CD25 Antibody

Abstract

Provided herein are humanized CD25 antibodies, methods for making and using them. In some embodiments, the CD25 antibodies are useful for treating cancer or autoimmune diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 113,784, filed November 13, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates generally to the field of MHC class II restricted T cell receptors, and more specifically to T cell receptors specific for the KRAS G12>V activating mutation.

[0003] INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on November 11, 2021, is named RUBR2002WO.txt and is 10,956 bytes in size. [Background technology]

[0004] CD25 is the alpha chain of the interleukin-2 (IL-2) receptor, a transmembrane protein present on regulatory T cells and activated T cells. Under normal conditions, regulatory T cells constitutively express CD25 and act to suppress the expansion of effector T cells. Regulatory T cells maintain a healthy state and inhibit effector T cells from reacting against self-antigens or overreacting to foreign antigens. In a normal protective immune response, effector T cells increase after contact with foreign antigens and overcome the inhibition by regulatory T cells. However, in the case of proliferative diseases, cancer cells neutralize healthy immune responses by increasing the amount of regulatory T cells, thereby limiting the generation of effector T cells against cancer cells. For example, to weaken the immune system for use in cancer therapy or to upregulate the immune system for use in autoimmune diseases, additional molecular tools are needed to modify the expansion of CD25-expressing regulatory T cells. Such tools are provided herein. Summary of the Invention [Means for solving the problem]

[0005] Provided herein are humanized antibodies (interchangeably referred to herein as anti-CD25 antibodies and CD25 antibodies) that specifically bind to CD25. In some embodiments, the antibodies are humanized. Also provided herein are methods of using and making the described antibodies. For example, CD25 antibodies can be used therapeutically to treat cancer, including administering the antibody or a pharmaceutical composition thereof to a subject in need thereof. Also provided are methods of producing the CD25 antibodies described herein.

[0006] As embodied and broadly described herein, one aspect of the disclosure relates to a humanized antibody that binds to human CD25, wherein the antibody comprises a variable heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or a sequence that comprises at least 70% sequence identity thereto; and a variable light chain amino acid sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:9, or a sequence that comprises at least 70% sequence identity thereto. In one aspect, the antibody comprises the variable heavy chain amino acid sequence of SEQ ID NO:2, or a sequence that comprises at least 70% sequence identity thereto; and the variable light chain amino acid sequence of SEQ ID NO:8 or 9, or a sequence that comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or even 99% sequence identity thereto. In another embodiment, the antibody comprises the variable heavy chain amino acid sequence of SEQ ID NO: 3, or a sequence that comprises at least 70% sequence identity thereto; and the variable light chain amino acid sequence of SEQ ID NO: 8 or 9, or a sequence that comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or even 99% sequence identity thereto. In another embodiment, the antibody comprises the variable heavy chain amino acid sequence of SEQ ID NO: 4, or a sequence that comprises at least 70% sequence identity thereto; and the variable light chain amino acid sequence of SEQ ID NO: 8 or 9, or a sequence that comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or even 99% sequence identity thereto. In another embodiment, the antibody comprises the variable heavy chain amino acid sequence of SEQ ID NO: 5, or a sequence that comprises at least 70% sequence identity thereto; and the variable light chain amino acid sequence of SEQ ID NO: 8 or 9, or a sequence that comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or even 99% sequence identity thereto. In another embodiment, the antibody comprises the variable heavy chain amino acid sequence of SEQ ID NO: 6, or a sequence that comprises at least 70% sequence identity thereto; and the variable light chain amino acid sequence of SEQ ID NO: 8 or 9, or a sequence that comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or even 99% sequence identity thereto.

[0007] As embodied and broadly described herein, one aspect of the disclosure relates to a humanized CD25 antibody comprising a variable light chain comprising the CDR sequences of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, wherein the antibody further comprises one or more mutations to the framework regions, including one or more of the bolded residues in SEQ ID NO:8 and SEQ ID NO:9.

[0008] As embodied and broadly described herein, one aspect of the disclosure relates to a humanized CD25 antibody comprising a variable heavy chain comprising the CDR sequences of SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, wherein the antibody further comprises one or more mutations to the framework regions, including one or more of the bolded residues in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0009] As embodied and broadly described herein, one aspect of the disclosure relates to a humanized CD25 antibody comprising a variable heavy chain comprising the CDR sequences of SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, wherein the antibody further comprises one or more mutations to the framework region, including one or more of the bolded residues in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; and a variable light chain comprising the CDR sequences of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, wherein the antibody further comprises one or more mutations to the framework region, including one or more of the bolded residues in SEQ ID NO:8 and SEQ ID NO:9. In another aspect, the antibody binds to human CD25 and cynomolgus CD25. In another aspect, the EC50 ratio for binding to human CD25 and cynomolgus CD25 is about 0.75 to about 1.25. In another aspect, the antibody is defucosylated.

[0010] As embodied and broadly described herein, one aspect of the disclosure relates to a pharmaceutical composition comprising a humanized antibody that binds to human CD25, wherein the antibody comprises a variable heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or a sequence that has at least 70% sequence identity thereto; and a variable light chain amino acid sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:9, or a sequence that has at least 70% sequence identity thereto.

[0011] As embodied and broadly described herein, one aspect of the disclosure relates to a nucleic acid sequence encoding a humanized antibody that binds human CD25, wherein the antibody comprises a variable heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or a sequence that comprises at least 70% sequence identity thereto; and a variable light chain amino acid sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:9, or a sequence that comprises at least 70% sequence identity thereto.

[0012] As embodied and broadly described herein, one aspect of the disclosure relates to a vector comprising a nucleic acid sequence of a humanized antibody that binds to human CD25, wherein the antibody comprises a variable heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or a sequence that comprises at least 70% sequence identity thereto; and a variable light chain amino acid sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:9, or a sequence that comprises at least 70% sequence identity thereto.

[0013] As embodied and broadly described herein, one aspect of the disclosure relates to a method of treating a subject in need thereof comprising administering to the subject a therapeutically effective amount of a humanized antibody that binds human CD25, wherein the antibody comprises a variable heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or a sequence that comprises at least 70% sequence identity thereto; and a variable light chain amino acid sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:9, or a sequence that comprises at least 70% sequence identity thereto.

