Fc-optimized anti-CD25 for tumor-specific cell depletion

Abstract

The present disclosure relates to the use of anti-CD25 antibodies that do not inhibit IL-2-CD25 interactions and, together with enhanced binding to activating FcγRs, result in effective depletion of tumor-infiltrating Treg cells and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies further improves tumor rejection. [Selection diagram] None

Description

[Technical Field]

[0001] The present invention relates to the field of cancer immunotherapy and to methods for treating cancer, including methods for treating solid tumors, the methods involving the use of antibodies against CD25. [Background technology]

[0002] Cancer immunotherapy involves the use of the subject's own immune system to treat or prevent cancer. Immunotherapy takes advantage of the fact that cancer cells often have subtly different molecules on their surface that can be detected by the immune system. These molecules, or cancer antigens, are most commonly proteins, but also include molecules such as carbohydrates. Therefore, immunotherapy involves inducing the immune system to attack tumor cells via these target antigens. However, malignant tumors, particularly solid tumors or hematological malignancies, can evade immune surveillance using various mechanisms inherent to tumor cells and mediated by components of the tumor microenvironment. Among the latter, tumor infiltration by regulatory T cells (Treg cells or Tregs), more specifically, an unfavorable balance of effector T cells (Teffs) to Tregs (i.e., a low ratio of Teffs to Tregs), has been proposed as a decisive factor (Non-Patent Literature 1).

[0003] Since their discovery, Tregs have been found to be important in mediating immune homeostasis and promoting the establishment and maintenance of peripheral tolerance. However, the role of Tregs in cancer is more complex. Because cancer cells express both autoantigens and tumor-associated antigens, the presence of Tregs that attempt to suppress effector cell responses can contribute to tumor progression. Therefore, Treg infiltration in established tumors is one of the main obstacles to effective antitumor responses and cancer treatment in general. The inhibitory mechanisms employed by Tregs are thought to significantly contribute to the limitations, or even failure, of current therapies, particularly immunotherapies that rely on the induction or enhancement of antitumor responses (Non-Patent Literature 2).

[0004] Treg depletion as a therapeutic approach for cancer treatment is supported by studies demonstrating the contribution of Tregs to tumor engraftment and progression in mouse models. Furthermore, Treg-mediated tumor invasion has been associated with a worse prognosis in several human cancers (Non-Patent Literature 3). It has been demonstrated that Treg cells contribute to tumor engraftment and progression in mouse models, and that the absence of Treg cells slows tumor progression (Non-Patent Literature 4, 5, 6, 7, and 8). In humans, high tumor invasion by Treg cells, and more importantly, a low ratio of effector T (Teff) cells to Treg cells, is associated with poor outcomes in several human cancers (Non-Patent Literature 3). Conversely, a high Teff / Treg cell ratio is associated with a favorable response to immunotherapy in both humans and mice (Non-Patent Literature 9 and 10). However, Treg depletion in tumors is complex, and the results of studies in this area are inconsistent.

[0005] CD25 is one of the molecular targets that may achieve Treg depletion. CD25, also known as the interleukin-2 high-affinity receptor α chain (IL-2Ra), is a promising target for Treg depletion because it is constitutively expressed at high levels on Treg cells and is absent or expressed at low levels on T effector cells. IL-2 / CD25 interaction has been the subject of several studies in mouse models, mostly involving the use of PC61, a rat anti-mouse CD25 mouse antibody (Non-Patent Literature 11). The CD25 binding and functional activity of this antibody have been compared with a panel of monoclonal antibodies produced by different authors (Non-Patent Literature 12, 13, 14, 15). While the original studies demonstrated the prophylactic rather than therapeutic activity of PC61, recent studies have shown that an Fc-optimized version of this anti-CD25 antibody leads to intratumoral Treg depletion in several mouse tumor models, resulting in significant therapeutic utility (Non-Patent Literature 16). Available anti-CD25 antibodies, such as PC61, block or inhibit the binding of IL-2 to CD25, similar to most antibodies disclosed as many other anti-mouse and anti-human CD25 antibodies. See, for example, Patent Documents 1, 2, 3, 4, and 5. For example, basiliximab and daclizumab are anti-human CD25 antibodies developed to inhibit the binding of IL-2 to CD25 and reduce the activation of T effector cells. Basiliximab is a chimeric mouse-human CD25 antibody currently approved for graft-versus-host disease, and daclizumab is a humanized CD25 antibody approved for the treatment of multiple sclerosis. However, other anti-CD25 antibodies, such as clone 7D4 (anti-mouse CD25), clone MA251 (anti-human CD25), or 7G7B6 (anti-human CD25), still allow the binding of IL-2 to CD25 (Non-Patent Documents 17 and 18). 7G7B6 has been used as a research antibody and has been proposed as a target portion for targeting radionuclides to CD25-expressing lymphomas (Non-Patent Literature 19).

[0006] For example, 7D4 is a rat IgM anti-mouse CD25 antibody widely used to detect CD25-positive cells in the presence of or after treatment with PC61 or an antibody having similar binding properties (Non-Patent Document 7). Very few documents disclose any functional properties of the 7D4-IgM antibody alone or compared to PC61 (Non-Patent Documents 20, 21, 22, 23, 11, and 24). In fact, the possibility of adapting or modifying the isotype or other structural features of 7D4 to obtain improved antibodies for use in cancer therapy has not been taught in the prior art.

[0007] However, the ability of 7D4-IgM (either alone or as a modified antibody), or any anti-human CD25 such as 7G7B6 or M-A251, designed or characterized to have CD25 binding characteristics similar to those of 7D4 to mouse CD25, has not been evaluated in detail with respect to the optimized depletion of Treg cells in tumors, either alone or in combination with other antibodies or other anticancer compounds. As discussed above, Treg cell infiltration in tumors, particularly a low ratio of Teff cells to Treg cells, can lead to poor clinical outcomes. Since CD25 has been identified as a Treg marker, it may be an interesting target for therapeutic antibodies aimed at depleting Treg cells. Importantly, CD25 is the α subunit of the IL-2 receptor, and IL-2 is a cytokine crucial for the Teff response. Anti-CD25 antibodies that have been clinically tested to date deplete Treg cells while blocking CD25-mediated IL-2 signaling. The inventors have now found that blocking IL-2 signaling limits the Teff response, and that anti-CD25 antibodies that do not block IL-2 signaling may provide antibodies that effectively deplete Treg cells while allowing IL-2 to stimulate Teff cells, thereby exhibiting a strong anti-cancer effect. Therefore, there is a need in the art for methods of treating cancer, particularly those involving depleting Treg cells while allowing IL-2 to stimulate Teff cells, especially methods using appropriate anti-CD25 antibodies. [Advanced Technology Documents] [Chartered documents]

[0008]

Patent Document 1

Patent document 2

Patent document 3

Patent document 4

Patent document 5

Non-licensed literature

[0009] [Non-licensed document 1] Smyth M et al., 2014, Immunol Cell Biol. 92, 473-4 [Non-licensed document 2] Onishi H et al, 2012 Anticanc. Res. 32, 997-1003 [Non-licensed document 3] Shang B et al., 2015, Sci Rep. 5:15179

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Summary of the Invention

[0010] This invention provides anti-CD25 antibodies and their uses, characterized by structural elements that bind to CD25 without substantially blocking interleukin-2 (IL-2) binding to CD25 or IL-2 signaling via CD25, and that efficiently deplete Tregs, particularly within tumors. The structural and functional characteristics of 7D4-IgM (described with respect to mouse CD25) were modified to provide an antibody that exhibits remarkably improved characteristics in terms of its use for depleting Tregs and its efficacy against tumors, either alone or in combination with other anticancer agents. Further structural and functional characteristics of anti-CD25 antibodies that efficiently deplete Tregs without blocking interleukin-2 binding to CD25 (and furthermore, without blocking IL-2 signaling via CD25) were also characterized. These findings can be used to identify and generate further anti-human CD25 antibodies that produce equivalent efficacy against tumors in human subjects. In this specification, references to "anti-CD25 antibodies," etc., include their antigen-binding fragments and variants (including affinity-matured variants), unless otherwise indicated by the context.

[0011] In its main aspects, the present invention provides a method for treating a human subject having cancer, comprising the step of administering an anti-CD25 antibody to the subject, wherein the subject has a tumor (preferably a solid tumor), and the antibody does not inhibit the binding of interleukin-2 (IL-2) to CD25.

