thereof targeting CD25 according to any one of claims 1 to 4, a chimeric antigen receptor according to claim 5, an isolated nucleic acid according to claim 6, a recombinant expression vector according to claim 7, a transformant according to claim 8, an immune cell according to claim 9, an antibody-drug conjugate according to claim 11 and / or a pharmaceutical composition according to claim 12.

18. A method for reducing the number of intratumoral or tumor-infiltrating Treg cells in a subject, the method comprising administering to the subject an effective amount of an antibody targeting CD25 or a variant thereof according to any one of claims 1 to 4, a chimeric antigen receptor according to claim 5, an isolated nucleic acid according to claim 6, a recombinant expression vector according to claim 7, a transformant according to claim 8, an immune cell according to claim 9, an antibody-drug conjugate according to claim 11, and / or a pharmaceutical composition according to claim 12.

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