[0014] As embodied and broadly described herein, one aspect of the disclosure relates to a method of depleting the number of regulatory T cells in a subject, comprising administering to the subject a therapeutically effective amount of a humanized antibody that binds to human CD25, wherein the antibody comprises a variable heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or a sequence comprising at least 70% sequence identity thereto; and a variable light chain amino acid sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:9, or a sequence comprising at least 70% sequence identity thereto, or a pharmaceutical composition thereof. In one aspect, the subject suffers from cancer. In another aspect, the subject suffers from an autoimmune-related disease or disorder.

[0015] As embodied and broadly described herein, one aspect of the disclosure relates to a method of depleting the number of regulatory T cells in a sample comprising peripheral blood mononuclear cells, comprising contacting said sample with a humanized antibody that binds to human CD25, wherein the antibody comprises a variable heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or a sequence that comprises at least 70% sequence identity thereto; and a variable light chain amino acid sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:9, or a sequence that comprises at least 70% sequence identity thereto.

[0016] As embodied and broadly described herein, one aspect of the disclosure relates to a kit comprising a humanized antibody that binds to human CD25, wherein the antibody comprises a variable heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or a sequence that has at least 70% sequence identity thereto; and a variable light chain amino acid sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:9, or a sequence that has at least 70% sequence identity thereto, or a pharmaceutical composition thereof. [Brief description of the drawings]

[0017] The present application can be understood by reference to the following description taken in conjunction with the accompanying drawings. [Figure 1] Figure 1 shows nanoDSF-based measurements of the Tm of D11 and D11 variants of the present disclosure across four separate manufacturing batches. The variants show a trend for higher Tm when compared to D11. [Diagram 2] Figure 2 shows nanoDSF-based measurements of Tagg for D11 and the disclosed D11 variants across two separate manufacturing batches. No significant aggregation was detected at lower temperatures, and Tagg for the humanized variants was generally higher than Tagg for the starting D11. [Diagram 3] Figure 3 shows the results of uPLC-based measurements of purity and aggregation of D11 and the disclosed D11 variants across four separate production batches. The data support consistent purification of D11 and variants at greater than 90% purity in a single-step purification. [Figure 4] FIG. 4 shows that the binding of D11 variants of the present disclosure exhibits no difference in binding to CD25-expressing SUDHL1 cells when compared to D11. [Diagram 5] FIG. 5 shows that the binding of D11 variants of the present disclosure exhibits no difference in binding to CD25-expressing SR786 cells when compared to D11. [Figure 6]FIG. 6 shows that the D11 variant H3h-L2 (interchangeably referred to herein as H3-L2) maintains approximately equal cross-reactivity with both human and cynomolgus CD25. [Figure 7] FIG. 7 shows that the D11 variants of the present disclosure do not exhibit differential ADCC activity in SUDHL1 cancer cells when compared to D11. [Figure 8] FIG. 8 shows that afucosylated D11 (D11 kif) exhibits higher ADCC activity in regulatory T cells (Treg), a target cell type that expresses lower levels of the CD25 antigen. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Provided herein are antibodies that specifically bind to CD25. The antibodies can be human, chimeric, or humanized. Also provided herein are methods of using and making the described antibodies. For example, CD25 antibodies can be used therapeutically to treat cancer, including administering the antibody or a pharmaceutical composition thereof to a subject in need thereof. Also provided are methods of producing the CD25 antibodies described herein.

[0019] definition Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

[0020] The headings provided herein are not intended to be limitations of the various aspects or embodiments of the invention. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0021] Numeric ranges are inclusive of the numbers defining the range.

[0022] The term antibody, as used herein, includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, non-human antibodies, chimeric antibodies, monovalent antibodies, and antigen-binding fragments of antibodies (e.g., Fab fragments, Fab'2 fragments, or scFV). Also provided herein are antibody-drug conjugates, bispecific antibodies, and multispecific antibodies that exhibit specificity for CD25. Non-human antibodies (e.g., murine antibodies) may be "humanized" using conventional techniques (e.g., by introducing changes in the framework regions while retaining the murine CDRs).

[0023] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, which may be ribonucleotides or deoxyribonucleotides. These terms include, but are not limited to, single-stranded, double-stranded, or multistranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms encompass nucleic acids that contain known analogs of natural nucleotides, have similar binding properties, and are metabolized in a manner similar to naturally occurring nucleotides, unless otherwise limited or stated.

[0024] When a nucleic acid or amino acid sequence is said to have a certain percentage "sequence identity" or "identity" with another nucleic acid or amino acid sequence, or is a certain percentage "identical," when the sequences are aligned, that percentage of bases or amino acids are the same and in the same relative positions when the two sequences are compared.

[0025] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to any subject for whom treatment or therapy is desired. The subject can be a mammalian subject. Mammalian subjects include, for example, humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, etc.), and the like. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a companion animal (e.g., cat, dog).

[0026] antibody Provided herein are humanized antibodies that specifically bind to CD25. The antibodies can bind to the CD25 antigen either alone or when the CD25 antigen associates with other molecules to form the high affinity IL-2 receptor that is normally present on regulatory T cells.

[0027] The CD25 antibody of the present disclosure can be any of human IgA, IgD, IgE, IgG, or IgM antibodies. The IgA antibody can be an IgA1 antibody or an IgA2 antibody. The IgG antibody can be an IgG1, IgG2, IgG2a, IgG2b, IgG3, or IgG4 antibody. Any combination of these antibodies can also be made and used. In some embodiments, the constant region is of the IgG type, e.g., of human IgG type. In some embodiments, the constant region is of the IgG1 type, e.g., of human IgG1 type.

[0028] In some embodiments, the CD25 antibody exhibits cross-reactivity to more than one species, e.g., specifically binds to both human CD25 and non-human CD25, e.g., specifically binds to both human CD25 and cynomolgus CD25. In exemplary embodiments, the binding affinity to both human CD25 and cynomolgus CD25 is approximately equal. In some embodiments, the EC50 ratio of binding of the humanized CD25 antibody to human CD25 and cynomolgus CD25 is about 0.75 to about 1.25, or about 0.8 to about 1.2, or about 0.9 to about 1.1. In some embodiments, the ratio is approximately 1.

[0029] The affinity constant (KD) of the antibodies provided herein is about 10 -5 nM~about 10 -14 In some embodiments, the KD of the antibodies provided herein can be in the range of about 10 -8 nM~about 10 -12 In an exemplary embodiment, the KD of the CD25 antibody is in the range of at least about 10 -5 nM, about 10 -6 nM, about 10 -7 nM, about 10 -8 nM, about 10 -9 nM, about 10 -10 nM, about 10 -11 nM, about 10 -12 nM, about 10 -13 nM, or even about 10 -14 nM.