[0012] In this specification, references to “non-blocking,” “non-blocking,” “non-IL-2 blockade,” “without blockade,” and similar terms (referring to the fact that IL-2 binding to CD25 is not blocked in the presence of an anti-CD25 antibody) include embodiments in which the anti-CD25 antibody does not block IL-2 signaling via CD25. That is, the anti-CD25 antibody of the present invention inhibits IL-2 signaling via CD25 by less than 50% compared to IL-2 signaling in the absence of the antibody. Preferably, the anti-CD25 antibody inhibits IL-2 signaling by about 40%, 35%, less than 30%, and preferably less than about 25%, compared to IL-2 signaling in the absence of the antibody.

[0013] In one embodiment, the anti-CD25 antibody competes with antibody 7G7B6 for binding to human CD25 and / or with antibody MA251 for binding to human CD25.

[0014] In one embodiment, the anti-CD25 antibody binds to the same epitope recognized by antibody 7G7B6 and / or the same epitope recognized by antibody MA251.

[0015] In one embodiment, the anti-CD25 antibody specifically binds to the epitope of human CD25, and the epitope contains one or more amino acid residues included in one or more amino acid stretches selected from amino acids 150-163 (YQCVQGYRALHRGP), amino acids 166-186 (SVCKMTHGKTRWTQPQLICTG), amino acids 42-56 (KEGTMLNCECKRGFR), and amino acids 70-88 (NSSHSSWDNQCQCTSSATR) of SEQ ID NO: 1. The epitope preferably contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen or more amino acid residues included in one or more amino acid stretches selected from amino acids 150-163 (YQCVQGYRALHRGP), amino acids 166-186 (SVCKMTHGKTRWTQPQLICTG), amino acids 42-56 (KEGTMLNCECKRGFR), and / or amino acids 70-88 (NSSHSSWDNQCQCTSSATR) of SEQ ID NO: 1.

[0016] In one embodiment, the anti-CD25 antibody specifically binds to the epitope of human CD25, and the epitope includes at least one sequence selected from amino acids 150-158 (YQCVQGYRA), amino acids 176-180 (RWTQP), amino acids 42-56 (KEGTMLNCECKRGFR), and amino acids 74-84 (SSWDNQCQCTS) of SEQ ID NO: 1.

[0017] In one embodiment, the anti-CD25 antibody specifically binds to the epitope of human CD25, and the epitope includes at least one sequence selected from amino acids 150-158 (YQCVQGYRA), amino acids 166-180 (SVCKMTHGKTRWTQP), amino acids 176-186 (RWTQPQLICTG), amino acids 42-56 (KEGTMLNCECKRGFR), and amino acids 74-84 (SSWDNQCQCTS) of SEQ ID NO: 1.

[0018] In one embodiment, the anti-CD25 antibody specifically binds to an epitope of human CD25, and the epitope includes at least one sequence selected from amino acids 42-56 (KEGTMLNCECKRGFR), amino acids 70-84 (NSSHSSWDNQCQCTS), and amino acids 150-158 (YQCVQGYRA) of SEQ ID NO: 1.

[0019] In one embodiment, the anti-CD25 antibody binds to an epitope containing the sequence of amino acids 42-56 (KEGTMLNCECKRGFR) of SEQ ID NO: 1. In another embodiment, the anti-CD25 antibody binds to an epitope containing the sequence of amino acids 42-56 (KEGTMLNCECKRGFR) and amino acids 150-160 (YQCVQGYRALH) of SEQ ID NO: 1. In yet another embodiment, the anti-CD25 antibody binds to an epitope containing the sequence of amino acids 42-56 (KEGTMLNCECKRGFR) and amino acids 74-88 (SSWDNQCQCTSSATR) of SEQ ID NO: 1. In another embodiment, the anti-CD25 antibody binds to an epitope containing the sequences of amino acids 150-163 (YQCVQGYRALHRGP), 166-180 (SVCKMTHGKTRWTQP), 42-56 (KEGTMLNCECKRGFR), and 74-88 (SSWDNQCQCTSSATR) of SEQ ID NO: 1.

[0020] In one embodiment, the anti-CD25 antibody binds to an epitope containing the sequence of amino acids 176-180 (RWTQP) of SEQ ID NO: 1. In another embodiment, the anti-CD25 antibody binds to an epitope containing the sequence of amino acids 166-180 (SVCKMTHGKTRWTQP) of SEQ ID NO: 1. In yet another embodiment, the anti-CD25 antibody binds to an epitope containing the sequence of amino acids 176-186 (RWTQPQLICTG) of SEQ ID NO: 1.

[0021] In one embodiment, the anti-CD25 antibody specifically binds to an epitope containing the sequences of amino acids 150-158 (YQCVQGYRA) and 176-180 (RWTQP) of SEQ ID NO: 1. In another embodiment, the anti-CD25 antibody specifically binds to an epitope containing the sequences of amino acids 150-158 (YQCVQGYRA) and 176-186 (RWTQPQLICTG) of SEQ ID NO: 1. In yet another embodiment, the anti-CD25 antibody specifically binds to an epitope containing the sequences of amino acids 150-163 (YQCVQGYRALHRGP) and 166-180 (SVCKMTHGKTRWTQP) of SEQ ID NO: 1.

[0022] In one embodiment, the anti-CD25 antibody binds to an epitope containing the sequence of amino acids 74-84 (SSWDNQCQCTSSATR) of SEQ ID NO: 1. In another embodiment, the anti-CD25 antibody binds to an epitope containing the sequence of amino acids 70-84 (NSSHSSWDNQCQCTS) of SEQ ID NO: 1.

[0023] The inventors have surprisingly found that antibodies that bind to specific epitopes of CD25, including antibodies that compete with 7G7B6 and / or MA251 for binding to CD25, are useful in treating cancer, particularly solid tumors. Such antibodies still enable IL-2 signaling via CD25 to which the antibody binds, and the inventors have for the first time discovered that, in addition to depleting Treg cells, the antibodies used in the present invention enable Teff cells to exert their anti-cancer effects optimally by enabling IL-2 binding to CD25 expressed on Teff cells and CD25-mediated signaling, at least in part.

[0024] Such antibodies are 10 times stronger than CD25. -7 Dissociation constant less than M (K d ) and / or about 10 for at least one activated Fcγ receptor -6 It is preferable to have a dissociation constant of less than M. The antibody is 10 to CD25. -8 or 10 -9 or 10-10 or 10 -11 or 10 -12 or 10 -13 and a dissociation constant (K d ) within the range of or less than that is preferred. Most preferably, the antibody is a human IgG1 antibody that binds with high affinity to at least one activating Fcγ receptor and depletes tumor infiltrating regulatory T cells. Most preferably, the anti-CD25 is characterized by other characteristics related to the Fcγ receptor, and in particular, (a) binds to the Fcγ receptor with an activatory to inhibitory ratio (A / I) greater than 1, and / or, (b) binds to FcγRIIa with a higher affinity than it binds to FcγRIIb.

[0025] Considering the use of the anti-CD25 antibody in a treatment method, the anti-CD25 antibody may exhibit further preferred characteristics. The anti-CD25 antibody is preferably a monoclonal antibody, particularly a human, chimeric or humanized antibody. The antibody may be its affinity matured variant, optionally the humanized or affinity matured variant of 7G7B6 or MA251. Further, considering its interaction with immune cells and / or other components of the immune system for its activity to be exerted, the anti-CD25 antibody may further induce an enhancement of the CDC, ADCC and / or ADCP responses, preferably an increase in the ADCC and / or ADCP responses, more preferably an increase in the ADCC response, compared to the existing anti-human CD25 clinical antibodies daclizumab and basiliximab. In some embodiments, the anti-CD25 antibody may induce a decrease in the CDC response compared to the existing anti-human CD25 clinical antibodies daclizumab and basiliximab, and more preferably, the anti-CD25 antibody does not induce a CDC response.

[0026] The anti-CD25 antibody of the present invention (as generally defined above and further detailed in the embodiments for carrying out the invention) can be used in a method of treating a human subject, comprising administering the anti-CD25 antibody to the subject. In one embodiment, the subject has cancer. Preferably, the subject has an encumbred solid tumor (preferably in a method further comprising the step of identifying a subject having a solid tumor). Such a method may further comprise administering a further therapeutic agent to the subject. In one embodiment, the further active agent may be an immune checkpoint inhibitor against the subject, for example, in the form of an antibody that binds to and inhibits an immune checkpoint protein. Preferred immune checkpoint inhibitors are PD-1 antagonists, which may be anti-PD-1 antibodies or anti-PD-L1 antibodies. More generally, the anti-CD25 antibody can be used in a method of depleting regulatory T cells in a solid tumor in a subject, comprising the step of administering the anti-CD25 antibody to the subject.

[0027] In a further embodiment, the anti-CD25 antibody of the present invention can be used in the manufacture of a drug for the treatment of cancer in a human subject, preferably the subject having a tumor, preferably a solid tumor. The antibody can be administered in combination with further therapeutic agents, preferably further cancer therapeutic agents, such as immune checkpoint inhibitors, preferably PD-1 / PD-L1 pathway antagonists, cancer vaccines, and / or in combination with standard of care therapies such as chemotherapy or radiotherapy.