[0030] The dissociation rate constant (Kd) of the antibodies provided herein is about 10 -2 1 / s~approx. 10 -6 It can be in the range of 1 / s.

[0031] In some embodiments, the CD25 antibody exhibits the same affinity (KD) for the CD25 antigen at both physiological pH (about 7.4) and non-physiological pH. In some embodiments, the CD25 antibody exhibits the same dissociation rate (Kd) for the CD25 antigen at both physiological pH (about 7.4) and non-physiological pH.

[0032] In some embodiments, the CD25 antibody exhibits different affinities (different KD) for the CD25 antigen at physiological pH (about 7.4) and non-physiological pH. In some embodiments, the CD25 antibody exhibits different dissociation rate constants (different Kd) for the CD25 antigen at physiological pH (about 7.4) and non-physiological pH.

[0033] In some embodiments, the CD25 antibody exhibits a lower affinity (higher KD) for the CD25 antigen at physiological pH (about 7.4) than at lower pH, e.g., when the pH is 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, or less. In exemplary embodiments, the antibody exhibits a higher affinity for the CD25 antigen at a pH of about 6.5 compared to the affinity at a pH of about 7.4. In some embodiments, such antibodies are useful for retaining activity or exhibiting enhanced activity in acidic, hypoxic environments, e.g., tumor microenvironments.

[0034] In some embodiments, the antibody is a non-IL-2 blocking antibody (non-IL-2 blocker) - i.e., the binding of the antibody to CD25 does not interfere with or prevent the binding of an IL-2 ligand to CD25 (IL-2 alpha chain) and does not affect IL-2 mediated signal transduction, e.g., signal transduction through the IL-2 / JAK3 / STAT-5 signaling pathway. In some embodiments, the antibody does not interfere with the binding of an IL-2 ligand to CD25 (IL-2 alpha chain) and binds to a different epitope than the 7G7B6 antibody binds. In some embodiments, the antibody does not interfere with the binding of an IL-2 ligand to CD25 (IL-2 alpha chain) but interferes with the trimerization of the beta, gamma, and alpha (CD25) chains of the IL-2 receptor.

[0035] In some embodiments, the antibody is an IL-2 blocking antibody (IL-2 blocker), e.g., the antibody interferes with or prevents binding of an IL-2 ligand to the alpha, beta, and / or gamma chain of the receptor and reduces or inhibits IL-2 mediated signal transduction. In certain embodiments, the antibody interferes with or prevents binding of an IL-2 ligand to CD25. In some embodiments, the antibody interferes with or prevents binding of an IL-2 ligand to CD25 and binds to an epitope that is distinct from the epitope bound by either daclizumab or basiliximab.

[0036] In some embodiments, the CD25 antibody is a partial blocking antibody (partial IL-2 blocker) that partially, if not completely, interferes with binding of the IL-2 ligand to the alpha, beta, and / or gamma chains of the IL-2 receptor (CD25) and / or partially, if not completely, reduces IL-2-mediated signal transduction.

[0037] In some embodiments, the CD25 antibody disrupts or prevents heterotrimerization of the alpha, beta, and gamma IL-2 chains. In some embodiments, the antibody does not block the binding of IL-2 ligand to CD25, but disrupts or prevents heterotrimerization of the alpha, beta, and gamma IL-2 chains. In certain embodiments, the antibody selectively binds to regulatory T cells. In other embodiments, the antibody selectively binds to T effector cells.

[0038] In some embodiments, binding of CD25 antibodies results in depletion of regulatory T cells (Treg) while allowing expansion of effector T cells (Teff).

[0039] In some embodiments, the antibody binds to CD25 in a trans orientation. In other embodiments, the antibody binds to CD25 in a cis orientation. In yet further embodiments, the antibody can bind to CD25 in either the cis or trans configuration.

[0040] In some embodiments, the CD25 antibody exhibits greater binding affinity to CD25 compared to the binding of 7G7B6 (anti-human CD25 with mouse IgG2a Fc receptor; IL-2 non-blocker; BioXcell) to CD25.

[0041] Exemplary sequences of the CD25 antibodies described herein are provided in the table below. It should be noted that the complementarity determining region (CDR) and framework (FR) sequences shown are based on the IMGT convention for antibody annotation. However, a person skilled in the art can determine other explicit representations of CDR and framework sequences based on the VH and VL sequences provided using other algorithms / conventions for antibody annotation, such as Kabat and Chothia. Thus, the CDR and FR sequences of the present disclosure are not limited to the exemplary CDR and FR sequences annotated in the table below, but rather are the CDRs as understood and determined by a person skilled in the art when considering the sequences of the variable regions.

[0042] Humanized CD25 antibody variants were created herein. The parent (original) mouse CD25 antibody was D11 from WO2020 / 102591. D11 was selected and reformatted to contain human IgG1 and used as the parent clone for the next generation of variants.

[0043] The amino acid sequences of the variable heavy (VH) and variable light (VL) chains of parent D11 are provided as SEQ ID NO:1 and SEQ ID NO:7, respectively. The present disclosure provides further VH variant sequences of D11 comprising any one of the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or even 99% sequence identity thereto. The present disclosure also provides further VL variant sequences of D11 comprising any one of the amino acid sequences of SEQ ID NO:8 and SEQ ID NO:9, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or even 99% sequence identity thereto. In some embodiments, the antibody is defucosylated.

[0044] The variable heavy chain (VH) amino acid sequence of the parent D11, D11 VH (SEQ ID NO: 1), is shown in Table 1. This is followed by the amino acid sequences of exemplary humanized variants thereof: hD11-H1 (SEQ ID NO: 2); hD11-H2 (SEQ ID NO: 3); hD11-H3 (SEQ ID NO: 4; also referred to interchangeably throughout as hD11-H3h); hD11-H2b (SEQ ID NO: 5; and hD11-H3b (SEQ ID NO: 6). In comparison with the amino acid sequence immediately above, the underlined residues are the CDR sequences and the bolded residues are the modified residues.

[0045] [Table 1]

[0046] The variable light chain (VL) amino acid sequence of the parent D11, D11-VL (SEQ ID NO: 7), is shown in Table 2, followed by the amino acid sequences of its humanized variants: hD11-L1 (SEQ ID NO: 8); and hD11-L2 (SEQ ID NO: 9). Compared to the immediately preceding amino acid sequence, the underlined residues are the CDR sequences and the bolded residues are the modified residues.