[0028] In a further embodiment, the present invention provides a combination of the anti-CD25 antibody defined above and another anti-cancer compound (preferably an immune checkpoint inhibitor or another compound specified in the embodiment for carrying out the invention) for use in the treatment of cancer in a human subject, preferably the subject having a solid tumor, and the anti-cancer compound (e.g., an immune checkpoint inhibitor such as a PD-1 antagonist or a cytokine such as interleukin-2) can be administered simultaneously, separately, or sequentially. To this extent, the present invention also provides a kit for use in the treatment of cancer, comprising the anti-CD25 antibody defined above and an anti-cancer compound (e.g., an immune checkpoint inhibitor such as a PD-1 antagonist).

[0029] In a further embodiment, the present invention also provides a pharmaceutical composition comprising the anti-CD25 antibody defined above in a pharmaceutically acceptable medium. Such a composition may also contain an anticancer compound (e.g., an immune checkpoint inhibitor such as a PD-1 antagonist).

[0030] In further embodiments, the present invention is (a) A first antigen-binding portion that binds to CD25, (b) A second antigen-binding moiety that binds to another antigen, The present invention also provides a bispecific antibody comprising an anti-CD25 antibody that does not inhibit the binding of interleukin-2 (IL-2) to CD25, and preferably the bispecific antibody is an IgG1 antibody that binds with high affinity to at least one activated Fcγ receptor and depletes tumor-invasion regulatory T cells. Preferably, such a second antigen-binding moiety binds to an antigen selected from immune checkpoint proteins or tumor-associated antigens, or may be an anti-human activated Fc receptor antibody (anti-FcgRI, anti-FcgRIIa, anti-FcgRIII) or an antagonist anti-human FcγRIIb antibody, or based thereon. Thus, the second antigen-binding moiety can bind to FcRIIb. Alternatively, the second antigen-binding moiety may bind to FcgRI, FcgRIIa and / or FcgRIII with antagonistic activity.

[0031] Preferably, such a bispecific antibody includes a second antigen-binding moiety that binds to an immune checkpoint protein selected from the group consisting of PD-1, CTLA-4, BTLA, KIR, LAG3, VISTA, TIGIT, TIM3, PD-L1, B7H3, B7H4, PD-L2, CD80, CD86, HVEM, LLT1, GAL9, GITR, OX40, CD137, and ICOS. Such an immune checkpoint protein is preferably expressed on tumor cells. The immune checkpoint protein is preferably selected from PD-1, PD-L1, and CTLA-4. The second antigen-binding moiety that binds to the immune checkpoint protein may be included in commercially available antibodies that act as immune checkpoint inhibitors. For example, (a) In the case of PD-1, the anti-PD-1 antibody may be nivolumab or pembrolizumab. (b) In the case of PD-L1, the anti-PD-L1 agent is atezolizumab. (c) In the case of CTLA-4, the anti-CTLA-4 agent is ipilimumab.

[0032] Such bispecific antibodies can be supplied in any commercially available format, including Duobody, BiTE DART, CrossMab, Knobs-in-holes, Triomab, or in other suitable molecular formats, including bispecific antibodies and their fragments.

[0033] Alternatively, such a bispecific antibody may include a second antigen-binding moiety that binds to a tumor-associated antigen. In this alternative embodiment, such antigens and corresponding antibodies include, but are not limited to, CD22 (blinatumomab), CD20 (rituximab, tositumomab), CD56 (lorvotuzumab), CD66e / CEA (rabetuzumab), CD152 / CTLA-4 (ipilimumab), CD221 / IGF1R (MK-0646), CD326 / Epcam (edrecolomab), CD340 / HER2 (trastuzumab, pertuzumab), and EGFR (cetuximab, panitumumab).

[0034] The combination of the anti-CD25 antibody of the present invention with another anticancer compound, and the bispecific antibody defined above, can be used in a method for treating cancer, particularly when the subject has a solid tumor, which includes the step of administering the above combination or the bispecific antibody to the subject, and for use in the treatment of cancer in the subject.

[0035] Further objectives of the present invention, including the anti-human CD25 antibody of the present invention, and methods for treating cancer, pharmaceutical compositions, combinations with other anticancer compounds, and further definitions of their use in bispecific antibodies, are presented in the embodiments and examples for carrying out the invention. Item 1 A method for treating a human subject with cancer, comprising the step of administering an anti-CD25 antibody to the subject, wherein the subject has a solid tumor and the antibody does not inhibit the binding of interleukin-2 (IL-2) to CD25. Section 2 The method according to claim 1, wherein the anti-CD25 antibody competes with antibody 7G7B6 for binding to human CD25 and / or with antibody MA251 for binding to human CD25. Section 3 The method according to item 1 or 2, wherein the anti-CD25 antibody binds to the same epitope recognized by antibody 7G7B6 and / or the epitope recognized by antibody MA251. Section 4 The method according to any one of claims 1 to 3, wherein the anti-CD25 antibody specifically binds to an epitope of human CD25, and the epitope comprises one or more amino acid residues included in one or more amino acid stretches selected from amino acids 150-163 (YQCVQGYRALHRGP), amino acids 166-186 (SVCKMTHGKTRWTQPQLICTG), amino acids 42-56 (KEGTMLNCECKRGFR), and amino acids 70-88 (NSSHSSWDNQCQCTSSATR) of SEQ ID NO: 1. Section 5 The method according to claim 4, wherein the anti-CD25 antibody specifically binds to an epitope of human CD25, and the epitope comprises at least one sequence selected from amino acids 150-158 (YQCVQGYRA), amino acids 176-180 (RWTQP), amino acids 42-56 (KEGTMLNCECKRGFR), and amino acids 74-84 (SSWDNQCQCTS) of SEQ ID NO: 1. Section 6 The method according to claim 4, wherein the anti-CD25 antibody specifically binds to an epitope of human CD25, and the epitope comprises at least one sequence selected from amino acids 150-158 (YQCVQGYRA), amino acids 166-180 (SVCKMTHGKTRWTQP), amino acids 176-186 (RWTQPQLICTG), amino acids 42-56 (KEGTMLNCECKRGFR), and amino acids 74-84 (SSWDNQCQCTS) of SEQ ID NO: 1. Section 7 The method according to claim 4, wherein the anti-CD25 antibody specifically binds to an epitope of human CD25, and the epitope comprises at least one sequence selected from amino acids 42-56 (KEGTMLNCECKRGFR), amino acids 70-84 (NSSHSSWDNQCQCTS), and amino acids 150-158 (YQCVQGYRA) of SEQ ID NO: 1. Section 8 The method according to item 4, wherein the anti-CD25 antibody binds to an epitope containing the sequence of amino acids 42-56 (KEGTMLNCECKRGFR) of SEQ ID NO: 1. Section 9 The method according to item 4, wherein the anti-CD25 antibody binds to an epitope containing the sequences of amino acids 42-56 (KEGTMLNCECKRGFR) and amino acids 150-160 (YQCVQGYRALH) of SEQ ID NO: 1. Section 10 The method according to claim 4, wherein the anti-CD25 antibody binds to an epitope containing the sequences of amino acids 42-56 (KEGTMLNCECKRGFR) and amino acids 74-84 (SSWDNQCQCTSSATR) of SEQ ID NO: 1. Section 11 The method according to item 4, wherein the anti-CD25 antibody binds to an epitope containing the sequences of amino acids 150-163 (YQCVQGYRALHRGP), 166-180 (SVCKMTHGKTRWTQP), 42-56 (KEGTMLNCECKRGFR), and 74-84 (SSWDNQCQCTSSATR) of SEQ ID NO: 1. Section 12 The method according to item 4, wherein the anti-CD25 antibody binds to an epitope containing the sequences of amino acids 150-158 (YQCVQGYRA) and amino acids 176-180 (RWTQP) of SEQ ID NO: 1. Section 13 The method according to item 4, wherein the anti-CD25 antibody binds to an epitope containing the sequences of amino acids 150-158 (YQCVQGYRA) and amino acids 176-186 (RWTQPQLICTG) of SEQ ID NO: 1. Section 14 The method according to item 4, wherein the anti-CD25 antibody binds to an epitope containing the sequences of amino acids 150-163 (YQCVQGYRALHRGP) and amino acids 166-180 (SVCKMTHGKTRWTQP) of SEQ ID NO: 1. Section 15 The method according to item 4, wherein the anti-CD25 antibody binds to an epitope containing the sequence of amino acids 74-84 (SSWDNQCQCTS) of SEQ ID NO: 1. Section 16 The method according to item 4, wherein the anti-CD25 antibody binds to an epitope containing the sequence of amino acids 70-84 (NSSHSSWDNQCQCTS) of SEQ ID NO: 1. Section 17 The method according to item 4, wherein the anti-CD25 antibody binds to an epitope containing the sequence of amino acids 176-180 (RWTQP) of SEQ ID NO: 1. Section 18 The method according to item 4, wherein the anti-CD25 antibody binds to an epitope containing the sequence of amino acids 166-180 (SVCKMTHGKTRWTQP) of SEQ ID NO: 1. Section 19 The method according to item 4, wherein the anti-CD25 antibody binds to an epitope containing the sequence of amino acids 176-186 (RWTQPQLICTG) of SEQ ID NO: 1. Section 20 The method according to any one of claims 1 to 19, wherein the anti-CD25 antibody is an IgG1 antibody that binds with high affinity to at least one activated Fcγ receptor selected from FcγRI, FcγRIIc and / or FcγRIIIa, and depletes tumor-infiltrating regulatory T cells. Section 21 The aforementioned anti-CD25 antibody (a) Binds to the Fcγ receptor in an activating-to-inhibiting ratio (A / I) greater than 1, and / or (b) The method according to any one of claims 1 to 19, wherein the binding is to FcγRI, FcγRIIc and / or FcγRIIIa with a higher affinity than the binding to FcγRIIb. Section 22 The aforementioned antibody (a) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 3 and a light chain containing the amino acid sequence of SEQ ID NO: 4, (b) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 5 and a light chain containing the amino acid sequence of SEQ ID NO: 6 (c) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 10 and a light chain containing the amino acid sequence of SEQ ID NO: 14. (d) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 18 and a light chain containing the amino acid sequence of SEQ ID NO: 22, (d) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 23 and a light chain containing the amino acid sequence of SEQ ID NO: 25, (e) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 23 and a light chain containing the amino acid sequence of SEQ ID NO: 26, (f) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 24 and a light chain containing the amino acid sequence of SEQ ID NO: 25 (g) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 24 and a light chain containing the amino acid sequence of SEQ ID NO: 26, (h) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 27 and a light chain containing the amino acid sequence of SEQ ID NO: 30, (i) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 27 and a light chain containing the amino acid sequence of SEQ ID NO: 31, (j) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 27 and a light chain containing the amino acid sequence of SEQ ID NO: 32. (k) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 27 and a light chain containing the amino acid sequence of SEQ ID NO: 33. (l) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 28 and a light chain containing the amino acid sequence of SEQ ID NO: 30, (m) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 28 and a light chain containing the amino acid sequence of SEQ ID NO: 31. (n) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 28 and a light chain containing the amino acid sequence of SEQ ID NO: 32. (o) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 28 and a light chain containing the amino acid sequence of SEQ ID NO: 33. (p) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 29 and a light chain containing the amino acid sequence of SEQ ID NO: 30. (q) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 29 and a light chain containing the amino acid sequence of SEQ ID NO: 31. (r) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 29 and a light chain containing the amino acid sequence of SEQ ID NO: 32, and (s) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 29 and a light chain containing the amino acid sequence of SEQ ID NO: 33. A method according to any one of items 1 to 21, selected from the group consisting of the following. Section 23 The method according to any one of claims 1 to 22, wherein the anti-CD25 antibody is a human IgG2 antibody. Section 24 The method according to any one of claims 1 to 23, wherein the anti-CD25 antibody has a dissociation constant (Kd) of less than 10⁻⁷ M relative to CD25. Section 25 The method according to any one of claims 1 to 24, wherein the anti-CD25 antibody inhibits IL-2 signaling by less than 50%. Section 26 The method according to any one of claims 1 to 25, wherein the anti-CD25 antibody is a monoclonal antibody. Section 27 The method according to any one of claims 1 to 26, wherein the anti-CD25 antibody is a human antibody, a chimeric antibody, or a humanized antibody. Section 28 The method according to any one of items 1 to 27, wherein the antibody is an affinity-matured variant thereof. Section 29 The method according to any one of claims 1 to 28, wherein the antibody is a humanized and / or affinity-matured variant of 7G7B6 or MA251. Section 30 The method according to any one of claims 1 to 29, wherein the anti-CD25 antibody induces enhancement of the CDC, ADCC, and / or ADCP response, preferably an increase in the ADCC and / or ADCP response, more preferably an increase in the ADCC response. Section 31 The method according to any one of claims 1 to 30, wherein the anti-CD25 antibody is administered to a subject having a colonized tumor. Section 32 The method according to any one of claims 1 to 31, further comprising the step of identifying a subject having a solid tumor. Item 33 The method according to any one of claims 1 to 32, further comprising administering an immune checkpoint inhibitor to the subject. Section 34 The method according to item 33, wherein the immune checkpoint inhibitor is a PD-1 antagonist. Item 35 The method according to claim 34, wherein the PD-1 antagonist is an anti-PD-1 antibody or an anti-PD-L1 antibody. Section 36 The method according to any one of items 1 to 32, further comprising administering a cancer vaccine. Section 37 The method according to paragraph 36, wherein the cancer vaccine is a GVAX cancer vaccine. Section 38 An anti-CD25 antibody as defined in any one of items 1 to 30. Section 39 Anti-CD25 antibodies, as described in item 38, used in pharmaceuticals. Section 40 An anti-CD25 antibody as defined in any one of items 1 to 30, used for the treatment of cancer in a human subject, wherein the subject has a solid tumor. Section 41 Use of an anti-CD25 antibody as defined in any one of paragraphs 1 to 30 for the manufacture of a drug for the treatment of cancer in a human subject, wherein the subject has a solid tumor. Section 42 The use of the anti-CD25 antibody for the use described in item 40 or the anti-CD25 antibody described in item 41, wherein the antibody is intended for administration in combination with a further therapeutic agent. Section 43 The use of an anti-CD25 antibody for use as described in item 42, wherein the further therapeutic agent is an immune checkpoint inhibitor. Section 44 The use of an anti-CD25 antibody for the use described in item 43, wherein the immune checkpoint inhibitor is a PD-1 antagonist, or the use described in item 43. Section 45 The use of an anti-CD25 antibody for use as described in item 42 or as described in item 41, wherein the aforementioned further therapeutic agent is a cancer vaccine. Section 46 A combination of an anti-CD25 antibody as defined in any one of items 1 to 30 and a further therapeutic agent for use in the treatment of cancer in a human subject, wherein the subject has a solid tumor and the anti-CD25 antibody and the further therapeutic agent are administered simultaneously, separately, or sequentially. Section 47 A kit for the treatment of cancer comprising an anti-CD25 antibody as defined in any one of items 1 to 30 and an additional therapeutic agent. Section 48 A pharmaceutical composition comprising an anti-CD25 antibody as defined in any one of items 1 to 30 in a pharmaceutically acceptable medium. Section 49 The pharmaceutical composition according to item 48, further comprising a further therapeutic agent. Item 50 The combination for use described in item 46, the kit described in item 47, or the pharmaceutical composition described in item 49, wherein the further therapeutic agent is an immune checkpoint inhibitor. Section 51 The combination for use described in claim 46, the kit described in claim 47, or the pharmaceutical composition described in claim 49, wherein the immune checkpoint inhibitor is a PD-1 antagonist. Section 52 The combination for use described in item 46, the kit described in item 47, or the pharmaceutical composition described in item 49, wherein the further therapeutic agent is a cancer vaccine. Section 53 (a) A first antigen-binding portion that binds to CD25, (b) A second antigen-binding site that binds to an immune checkpoint protein, A bispecific antibody comprising a CD25 binding moiety that does not inhibit the binding of interleukin-2 (IL-2) to CD25, and preferably the bispecific antibody is an IgG1 antibody that binds with high affinity to at least one activated Fcγ receptor selected from FcgRI, FcgRIIa, and FcgRIII, thereby depleting tumor-invading regulatory T cells. Section 54 The bispecific antibody according to item 53, wherein the immune checkpoint protein is selected from the group consisting of PD-1, CTLA-4, BTLA, KIR, LAG3, VISTA, TIGIT, TIM3, PD-L1, B7H3, B7H4, PD-L2, CD80, CD86, HVEM, LLT1, GAL9, GITR, OX40, CD137, and ICOS. Section 55 A bispecific antibody according to item 53 or 54, wherein the aforementioned immune checkpoint protein is expressed on tumor cells. Section 56 The bispecific antibody according to item 53 or 54, wherein the immune checkpoint protein is PD-L1. Section 57 The bispecific antibody according to item 53, wherein the second antigen-binding moiety that binds to PD-L1 is contained in atezolizumab. Section 58 A method for treating cancer, comprising the step of administering a bispecific antibody described in any one of items 53 to 57 to a subject. Section 59 The method according to paragraph 57, wherein the subject has a solid tumor. Item 60 A bispecific antibody as described in any one of sub-sub Section 61 A bispecific antibody for use according to item 60, wherein the subject has a solid tumor. Section 62 A method for depleting regulatory T cells in a subject, comprising the step of administering an anti-CD25 antibody to the subject, wherein the antibody is as defined in any one of items 1 to 30. Section 63 The method according to item 62, wherein the subject has a solid tumor. [Brief explanation of the drawing]