[0047] [Table 2]

[0048] Thus, in some embodiments, humanized and / or stabilized CD25 antibodies are provided comprising the variable heavy chain CDR sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. Residues selected for humanization and / or stabilization include, but are not limited to, one or more of the bolded residues in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0049] In some embodiments, humanized and / or stabilized CD25 antibodies are provided comprising the variable light chain CDR sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. Residues selected for humanization and / or stabilization include, but are not limited to, one or more of the bolded residues in SEQ ID NO: 8 and SEQ ID NO: 9.

[0050] In some embodiments, a CD25 antibody is provided comprising the variable heavy chain CDR sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, and the variable light chain CDR sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, wherein the antibody is humanized and / or stabilized. Residues selected for humanization and / or stabilization include, but are not limited to, one or more of the bolded residues in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 9. In some embodiments, the antibody is defucosylated.

[0051] Exemplary antibody VH+VL combinations for humanized CD25 antibodies are provided in Table 3 below.

[0052] [Table 3]

[0053] In some embodiments, the CD25 antibodies are conjugated and are useful for a variety of purposes, including but not limited to, therapy and detection / diagnosis.

[0054] In some embodiments, the CD25 antibody is fucosylated.

[0055] In some embodiments, the CD25 antibody is defucosylated. In some embodiments, the CD25 antibody is defucosylated, resulting in higher ADCC activity in cells, such as regulatory T cells.

[0056] Also provided herein are nucleic acid sequences encoding any one of the CD25 antibodies provided herein. Also provided herein are vectors comprising any one of the nucleic acids encoding the antibodies, phages comprising such vectors, and host cells comprising such vectors.

[0057] Antibody generation and testing The CD25 antibodies described herein can be generated by injecting a full or partial immunogen of CD25 into an animal, e.g., a mouse or rabbit, followed by humanization. CD25 immunogen-positive B cells can be collected from the animal, from which a phage library can be generated. In some embodiments, the phage expresses Fab fragments of candidate CD25 antibodies. The phage can be subjected to multiple rounds of screening (referred to herein as phage panning), e.g., against successively lower concentrations of CD25 antigen, to select such Fab fragments that can bind to CD25 with high affinity to be selected for further variants. The phage can be screened, e.g., against beads or some other substrate coated with CD25 antigen. In some embodiments, screening is performed at physiological pH (e.g., about pH 7.4). In other embodiments, screening can be performed at a lower pH, e.g., about pH 6.5, to screen for Fab fragments that can bind to CD25 antigen at lower pH, e.g., for use in a therapeutic setting, e.g., for use in a hypoxic, acidic tumor microenvironment.

[0058] The CD25 antibodies generated herein can be tested for efficacy using a number of in vitro, in vivo, ex vivo, and / or cell-based assays.

[0059] In some embodiments, the CD25 antibodies herein can be assayed for and further selected based on their ability to deplete regulatory T cells. In certain embodiments, the CD25 antibodies herein can be assayed for and further selected based on their ability to deplete regulatory T cells in an acidic environment, e.g., at a pH lower than physiological pH, e.g., pH 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, or less.

[0060] In some embodiments, the CD25 antibodies herein can be assayed for in vitro assays of pSTAT5 and further selected on this basis to assay for signaling through the IL-2 / IL-2 receptor pathway, although maintenance of this signaling indicates that the antibody is not an IL-2 blocking antibody.

[0061] In some embodiments, the CD25 antibodies herein can be assayed for characterizing molecular interactions using biosensor screening.

[0062] In some embodiments, the CD25 antibodies herein can be assayed for competition for binding with other known CD25 antibodies by known mechanisms.

[0063] In some embodiments, the CD25 antibodies herein can be assayed for epitope specificity.

[0064] In some embodiments, the CD25 antibodies herein can be assayed for their capacity to be non-IL-2 blockers, IL-2 blockers, or partial IL-2 blockers.

[0065] therapeutic use Provided herein are CD25 antibodies for therapeutic uses, for example for use in proliferative diseases or disorders such as cancer, or for use in autoimmune diseases.

[0066] Thus, provided herein is a method of treating cancer, comprising administering a therapeutically effective amount of a therapeutic CD25 antibody to a subject in need thereof. In some embodiments, the cancer is a primary cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer involves a solid tumor; in other embodiments, the cancer involves a liquid tumor, e.g., a blood-based cancer. In an exemplary embodiment, the CD25 antibody is a non-IL-2 blocking antibody.

[0067] Accordingly, provided herein is a method of treating an autoimmune-related disease or disorder comprising administering to a subject in need thereof a therapeutically effective amount of a therapeutic CD25 antibody. In an exemplary embodiment, the CD25 antibody is an IL-2 blocking antibody.

[0068] As used herein, a subject refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo, sports, or pet animals, such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. A subject may be male or female.

[0069] Administration of any of the therapeutic CD25 antibodies provided herein may be performed in combination with other known drugs / treatments (e.g., small molecule drugs or biologics. Administration may be sequential or simultaneous.

[0070] In vivo administration of the therapeutic CD25 antibodies described herein can be performed intravenously, intratumorally, intracranially, intralesionally (e.g., intralesional injection, direct contact diffusion), intracavitary (intraperitoneally, intrapleurally, intrauterinely, intrarectally), intraperitoneally, intramuscularly, subcutaneously, topically, orally, transdermally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In an exemplary embodiment, the route of administration is by intravenous injection.

[0071] A therapeutically effective amount of the therapeutic antibody is administered. The appropriate dosage of the therapeutic antibody can be determined based on the severity of the cancer, the subject's clinical condition, the subject's medical history and response to treatment, and the discretion of the attending physician.

[0072] Dosages of CD25 antibodies provided herein can vary from about 1 ng / kg to about 1000 mg / kg or more of subject body weight per day, depending on the route of administration. In the case of repeated administration over several days or more, depending on the severity of the cancer, treatment can be maintained until a desired suppression of symptoms is achieved. Dosing regimens can be useful depending on the pattern of pharmacokinetic decay the physician wishes to achieve. For example, dosing an individual from once to 21 times per week is provided herein. In certain embodiments, the dosing frequency is 3 times per day, 2 times per day, once per day, once every 2 days, once per week, once every 2 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, or once a month, once every 2 months, once every 3 months or more. Progress of therapy can be monitored by conventional techniques and assays. Dosage regimens may vary over time regardless of the dose used.