[0036] [Figure 1]This figure shows the characterization of the 7D4 and PC61 anti-mouse CD25 antibodies used in the examples. The effect on IL-2 binding was induced using a tandem-format cross-blocking assay. Biotinylated mouse CD25 was loaded onto an SA sensor. The sensor was then exposed to 100 nM mouse IL-2, followed by either anti-mouse CD25 antibody at 150 seconds. Additional binding by the antibody after IL-2 association indicates that anti-mouse CD25 does not block IL-2 binding, and the absence of further binding indicates ligand blockade. PC-61 mIgG2a shows interference with the mouse IL-2-mouse CD25 interaction, in contrast to 7D4 (A). The mouse IL-2 / mouse CD25 interaction in the presence of recombinant anti-mouse CD25 7D4(mIgG1) was evaluated using a standard sandwich-format cross-blocking assay. 7D4(mIgG1) was loaded onto an AHQ sensor, and the unoccupied Fc binding sites on the sensor were blocked with an unrelated human IgG1 antibody. Next, the sensors were exposed to 100 nM recombinant mouse CD25 (R&D Systems; catalog number 2438-RM-050), followed by recombinant mouse IL-2 (Peprotech; catalog number 212-12). Additional binding by mouse IL-2 after 7D4(mIgG1)-mouse CD25 association showed an unoccupied epitope, and since both 7D4(mIgG1) and mouse IL-2 bind to mouse CD25 simultaneously, they do not compete for epitopes within mouse CD25 (B). Binding to mouse CD25 was determined using CHO cells expressing mouse CD25 for the anti-human CD25-binding antibody daclizumab (DAC), PC61(mIgG2a) antibody (original anti-CD25 obtained from clone PC-61 having mouse IgG2a and κ constant regions associated with ADCC), and a7D4(mIgG1) antibody (anti-D25 obtained from clone 7D4 having mouse IgG1 and κ constant regions). Anti-mouse CD25 IgG binds to mCD25 expressed in cells. CHO-mCD25 was dispensed into a 96-well assay plate (50,000 cells / well) and incubated with 0.1 mL of antibody-containing solution (PBS + 100 nM antibody in 0.1% bovine serum albumin) at 25°C for 15 minutes.Cells were washed three times with ice-cold PBS (+0.1% bovine serum albumin), labeled with goat anti-human IgG (γ-chain specific) R-PE (Southern Biotech, catalog number 2040-09), and analyzed by flow cytometry (dead cells were distinguished using propidium iodide). No binding was detected for DAC, but both PC61 (mIgG2a) and 7D4 (mIgG1) showed clear binding to the cells (C). [Figure 2]This figure shows the effect of anti-mouse CD25 antibody on the induction of granzyme B expression by CD4 T cells after stimulation with anti-CD3 and anti-CD28. All CD4-positive T cells were isolated from mouse lymph nodes and spleen using CD4 microbeads and labeled with CellTrace® Violet dye (ThermoFisher) for measurement of cell proliferation. Labeled T cells (105 cells) were seeded in a 96-well plate with feeder cells (105 cells; CD90.2 negative fraction, using the mouse pan-T Dynabeads kit). Anti-CD3 (clone 145-2C11, BioXcell catalog no. BE0001-1; 1 μg / ml) and anti-CD28 (clone 37.51, BioXcell catalog no. BE0015-1; 0.5 μg / ml) were added to the wells (excluding the control sample of unstimulated labeled T cells) to activate CD4 T cells and induce proliferation and granzyme B production. Next, to demonstrate the effect of blocking the interaction between IL-2 and its receptor on T cell activation, the following antibodies were added (at 25 μg / mL) to wells containing labeled CD4 T cells and anti-CD3 and anti-CD28 antibodies (wells containing only labeled T cells and anti-CD3 and anti-CD28 antibodies were used as negative controls): PC61 (mIgG2a), 7D4 (mIgG1), or the neutralizing anti-mouse IL-2 antibody used as a positive control (clone Jes6-1A12, BioXcell 6032988564). The labeled T cell samples were then incubated for approximately 84 hours. The cells were then fixed, permeabilized, and stained with anti-mouse granzyme B antibody (clone GB11, Invitrogen). The cells were then analyzed by flow cytometry for granzyme B expression and CellTrace violet dye dilution (BV450). The percentage of labeled T cells that proliferate and express granzyme B (GnzB) is shown in the upper left quadrant (Q9) of the specific graph for each treatment, and the percentage of labeled T cells that proliferate but do not express GnzB is shown in the lower left quadrant (Q12) of the specific graph for each treatment. [Figure 3]This figure shows the in vivo effects on immune cells of anti-mouse CD25 antibodies with the same isotype (mouse IgG2a) that either block or do not block the interaction between CD25 and IL-2, when administered in or without the presence of anti-mouse PD1 (aPD1; clone RMP1-14). Six mouse groups were subcutaneously injected with MCA205 tumor cells (5 × 10⁵ cells, 15 cells) on day 0 and treated separately as indicated in the graph. Four groups were intraperitoneally injected with anti-mouse CD25 antibody (either aPC61 mIgG2a or a7D4 mIgG2a; 200 μg) on ​​day 5. Three groups were intraperitoneally injected with aPD1 (100 μg) on ​​days 6 and 9. After collecting tumors and lymph nodes on day 12, cells were processed for staining according to the desired type and analyzed by flow cytometry using the following antibodies as specified in each panel: anti-CD3 (clone 17A2, Biolegend), anti-CD4 (clone RM4-5, BD biosciences), anti-CD8 (clone 53-6.7, Biolegend), and anti-FoxP3 (clone FJK-16s, eBiosciences). Intranuclear staining for FoxP3 was performed using the FoxP3 Transcription Factor Staining Buffer Set (eBioscience). The percentages of CD4-positive / Foxp3-positive regulatory T cells and CD4-positive / FoxP3-negative effector CD4 T cells (CD4 Teff) in LNs and TILs, as well as the ratios of effector CD8-positive T cells / Treg cells and CD4 Teff / Treg cells are shown. Data analysis was performed using Flowjo version 10.0.8 (Tree Star Inc.). Statistical analysis was performed using Prism 6 (GraphPad Software, Inc.). p-values ​​were calculated using Kruskal-Wallis ANOVA and Dunn's post-hoc test (ns=p>0.05; ****=p<0.0001). [Figure 4]This figure shows the effect of in vivo proliferation of T cells on the production of granzyme B by anti-mouse CD25 antibodies of the same isotype (IgG2a) that either block or do not block the interaction between CD25 and IL-2, in the presence or absence of anti-mouse PD1 (aPD1; clone RMP1-14). Cell samples were generated using an MCA205-based model in six treatment groups as specified in Figure 3. Tumor cells were stained according to the desired type and analyzed by flow cytometry using the following antibodies as specified for each panel: anti-CD3 (PeCy7, clone 145-2C11, eBioscience, 25003182), anti-CD4 (V500, clone RM4-5, BD biosciences, 560782), anti-CD8 (BV785, clone 53-6.7, Biolegend, 100750), anti-granzyme B (APC, clone GB11; Invitrogen, grb05), and Ki67 (V450, clone SolA15; eBiosciences, 48569882). Intranuclear staining for Ki67 and granzyme B was performed using the FoxP3 Transcription Factor Staining Buffer Set (eBioscience, 00-5523-00). The percentage of GnzB-positive wells as the total number of GnzB-positive proliferative (indicated by Ki67 positivity) CD4-positive or CD8-positive T cells was compared. Statistical analysis was performed as in Figure 3 (ns=p>0.05;*=p<0.05;**=p<0.01;***=p<0.001;****=p<0.0001). [Figure 5]This figure shows the effect of anti-mouse CD25 (IgG2a isotype), administered in combination with or without anti-PD-1 (clonal RMP1-14), on the eradication of settled tumors in a CT26 mouse model. 7D4m2a and PC61m2a are both anti-mouse CD25 Treg depletion antibodies, but one is non-IL-2 blockade (7D4m2a) and the other is IL-2 blockade (PC61m2). Individual mouse growth curves were established over time for each treatment group. The number of tumor-free survivors at 50 days is shown in each graph. CT26 cells used for transplantation were collected during the log phase growth and resuspended in cold PBS. On day 1 of the study (D1), 3 × 10⁵ cells (0.1 mL cell suspension) were subcutaneously injected into the right flank of each mouse. Anti-mouse CD25 was administered via ip injection (10 mg / kg) on ​​day 6 (if a palpable tumor was detected). Anti-mouse PD1 was administered via intravenous injection (100 μg / injection) on days 7, 10, 14, and 17. Tumor growth was monitored by measuring two dimensions of the tumor twice weekly with calipers. Tumor size (mm³) was calculated from tumor volume = (w² × l) / 2 (where w is the tumor width (mm) and l is the tumor length (mm)). The study endpoint was defined as a tumor volume of 4000 mm³ or 50 days, whichever came first (data points ending earlier or on different days are due to mouse death; the number of surviving animals at the end of the experiment is shown within each panel). [Figure 6] This figure shows the CT26 tumor growth curves of individual mice treated with an anti-mouse CD25 IgG2a antibody that either depletes Tregs, but is either non-IL-2 blocked (7D4m2a) or blocked (PC61m2), either untreated (PBS, vehicle only) or combined with or untreated with anti-mouse PD-L1 clone 10F.9G2 (aPDL1; clone 10F.9G2). The model, regimen, and data analysis are the same as in Figure 5. [Figure 7]This figure shows the effect of anti-mouse CD25 (IgG2a isotype), administered in combination with or without anti-PD-1 (clone RMP1-14), on the eradication of settled tumors in an MC38 mouse model. The antibodies tested are listed in Figure 5. Growth curves for individual mice were established over time for each treatment group. The number of tumor-free survivors at 35 days is shown in each graph. MC38 colon cancer cells used for transplantation were collected during the logarithmic growth phase and resuspended in cold PBS. 5 × 10⁵ tumor cells (0.1 mL cell suspension) were subcutaneously injected into the right flank of each mouse. Tumors were monitored when their volume approached the target range of 100 mm³ to 150 mm³. 22 days after tumor transplantation, on day 1 of the study, animals with individual tumor volumes ranging from 75 mm³ to 126 mm³ were sorted into nine groups (n=10) with an average tumor volume of approximately 10⁶ mm³. Treatment was initiated on day 1 in mice carrying established MC38 tumors. On days 1, 2, 5, 9, and 12, the effects of each treatment were compared to a vehicle-treated control group administered PBS intraperitoneally (ip). Anti-PD1 was administered ip at 100 μg / animal twice weekly (biwk×2) for two weeks, starting on day 2. 7D4m2a and PC61m2a were administered ip at 200 μg / animal once on day 1. Tumor measurements were performed twice weekly. The study endpoint was defined as tumor volume of 4000 mm3 or 35 days, whichever came first (data points ending earlier or on different days are due to mouse death; the number of surviving animals at the end of the experiment is shown within each panel). [Figure 8] This figure shows the MC38 tumor growth curves of individual mice treated with an anti-mouse CD25 IgG2a antibody that either depletes Tregs, but is either non-IL-2 blocked (7D4m2a) or IL-2 blocked (PC61m2), either untreated (PBS, vehicle only) or combined with or untreated with anti-mouse PD-L1 clone 10F.9G2 (aPDL1; clone 10F.9G2). The model, regimen, and data analysis are the same as in Figure 7. [Figure 9]This figure shows the evaluation of the therapeutic activity of 7D4 mIgG2a, an anti-mouse CD25 non-IL-2 blocking Treg depletion antibody, in female BALB / c mice carrying CT26 syngeneic colon tumors. This activity was evaluated using 7D4 mIgG2a alone (D), in combination with an IL-2 neutralizing antibody (ThermoFisher; JES6-1A12) (E), or in combination with a mouse IgG1 isotype IL-2 blocking non-depletion anti-mouse CD25 antibody (PC61 mouse IgG1) (F). The activity of mouse IgG2s control (A), IL-2 neutralizing antibody alone (B), and IL-2 blocking anti-CD25 antibody alone (C) was tested for comparison. [Figure 10] This figure shows the consensus sequence of human CD25 (Uniprot code P01589), referred to herein as Sequence ID No. 1. The extracellular domain of mature CD25 corresponding to amino acids 22-240 is underlined. The locations of epitopes from previously identified non-IL-blocking anti-CD25 antibodies are shown: Epitope 1 (complete and short epitope), Epitope 2 (complete and short epitope), Epitope 3, and Epitope 4 (complete and short epitope). The locations of the epitopes of basiliximab and daclizumab (shown as DAC) are also identified. [Figure 11] This figure shows the characterization of the binding of (A)7D4, (B)PC61, and (C)2E4 to CD25 expressed on CD25-expressing CHO cells, compared to mouse IgG2a isotype controls, at gradually increasing antibody concentrations. [Figure 12] This figure shows SPR-based analysis of purified antibody (mIgG2a) against his-tagged rmCD25 using Biacore 2000. (A) 7D4, (B) 2E4. [Figure 13] This figure shows the competitive analysis of anti-mCD25 antibodies against his-tagged rmCD25 in Octet96. It shows the capture of 7D4 on the sensor and binding by a second antibody after the antigen association step. Competitive binding to mCD25 is observed between 7D4 and 2E4 (A), but not between 7D4 and PC61 (B). [Figure 14]This figure shows the characterization of 7D4, PC61, and 2E4 T cells isolated from C57BL / 6 splenocytes for IL-2 signaling blockade in a STAT5 phosphorylation assay, compared to mouse IgG2a isotype control or in the absence of primary antibody. Cells were incubated with 50 μg / ml antibody, followed by 50 U / ml IL-2. Analysis was limited to the percentage of Treg cells phosphorylating STAT5. [Figure 15] This figure shows in vivo Treg depletion in balb / c mice carrying 4T1 tumors after administration of mouse anti-mouse CD25(7D4) antibody. (A)-(C): Non-CD4, CD4+, and CD25+FoxP3+ cells (%) in whole blood 3 days after administration, respectively. (D)-(F): Non-CD4, CD4+, and CD25+FoxP3+ cells (%) in tumors 3 days after administration, respectively. (G)-(I): Non-CD4, CD4+, and CD25+FoxP3+ cells (%) in whole blood 9 days after administration, respectively. (J)-(L): Non-CD4, CD4+, and CD25+FoxP3+ cells (%) in tumors 9 days after administration, respectively. [Figure 16] This figure shows the characterization of mouse (B) or chimeric (A, C, and D) anti-human CD25 clone 7G7B6 that binds to CD25 expressed on Karpas 299 cells (A), human in vitro differentiated Treg cells (B), SU-DHL-1 cells (C), or SR-786 cells (D), compared to human IgG1 isotype controls, at progressively increasing antibody concentrations. [Figure 17] This figure shows the characterization of 7G7B6 cells in a STAT5 phosphorylation assay using human-derived PBMCs, compared to mouse IgG2a isotype controls, human IgG1 isotype controls, and the absence of daclizumab or primary antibody, for blocking IL-2 signaling. Cells were incubated with 10 μg / ml antibody, followed by escalating concentrations of IL-2 (shown in the figure). Analysis was limited to the percentage of CD3-positive cells that phosphorylated STAT5. [Figure 18]This figure shows the functional characterization of chimeric 7G7B6 compared to a human IgG1 isotype control using pan-T cells, daclizumab, or a commercially available mouse anti-human IL-2 neutralizing antibody (clone: ​​AB12-3G4) as a positive control. Cells were incubated with 10 μg / ml antibody and then activated with CD3 / CD28 beads for 72 hours before flow cytometry analysis. The results show the percentage of granzyme B-positive proliferative CD4 T cells. [Figure 19] This figure shows the functional characterization of the chimeric 7G7B6 compared to human IgG1 isotype controls in terms of killing CD25-positive cell lines in the ADCC assay. High- and low-expression CD25 cells, SU-DHL-1 (A) or SR-786 (B), were co-cultured with purified NK cells in the presence of various concentrations of antibodies (shown in the figure). Target cell lysis was measured by calcein release into the supernatant 4 hours after addition to the NK cells. Data were normalized to saponin-treated controls. [Figure 20] This figure shows the functional characterization of chimeric 7G7B6 in vitro differentiated Treg cells in ADCP assays compared to human IgG1 isotype controls. Treg cells were co-cultured with MCSF-differentiated macrophages in the presence of various antibody concentrations (shown in the figure). Two-color flow cytometry analysis was performed using CD14+ stained macrophages and eFluor450-labeled Treg cells. Residual target cells were defined as cells that are eFluor450-labeled / CD14-. Double-labeled cells (eFluor450-labeled / CD14+) were considered to represent phagocytosis of the target by macrophages. The phagocytosis of target cells was calculated using the following formula: Phagocytosis (%) = 100 × [(Double Positive Rate) / (Double Positive Rate + Residual Target Rate)]. [Figure 21] This figure shows the characterization of MA-251, which binds to CD25 expressed on Karpas 299 cells, compared to a mouse IgG1 isotype control, at progressively increasing antibody concentrations. [Figure 22]This figure shows the characterization of MA-251 compared to mouse IgG1 isotype control, human IgG1 isotype control, and the absence of daclizumab or primary antibody. Blockade of IL-2 signaling in the STAT5 phosphorylation assay was assessed using human-derived PBMCs. Cells were incubated with 10 μg / ml antibody, followed by escalating concentrations of IL-2 (shown in the figure). Analysis was limited to the percentage of CD3-positive cells phosphorylating STAT5. [Figure 23] This figure shows the characterization of MA-251 and IL-2 binding to CD25. Interference with IL-2 ligands binding to CD25 was performed on a Forte Bio Octet Red384 system (Pall Forte Bio Corp., USA) using a standard sandwich