[0073] Diagnostic Uses The CD25 antibodies provided herein can be used for diagnostic and detection purposes. Depending on the application, the CD25 antibodies can be detected and quantified in vivo or in vitro.

[0074] The CD25 antibodies provided herein can be calibrated for use in a variety of immunoassays, including, but not limited to, enzyme-linked immunosorbent assay (ELISA), Western blot, radioimmunoassay (RIA), flow cytometry, certain radioimmunoassays, immunofluorescence assays, spectrophotometry, radiography, electrophoresis, high performance liquid chromatography (HPLC), or thin layer chromatography (TLC).

[0075] The CD25 antibodies provided herein can include a detectable label, such as a label detectable by spectroscopic, photochemical, biochemical, immunological, fluorescent, electrical, optical, or chemical means. Labels useful in this disclosure include, but are not limited to, fluorescent dyes, radiolabels, enzymes, colorimetric labels, avidin, or biotin.

[0076] In some embodiments, the CD25 antibody is radiolabeled with an isotope useful for imaging with nuclear medicine devices (SPECT, PET, or scintigraphy).

[0077] Pharmaceutical Compositions The present disclosure provides a composition comprising a therapeutic CD25 antibody. In some embodiments, the composition is sterile. The pharmaceutical composition as a whole comprises an effective amount of a therapeutic antibody in a pharma- ceutically acceptable excipient.

[0078] Kits and Products The present disclosure also provides kits comprising any one of the CD25 antibodies described herein, for example, for either therapeutic or diagnostic use. In some embodiments, the kit further contains a component selected from a secondary antibody, a reagent for immunohistochemistry analysis, a pharma- ceutically acceptable excipient, and instructions for use, and any combination thereof. In some embodiments, the kit comprises any one or more therapeutic compositions described herein, together with one or more pharma- ceutically acceptable excipients.

[0079] The present application also provides an article of manufacture comprising any one of the therapeutic or diagnostic compositions or kits described herein. An example article of manufacture includes a vial (e.g., a sealed vial).

[0080] The description provided herein sets forth numerous example structures, methods, parameters, etc. However, it should be understood that such description is not intended as a limitation on the scope of the present disclosure, but is provided instead as an illustration of an example embodiment.

[0081] The following examples are included for illustrative purposes and are not intended to limit the scope of the invention. [Example] EXAMPLES

[0082] Generation of CD25 antibody variants BalbC mice were injected with full-length human CD25 via the tail vein. The immunization protocol and subsequent selection and characterization of the resulting antibodies are described in WO 2020 / 102591, the contents of which are incorporated by reference in their entirety.

[0083] A Fab, D11, from WO2020 / 102591 was selected, reformatted to contain human IgG1, and used as a parent clone for the next generation of variants. Humanized variants were created. In the data provided herein, reference to the original D11, or parent D11, or simply D11, refers to an antibody comprising a human IgG1 Fc and a variable heavy chain (VH) amino acid sequence comprising SEQ ID NO:1 and a variable light chain (VL) amino acid sequence comprising SEQ ID NO:7.

[0084] Table 1 shows the variable heavy chain (VH) amino acid sequence of the parent D11, D11 VH (SEQ ID NO: 1), followed by the amino acid sequences of its humanized variants: hD11-H1 (SEQ ID NO: 2); hD11-H2 (SEQ ID NO: 3); hD11-H3 (SEQ ID NO: 4); hD11-H2b (SEQ ID NO: 5); and hD11-H3b (SEQ ID NO: 6). Compared to the amino acid sequence immediately above, the underlined residues are the CDR sequences and the bolded residues are the modified residues.

[0085] Table 2 shows the variable light chain (VL) amino acid sequence of the parent D11, D11-VL (SEQ ID NO: 7), followed by the amino acid sequences of its humanized variants: hD11-L1 (SEQ ID NO: 8); and hD11-L2 (SEQ ID NO: 9). Compared to the immediately preceding amino acid sequence, the underlined residues are the CDR sequences and the bolded residues are the modified residues.

[0086] For the antibody characterization and assays described herein, the following antibodies were used, as set forth in Table 4.

[0087] [Table 4] EXAMPLES

[0088] Characterization of CD25 antibody variants The melting behavior (Tm) and aggregation behavior (Tagg) of the antibodies were measured using a Prometheus NT.Plex nanoDSF instrument, which uses tyrosine and phenylalanine residues to measure protein autofluorescence. Prometheus NT.Plex High Sensitivity capillaries (Cat.PR-AC006) were loaded with 10 μl / capillary of samples at 0.2 mg / mL of antibody diluted in PBS pH 7.4 and run in duplicate. The temperature range was 20-95°C with a gradient of 1°C / min over 75 minutes. Fluorescence was detected at 330 nm, 350 nm and the inflection point of the ratio 350 / 330 nm was used to determine Tm. Aggregation optics allowed for the measurement of scattered light and the inflection point of the backscattered light was used to determine Taggr. Figure 1 shows the nanoDSF-based measurements of Tm for D11 and D11 variants of the present disclosure across four separate manufacturing batches. The variants show a trend towards higher Tm when compared to D11. Figure 2 shows nanoDSF-based measurements of Tagg for D11 and the disclosed D11 variants across two separate production batches. No significant aggregation was detected at lower temperatures, and Tagg for the humanized variants was generally higher than Tagg for the starting D11.

[0089] The humanized antibody hD11-H3h-L2 showed higher Tagg than most, indicating better stability and resistance to aggregation.

[0090] uPLC-based measurements of purity and degree of aggregation of humanized D11 variants were performed using a Dionex UltiMate® 3000-RS UHPLC system run by Chromeleon® (version 7.3) software. Antibodies were separated using a size-exclusion column TSKgel UP-SW3000, 2 μm, 4.6 mm ID×15 cm (Tosoh) at 28° C. The mobile phase was 50 mmol monosodium phosphate, 300 mmol sodium chloride, 5% acetonitrile; pH 6.5, run in isocratic mode at a flow rate of 0.2 mL / min. Proteins were detected using UV absorbance at 280 nm. All samples were injected at a concentration of 0.1 mg / mL in a volume of 20.0 μL. FIG. 3 shows the results of uPLC-based measurements of purity and aggregation of D11 and the disclosed D11 variants across four separate production batches. The data supports consistent purification of D11 and variants at greater than 90% purity in a single step purification. Table 5 below shows the data in tabular form.

[0091] [Table 5] EXAMPLES

[0092] Cell binding assay To verify the binding and specificity of the antibodies to CD25 on cells, two cell lines, SUDHL-1 (human large diffuse histiocytic lymphoma cell line (ATCC)) and SR786 cells (anaplastic large T-cell lymphoma cells), were used to test for CD25 binding. Both are CD25 expressing cell lines. For each cell line, 100,000 cells were plated in 96-well round bottom plates in cell buffer (PBS + 2% HI FBS) and centrifuged. Antibody concentrations of 1-10ug / mL were used to test for binding of each antibody. Cells were resuspended in 100uL / well of antibody and incubated on ice for 20 minutes. After incubation, cells were centrifuged at 300xg for 5 minutes at room temperature, resuspended and washed with ice-cold cell buffer. Cells were then stained with anti-human Fc secondary antibody conjugated to AF647 (Biolegend) and incubated for 20 min on ice in the dark. Cells were centrifuged, washed, resuspended in cold cell buffer containing DAPI, and analyzed using flow cytometry (Cytoflex, Becton Dickinson). DAPI+ cells were excluded from the analysis. Mean fluorescence intensity was calculated using the median (FlowJo, TreeStar).

[0093] Figure 4 shows that the binding of D11 variants of the present disclosure does not exhibit differences in binding to CD25-expressing SUDHL1 cells when compared to parent D11. Figure 5 shows that the binding of D11 variants of the present disclosure does not exhibit differences in binding to CD25-expressing SR786 cells when compared to D11. Binding was also compared to commercial control antibodies, 767B6, daclizumab, and basiliximab, and no significant differences were observed. EXAMPLES

[0094] Binding of CD25 antibodies to recombinant cynomolgus monkey CD25 To test for binding to recombinant cyno (cyno) monkey CD25, microtiter plates were coated overnight at 4°C with 80ul of 1ug / ml Cyno CD25 in 50mM sodium carbonate pH 9.6. The next day, the protein was removed from the wells and blocked with 200ul of PBS / 0.1%BSA / 0.05% tween20. Plates were incubated for 1 hour at room temperature. Antibodies, starting at 25nM, were serially diluted 3-fold in PBT buffer and added to the plates for 1 hour incubation at RT. Plates were then washed and then a 1:2500 dilution of HRP-conjugated anti-Fab antibody was added. Plates were washed 5-10 times with PBS / tween20 to remove non-specific binders. TMB peroxidase substrate and peroxidase solution were added and incubated for the time required for color development, at the end of which 80 uL of ELISA stop solution was added.OD readings were then taken at 450 nM using a plate reader (SpectraMax iD3 Plate Reader, Molecular Devices).

[0095] Figure 6 shows that D11 variant H3h-L2 (H3-L2) maintains cross-reactivity with both human and cynomolgus CD25. Table 6 shows the quantification of cyno / human binding ratios along with their quantification. Dac stands for daclizumab. The ratios indicate preference in binding to either cyno or human CD25. Variant H3h-L2 (H3-L2) shows equal cross-reactivity, while other variants show a clear preference for one species or the other (e.g. variant H1-L2).

[0096] [Table 6] EXAMPLES

[0097] Antibody-dependent cell cytotoxicity (ADCC) Functional cell killing assay ADCC One of the mechanisms for Treg (regulatory T cells) depletion is through antibody-dependent cellular cytotoxicity (ADCC). This is a cell-mediated immune defense mechanism that is triggered by immune / effector cell recognition of antibodies bound to specific antigens on target cells, usually resulting in cell death. To trigger ADCC, an antibody with a human Fc subclass, IgG1, is generally selected for its effector function, which has the ability to bind to three Fc receptors: FcγRI (CD64), FcγRII (CD32), and FcγRIIIA (CD16), which are expressed on immune cells such as NK cells, monocytes, and granulocytes. NK cells predominantly express FcγRIIIA and are believed to be the major effector cells in ADCC.

[0098] ADCC was quantified using a lactate dehydrogenase (LDH) cytotoxicity plate-based colorimetric assay (Thermo Fisher). In this assay, the release of LDH is proportional to the amount of cell death. LDH is a cytoplasmic enzyme present in all cells and is released when the plasma membrane is damaged. Extracellular LDH in the medium is quantified by a coupled enzymatic reaction in which LDH catalyzes the conversion of lactate to pyruvate via NAD+ reduction to NADH. Upon addition of diaphorase, NADH is reduced to a tetrazolium salt (INT) to form a formazan product that can be measured at 490 nm. Controls for the assay include the use of rituximab (an anti-CD20 antibody) on the human Raji lymphoma cell line and daclizumab on SUDHL-1 cells (both antibodies are known to induce ADCC). Additional controls may include: non-glycosylated human IgG1 as a negative control, and non-fucosylated human IgG1 as a positive control. As a first initial screen, reformatted clones were tested in an ADCC assay at one concentration (10ug / mL) using PBMCs from a single donor. Human PBMCs (ASTARTE Biologics) were thawed the day before and cultured overnight at 37°C in a 5% CO2 incubator with X-VIVO 15 or 20 medium (Lonza). On the day of the experiment, PBMCs and SUDHL-1 target cells were counted and resuspended in X-VIVO 15 (Lonza, phenol red) serum-free medium. Antibody dilutions were prepared in 5-fold serial dilutions starting at 10ug / ml. Target cells were then plated in 96-well white flat-bottom opaque plates (CORNING Ref#3917) at 50 μL / well, and 10 μL / well of antibody dilutions were added to the target cells and incubated for 30 minutes at 37° C. in a 5% CO2 incubator. After incubation, PBMCs were added to the target cells (50 μL / well). For control target cells for spontaneous and maximum LDH release calculations, 50 μL / well of assay medium (X-VIVO15, no phenol red) was added.Co-incubation (ADCC induction) was performed by incubating the plates for 4 hours in a 37°C, 5% CO2 incubator, and 45 minutes before the co-incubation was completed, 10% Triton X100 solution (in PBS) was added to the wells to calculate the maximum LDH release (10 μL / well, 11× dilution ratio). After incubation, 50 μL / well of reaction substrate was plated in a 96-well plate (clear flat bottom), and then 50 μL / well of the assay plate supernatant was transferred to the reaction substrate. The plate was developed in the dark at RT for 30 minutes. After incubation, 50 μL / well of stop solution was added, and the absorbance at 490 nm and 680 nm was measured by using a plate reader (SpectraMax iD3 Plate Reader, Molecular Devices).