binning assay. MA251 antibody was loaded onto an AHQ sensor, and the unoccupied Fc binding site on the sensor was blocked with unrelated human IgG1 antibody. The sensor was exposed to 100 nM human CD25, followed by 100 nM human IL-2. Data were processed using Forte Bio Data Analysis Software 7.0. Additional binding by human IL-2 after antigen association indicates an unoccupied epitope (non-competitor), while the absence of binding indicates epitope blockade (competitor). [Figure 24] This figure shows the competitive analysis of anti-CD25 antibodies in Octet. The first Ab is conjugated to immobilized rhCD25, followed by either the first Ab (control) or the second Ab again. mAbs, which are non-blockers of the IL-2 signaling pathway, compete with each other or with 7G7B6 and MA251, but not with research-grade daclizumab or research-grade basiliximab (Figure 24(A)-(N)). (A)-(C) Competitive analysis of 7G7B6; (D)-(F) Competitive analysis of MA251; (G)-(I) and (N) Competitive analysis of antibody 3; (J)-(M) Competitive analysis of antibody 1. The IL-2 signaling blocker mAb (TSK031) competes with research-grade daclizumab and research-grade basiliximab, but not with 7G7B6 (Figure 24(O)-(Q)). [Figure 25]This figure shows an in vivo model demonstrating the suppression of tumor growth after administration of vehicles (A) and (C), or antibodies 1 (B), (D), and (E). [Figure 26] This figure shows the affinity determination of purified antibody (IgG1) against his-tagged rhCD25 by SPR-based analysis using Biacore 2000. A) 7g7B6ch, B) MA251ch, C) Antibody 1, D) Antibody 3 and E) Daclizumab (control), or by biolayer interferometry using an Octet Red 96 instrument (F). [Figure 27] This figure shows the characterization of antibody 1 that binds to CD25, expressed on human in vitro differentiated Treg cells (A), SU-DHL-1 cells (B), or SR-786 cells (C), compared to human IgG1 isotype controls, at gradually increasing antibody concentrations. [Figure 28] This figure shows the characterization of antibody 1, which binds to CD25, expressed on human (A) and (B) pan-T cells activated with CD3 / CD28 beads and gated on CD4+ and CD8+ T cells, compared to human IgG1 isotype controls at gradually increasing antibody concentrations. [Figure 29] This figure shows non-competitive binding of antibody 1 to IL-2 and competitive binding of an IL-2 competitive antibody to IL-2 (B) using biolayer interference with Octet Red384 via a standard sandwich format binning assay. Antibody 1, an anti-human CD25 antibody, was loaded onto an AHQ sensor. The sensor was then exposed to 100 nM human CD25, followed by human IL-2. Additional binding by human IL-2 after antigen association indicates an unoccupied epitope (non-competitive substance), while the absence of binding indicates epitope blockade (competitive substance). [Figure 30]This figure shows the non-competitive binding of antibody 1 and daclizumab to CD25 by biolayer interference on an Octet Red384 system using a standard sandwich format binning assay. A reference monoclonal anti-human CD25 antibody, daclizumab, was loaded onto an AHQ sensor. The sensor was then exposed to 100 nM human CD25 antigen, followed by anti-human CD25 antibody (antibody 1). Additional binding by the second antibody after antigen association indicates an unoccupied epitope (non-competitive substance), while the absence of binding indicates epitope blockade (competitive substance). [Figure 31] This figure shows the characterization of antibody 1 in a STAT5 phosphorylation assay using human-derived PBMCs, compared to the absence of a human IgG1 isotype control, daclizumab, or primary antibody, for blocking IL-2 signaling. Cells were incubated with 10 μg / ml antibody, followed by escalating concentrations of IL-2 (shown in the figure). Analysis was limited to the percentage of CD3-positive cells that phosphorylated STAT5. [Figure 32] This figure shows the functional characterization of antibody 1 compared to a human IgG1 isotype control using pan-T cells, daclizumab, or a commercially available mouse anti-human IL-2 neutralizing antibody (clone: ​​AB12-3G4) as a positive control. Cells were incubated with 10 μg / ml antibody and then activated with CD3 / CD28 beads for 72 hours before flow cytometry analysis. The results show the percentage of granzyme B-positive proliferative CD4(A) or CD8(B) T cells. [Figure 33] This figure shows the functional characterization of antibody 1 compared to human IgG1 isotype controls for killing CD25-positive cell lines in the ADCC assay. CD25-high and low-expression cells, SU-DHL-1 (A) or SR-786 (B), were co-cultured with purified NK cells in the presence of various concentrations of antibody (shown in the figure). Target cell lysis was measured by calcein release into the supernatant 4 hours after addition to NK cells. Data were normalized to saponin-treated controls. [Figure 34]This figure shows the functional characterization of antibody 1 compared to human IgG1 isotype controls regarding phagocytosis of differentiated Treg cells in an ADCP assay. Treg cells were co-cultured with MCSF-differentiated macrophages in the presence of various concentrations of antibody (shown in the figure). Two-color flow cytometry analysis was performed using CD14+ stained macrophages and eFluor450-labeled Treg cells. Residual target cells were defined as cells that are eFluor450-labeled / CD14-. Double-labeled cells (eFluor450-labeled / CD14+) were considered to represent phagocytosis of the target by macrophages. Phagocytosis of target cells was calculated using the following formula: Phagocytosis (%) = 100 × [(Double Positive Rate) / (Double Positive Rate + Residual Target Rate)]. [Figure 35] This figure shows the characterization of antibody 3 that binds to CD25 expressed on human in vitro differentiated Treg cells (A), SU-DHL-1 cells (B), or SR-786 cells (C) at gradually increasing antibody concentrations, compared to human IgG1 isotype controls. [Figure 36] This figure shows the characterization of antibody 3, which binds to CD25, expressed on human (A) and (B) or cynomolgus monkey (C) and (D) pan-T cells activated with CD3 / CD28 beads, and gated in CD4+ and CD8+ T cells, compared to human IgG1 isotype controls at gradually increasing antibody concentrations. [Figure 37] This figure shows non-competitive binding of antibody 3 to IL-2 by biolayer interference in Octet Red384 using a standard sandwich format binning assay. Antibody 3, an anti-human CD25 antibody, was loaded onto an AHQ sensor. The sensor was then exposed to 100 nM human CD25, followed by human IL-2. Additional binding by human IL-2 after antigen association indicates a non-occupied epitope (non-competitive substance). [Figure 38]This figure shows the non-competitive binding of antibody 3 and daclizumab to CD25 by biolayer interference on an Octet Red384 system using a standard sandwich format binning assay. A reference monoclonal anti-human CD25 antibody, daclizumab, was loaded onto an AHQ sensor. The sensor was then exposed to 100 nM human CD25 antigen, followed by anti-human CD25 antibody (antibody 3). Additional binding by the second antibody after antigen association indicates an unoccupied epitope (non-competitive substance), while the absence of binding indicates epitope blockade (competitive substance). [Figure 39] This figure shows the characterization of antibody 3 for blocking IL-2 signaling in a STAT5 phosphorylation assay using human-derived PBMCs, compared to the absence of a human IgG1 isotype control, daclizumab, or primary antibody. Cells were incubated with 10 μg / ml antibody, followed by escalating concentrations of IL-2 (shown in the figure). Analysis was limited to the percentage of CD3-positive cells that phosphorylated STAT5. [Figure 40] This figure shows the functional characterization of antibody 3 compared to a human IgG1 isotype control using pan-T cells, daclizumab, or a commercially available mouse anti-human IL-2 neutralizing antibody (clone: ​​AB12-3G4) as a positive control. Cells were incubated with 10 μg / ml antibody and then activated with CD3 / CD28 beads for 72 hours before flow cytometry analysis. The results show the percentage of granzyme B-positive proliferative CD4(A) or CD8(B) T cells. [Figure 41] This figure shows the functional characterization of antibody 3 compared to human IgG1 isotype controls for killing CD25-positive cell lines in the ADCC assay. CD25-high or low-expression cells, SU-DHL-1 (A) or SR-786 (B), were co-cultured with purified NK cells in the presence of various concentrations of antibody (shown in the figure). Target cell lysis was measured by calcein release into the supernatant 4 hours after addition to the NK cells. Data were normalized to saponin-treated controls. [Figure 42]This figure shows