[0099] FIG. 7 shows that the D11 variants of the present disclosure exhibit no difference in ADCC activity in cancer cells when compared to D11, thereby indicating that, as a general matter, their functionality is maintained.

[0100] Table 8 shows that certain D11 variants exhibit higher ADCC activity when tested on regulatory T cells. Certain variant antibodies, such as D11-H3h-L2 and D11-H3b-L2, exhibited higher ADCC activity (lower EC50) when compared to the D11 parent antibody and 7G7B6.

[0101] [Table 7]

[0102] Figure 8 further shows that defucosylated D11 exhibits higher Treg ADCC activity (D11 kif) when produced in cell culture medium containing kifunensine, resulting in defucosylation of the antibody. Assays were performed at 20,000 cells / well in 384-well plates.

[0103] A functional antibody-dependent cell phagocytosis (ADCP) assay can also be performed to test the activity of macrophages to phagocytose Treg cells when antibodies are bound. This could be an additional mechanism of action for Treg depletion. The Fc receptor FCγRIIa (CD32b) on macrophages is thought to be the predominant inducer for ADCP. In this assay, primary Tregs are used as target cells with human monocyte-derived macrophages as effector cells. PBMCs are isolated from leukapheresis system chambers (Stanford Blood Center) for monocyte isolation using CD14 microbeads (Miltenyi Biotec) and for Tregs using Regulatory Human CD4+CD25+ T cell kit (Dynabeads) and Human Treg Cell Differentiation kit (R&D Systems). Monocytes are cultured with human serum or M-CSF in culture medium for 5-7 days. On days 5-7, macrophages are co-cultured with pre-labeled Tregs (labeled with fixable viability dye (Invitrogen)) at an effector:target ratio of 10:1 in the presence of anti-CD25 antibodies and controls for 2-4 hours and fixed with fixation buffer (BD Biosciences). To distinguish macrophages from labeled Tregs, macrophages are stained for CD14 and analyzed using flow cytometry for phagocytic populations defined as CD14+ and Treg labeled+ populations. Anti-CD25 antibodies are expected to also induce ADCP. EXAMPLES

[0104] Functional characterization of CD25 antibodies In vitro characterization Subsequent in vitro characterization may include T cell activation and Treg suppression studies to assess the impact of CD25 antibodies on T effector cell responses in the absence and presence of Treg cells. Activation readouts may include intracellular granzyme B, proliferation, and cytokine release (e.g., IL-2, IFNγ, TNF-α). Specifically, human primary conventional T cells (T conv cells) from healthy donors and donors with specific antigen responses (e.g., human cytomegalovirus or influenza flu antigens) are labeled with a cell proliferation dye (e.g., ThermoFisher), treated with various concentrations of CD25 and control antibodies (1-10ug / mL), followed by activation using CD3 / CD28 beads and incubation with or without Tregs for 48-72 hours of incubation at 37°C, 5% CO2. To assess T cell activation, supernatants are collected for cytokine analysis and cells are stained with fixable viability dyes (e.g., Thermofisher) and with surface anti-human T cell markers: CD3, CD4, CD8, CD45RA, CD25, followed by fixation and permeabilization for staining of intracellular Granzyme B and Foxp3. Cells are analyzed for Granzyme B positive cells and proliferating cells using flow cytometry.

[0105] In vivo characterization A mouse xenograft tumor model can be used to evaluate the in vivo activity of CD25 antibodies. This experiment helps distinguish between CD25 antibodies based on CD25+ tumor depletion via ADCC and / or ADCP. Variant antibodies can be produced as mouse IgG2a isotype antibodies and used in immunodeficient RAG- / - knockout mice (which have functional NK and APC cells but no mature B or T lymphocytes). Animals are subcutaneously implanted with CD25+ human cell lines (e.g., SUDHL-1, CD25+ anaplastic large cell lymphoma). Tumors of various sizes (palpable, 100-500 mm3) can be used to distinguish between antibodies. Animals are treated with different doses of CD25 antibodies (e.g., 1-10 mg / kg, 3x per week or once per day) and monitored for changes in body weight and TGI.

[0106] Drug efficacy and MOA studies in mouse xenograft tumor models in humanized mice The variant antibodies that are more effective at inhibiting tumor growth through Treg depletion and increasing intratumoral Teff / Treg ratios with increased T effector cell activity are tested in various human tumor models (e.g., liver cancer, breast cancer, melanoma, gastric cancer, NSCLC, and colon cancer) to demonstrate efficacy and mechanism of action. Some studies may include using triple-negative breast cancer cell line MDA-MB-231, gastric cell line, melanoma cell line A375, and liver cancer cell line Huh-7, as well as human PDX models that have been well characterized and have shown a correlation between infiltrated Tregs and tumor growth, and / or have been treated with PD-1 antibody combinations in humanized mice engrafted with human PBMCs and / or CD34+ cells. In these models, tumors are implanted subcutaneously and treated with various doses of CD25 antibodies and dosing schedules (e.g., 1-10 mg / kg, 3x per week or once daily) until tumors reach a size range of palpable to 100-500 mm3. Animal health scoring, body weight, tumor growth, and blood and tumor immunophenotyping are performed to characterize tumor and immune cell composition and tumor infiltrating cells, as well as cytokine secretion using flow cytometry, Meso Scale Discovery multiplates, and histology.

[0107] Combination studies in mouse xenograft tumor models in humanized mice. Once lead candidates and indications are validated based on efficacy and MOA studies, we will conduct combination studies using a broad range of agents (e.g., chemotherapy, checkpoint inhibitors, TLR agonists, vaccines) to improve immunogenicity and activation of other pathways for immune activation. Combination experiments will be performed in similar tumor models used in efficacy and MOA studies.

[0108] All patents, patent applications, publications, documents, web links, and articles cited herein are hereby incorporated by reference in their entirety.

Claims

1. A humanized CD25 antibody that binds to human CD25, a variable heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 10, a variable heavy chain CDR2 of SEQ ID NO: 11, and a variable heavy chain CDR3 of SEQ ID NO: 12; a variable light chain CDR1 of SEQ ID NO: 13, a variable light chain CDR2 of SEQ ID NO: 14, and a variable light chain CDR3 of SEQ ID NO: 15; and 1) a variable heavy chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2 and having the 2nd V residue, the 5th V residue, the 7th S residue, the 11th V residue, the 12th K residue, the 13th K residue, the 16th A residue, the 18th V residue, the 20th V residue, the 24th A residue, the 40th A residue, the 41st P residue, the 42nd G residue, the 43rd Q residue, the 44th G residue, the 48th M residue, the 61st A residue, the 65th Q residue, the 67th R residue, the 68th V residue, the 72nd R residue, the 74th T residue, the 76th T residue, the 79th V residue, the 81st M residue, the 84th S residue, the 85th S residue, the 87th R residue, the 89th E residue, the 91st T residue, and the 93rd V residue of SEQ ID NO:2; a variable light chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:8 and having the following residues of S at position 7, P at position 8, V at position 15, P at position 40, A at position 43, K at position 45, I at position 48, D at position 70, T at position 72, T at position 74, S at position 76, A at position 84, T at position 85, and Q at position 100 of SEQ ID NO:8; 2) a variable heavy chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2 and having the 2nd V residue, the 5th V residue, the 7th S residue, the 11th V residue, the 12th K residue, the 13th K residue, the 16th A residue, the 18th V residue, the 20th V residue, the 24th A residue, the 40th A residue, the 41st P residue, the 42nd G residue, the 43rd Q residue, the 44th G residue, the 48th M residue, the 61st A residue, the 65th Q residue, the 67th R residue, the 68th V residue, the 72nd R residue, the 74th T residue, the 76th T residue, the 79th V residue, the 81st M residue, the 84th S residue, the 85th S residue, the 87th R residue, the 89th E residue, the 91st T residue, and the 93rd V residue of SEQ ID NO:2; a variable light chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:9 and having a V residue at position 48 and a G residue at position 84 of SEQ ID NO:9; 3) a variable heavy chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:3 and having the following residues: G at position 24, V at position 72, and A at position 79 of SEQ ID NO:3; a variable light chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:8 and having the following residues of S at position 7, P at position 8, V at position 15, P at position 40, A at position 43, K at position 45, I at position 48, D at position 70, T at position 72, T at position 74, S at position 76, A at position 84, T at position 85, and Q at position 100 of SEQ ID NO:8; 4) a variable heavy chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:3 and having the following residues: G at position 24, V at position 72, and A at position 79 of SEQ ID NO:3; a variable light chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:9 and having a V residue at position 48 and a G residue at position 84 of SEQ ID NO:9; 5) a variable heavy chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:4, and having the following residues of L at position 18, I at position 20, G at position 24, I at position 48, A at position 68, V at position 72, A at position 79, and L at position 81 of SEQ ID NO:4; a variable light chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:8 and having the following residues of S at position 7, P at position 8, V at position 15, P at position 40, A at position 43, K at position 45, I at position 48, D at position 70, T at position 72, T at position 74, S at position 76, A at position 84, T at position 85, and Q at position 100 of SEQ ID NO:8; 6) a variable heavy chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:4, and having the following residues: L at position 18, I at position 20, G at position 24, I at position 48, A at position 68, V at position 72, A at position 79, and L at position 81 of SEQ ID NO:4; a variable light chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:9 and having a V residue at position 48 and a G residue at position 84 of SEQ ID NO:9; 7) a variable heavy chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:5, and having the following residues: G at position 24, V at position 72, K at position 74, and A at position 79 of SEQ ID NO:5; a variable light chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:8 and having the following residues of S at position 7, P at position 8, V at position 15, P at position 40, A at position 43, K at position 45, I at position 48, D at position 70, T at position 72, T at position 74, S at position 76, A at position 84, T at position 85, and Q at position 100 of SEQ ID NO:8; 8) a variable heavy chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:5, and having the following residues: G at position 24, V at position 72, K at position 74, and A at position 79 of SEQ ID NO:5; a variable light chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:9 and having a V residue at position 48 and a G residue at position 84 of SEQ ID NO:9; 9) a variable heavy chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:6, and having the following residues: L at position 18, I at position 20, G at position 24, I at position 48, A at position 68, V at position 72, K at position 74, A at position 79, and L at position 81 of SEQ ID NO:6; or a variable light chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:8 and having the following residues of S residue 7, P residue 8, V residue 15, P residue 40, A residue 43, K residue 45, I residue 48, D residue 70, T residue 72, T residue 74, S residue 76, A residue 84, T residue 85, and Q residue 100 of SEQ ID NO:8; or 10) a variable heavy chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:6, and having the following residues of L at position 18, I at position 20, G at position 24, I at position 48, A at position 68, V at position 72, K at position 74, A at position 79, and L at position 81 of SEQ ID NO:6; A variable light chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:9 and having a V residue at position 48 and a G residue at position 84 of SEQ ID NO:

9. The humanized CD25 antibody.

2. The humanized CD25 antibody of claim 1 , which binds to human CD25 and cynomolgus monkey CD25.

3. 3. The humanized CD25 antibody of claim 2, which has an EC50 ratio of binding to human CD25 and cynomolgus monkey CD25 of 0.75 to 1.

25.

4. The humanized CD25 antibody of any one of claims 1 to 3, which is defucosylated.

5. A pharmaceutical composition comprising any one of the humanized CD25 antibodies according to claims 1 to 4.

6. A nucleic acid encoding any one of the humanized CD25 antibodies according to claims 1 to 4.

7. A vector comprising the nucleic acid of claim 6.

8. A therapeutic agent for a subject in need thereof, comprising any one of the humanized CD25 antibodies according to claims 1 to 4 or the pharmaceutical composition according to claim 5.

9. A method for depleting the number of regulatory T cells in a subject, comprising administering to a subject a humanized CD25 antibody according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 5.

10. The method according to claim 8 or 9, wherein the subject is suffering from cancer.

11. The method according to claim 8 or 9, wherein the subject is suffering from an autoimmune-related disease or disorder.

12. 13. Use of a humanized CD25 antibody for depleting the number of regulatory T cells in a sample comprising peripheral blood mononuclear cells, said use comprising a step of contacting said sample with any one of the humanized CD25 antibodies according to claims 1 to 4 or the pharmaceutical composition according to claim 5.

13. A kit comprising any one of the humanized CD25 antibodies of claims 1 to 4 or the pharmaceutical composition of claim 5.

Images