the functional characterization of antibody 3 compared to human IgG1 isotype controls regarding phagocytosis of differentiated Treg cells in an ADCP assay. Treg cells were co-cultured with MCSF-differentiated macrophages in the presence of various antibody concentrations (shown in the figure). Two-color flow cytometry analysis was performed using CD14+ stained macrophages and eFluor450-labeled Treg cells. Residual target cells were defined as cells that are eFluor450-labeled / CD14-. Double-labeled cells (eFluor450-labeled / CD14+) were considered to represent phagocytosis of the target by macrophages. Phagocytosis of target cells was calculated using the following formula: Phagocytosis (%) = 100 × [(Double Positive Rate) / (Double Positive Rate + Residual Target Rate)]. [Figure 43] This figure shows the characterization of antibody 4 that binds to CD25 expressed on human in vitro differentiated Treg cells (A), SU-DHL-1 cells (B), or SR-786 cells (C) at gradually increasing antibody concentrations, compared to human IgG1 isotype controls. [Figure 44] This figure shows the characterization of antibody 4, which binds to unmodified CHO-S cells (negative control) (A) or cyno-CD25-CHO-S cells (B) at a 100 nM antibody concentration, compared to a human IgG1 isotype control. [Figure 45] This figure shows non-competitive binding of antibody 4 to IL-2 by biolayer interference in Octet Red384 using a standard sandwich format binning assay. Antibody 4, an anti-human CD25 antibody, was loaded onto an AHQ sensor. The sensor was then exposed to 100 nM human CD25, followed by human IL-2. Additional binding by human IL-2 after antigen association indicates a non-occupied epitope (non-competitive substance). [Figure 46]This figure shows the non-competitive binding of antibody 4 and daclizumab to CD25 by biolayer interference on an Octet Red384 system using a standard sandwich format binning assay. A reference monoclonal anti-human CD25 antibody, daclizumab, was loaded onto an AHQ sensor. The sensor was then exposed to 100 nM human CD25 antigen, followed by anti-human CD25 antibody (antibody 4). Additional binding by the second antibody after antigen association indicates an unoccupied epitope (non-competitive substance), while the absence of binding indicates epitope blockade (competitive substance). [Figure 47] This figure shows the characterization of antibody 4 for blocking IL-2 signaling in a STAT5 phosphorylation assay using human-derived PBMCs, compared to the absence of a human IgG1 isotype control, daclizumab, or primary antibody. Cells were incubated with 10 μg / ml antibody, followed by escalating concentrations of IL-2 (shown in the figure). Analysis was limited to the percentage of CD3-positive cells that phosphorylated STAT5. [Figure 48] This figure shows the functional characterization of antibody 4 compared to a human IgG1 isotype control, daclizumab, or a commercially available mouse anti-human IL-2 neutralizing antibody (clone: ​​AB12-3G4) as a positive control using pan-T cells. Cells were incubated with 10 μg / ml antibody and then activated with CD3 / CD28 beads for 72 hours before flow cytometry analysis. The results show the percentage of granzyme B-positive proliferative CD4(A) or CD8(B) T cells. [Figure 49] This figure shows the functional characterization of antibody 4 compared to human IgG1 isotype controls for killing CD25-positive cell lines in the ADCC assay. CD25-high or low-expression cells, SU-DHL-1 (A) or SR-786 (B), were co-cultured with purified NK cells in the presence of various concentrations of antibody (shown in the figure). Target cell lysis was measured by calcein release into the supernatant 4 hours after addition to the NK cells. Data were normalized to saponin-treated controls. [Figure 50]This figure shows the functional characterization of antibody 4 compared to human IgG1 isotype controls for ADCP induction in the Reporter Bioassay. CD25-expressing SU-DHL-1 cells were co-cultured with Jurkat T cells genetically engineered to express the NFAT response element (NFAT-RE-luc2) that drives FcγRIIa and luciferase expression, in the presence of various antibody concentrations (shown in the figure). [Figure 51] This figure shows the characterization of antibody 2 that binds to CD25 expressed on human in vitro differentiated Treg cells (A), SU-DHL-1 cells (B), or SR-786 cells (C) at gradually increasing antibody concentrations, compared to human IgG1 isotype controls. [Figure 52] This figure shows the characterization of antibody 2, which binds to CD25, expressed on human (A) and (B) or cynomolgus monkey (C) and (D) pan-T cells activated with CD3 / CD28 beads, and gated in CD4+ and CD8+ T cells, compared to human IgG1 isotype controls at gradually increasing antibody concentrations. [Figure 53] This figure shows non-competitive binding of antibody 2 to IL-2 and competitive binding of an IL-2 competitive antibody to IL-2 (B) using biolayer interference with Octet Red384 via a standard sandwich format binning assay. Antibody 2, an anti-human CD25 antibody, was loaded onto an AHQ sensor. The sensor was then exposed to 100 nM human CD25, followed by human IL-2. Additional binding by human IL-2 after antigen association indicates a non-occupied epitope (non-competitive substance). [Figure 54] This figure shows the non-competitive binding of antibody 2 and daclizumab to CD25 by biolayer interference on an Octet Red384 system using a standard sandwich format binning assay. A reference monoclonal anti-human CD25 antibody, daclizumab, was loaded onto an AHQ sensor. The sensor was then exposed to 100 nM human CD25 antigen, followed by anti-human CD25 antibody (antibody 2). Additional binding by the second antibody after antigen association indicates an unoccupied epitope (non-competitive substance), while the absence of binding indicates epitope blockade (competitive substance). [Figure 55] This figure shows the characterization of antibody 2 for blocking IL-2 signaling in a STAT5 phosphorylation assay using human-derived PBMCs, compared to the absence of a human IgG1 isotype control, daclizumab, or primary antibody. Cells were incubated with 10 μg / ml antibody, followed by escalating concentrations of IL-2 (shown in the figure). Analysis was limited to the percentage of CD3-positive cells that phosphorylated STAT5. [Figure 56] This figure shows the functional characterization of antibody 2 compared to human IgG1 isotype controls for killing CD25-positive cell lines in the ADCC assay. CD25-high or low-expression cells, SU-DHL-1 (A) or SR-786 (B), were co-cultured with purified NK cells in the presence of various concentrations of antibody (shown in the figure). Target cell lysis was measured by calcein release into the supernatant 4 hours after addition to NK cells. Data were normalized to saponin-treated controls. [Figure 57] This figure shows the functional characterization of antibody 2 compared to human IgG1 isotype control regarding phagocytosis of differentiated Treg cells in an ADCP assay. Treg cells were co-cultured with MCSF-differentiated macrophages in the presence of various concentrations of antibody (shown in the figure). Two-color flow cytometry analysis was performed using CD14+ stained macrophages and eFluor450 dye-labeled Treg cells. Residual target cells were defined as cells that are eFluor450 dye+ / CD14-. Double-labeled cells (eFluor450 dye+ / CD14+) were considered to represent phagocytosis of the target by macrophages. Phagocytosis of target cells was calculated using the following formula: Phagocytosis (%) = 100 × [(Double Positive Rate) / (Double Positive Rate + Residual Target Rate)]. [Figure 58] This figure shows the characterization of antibody 5, which binds to CD25 expressed on Karpas 299 cells, compared to a human IgG1 isotype control, at gradually increasing antibody concentrations. [Figure 59]This figure shows the characterization of antibody 5 for blocking IL-2 signaling in a STAT5 phosphorylation assay using human-derived PBMCs. Mouse anti-human antibody MA-251 was used as a non-blocking control, and clinical-grade high-yield synthetic daclizumab (Daclizumab High Yield Process; DAC HYP) was used as a blocking control, compared to mouse IgG1 isotype control, human IgG1 isotype control, or the absence of the primary antibody. Cells were incubated with 10 μg / ml antibody, followed by 10 U / ml IL-2. Analysis was limited to the percentage of CD3-positive cells that phosphorylated STAT5. [Figure 60] This figure shows a competitive assay in Octet. Antibody 1 is conjugated to immobilized rhCD25, followed by either a first Ab (antibody 1 as a control) or a second Ab (either an IL-2 competitor, e.g., research-grade daclizumab and basiliximab, or an IL-2 non-competitive, e.g., 7G7B6). Antibody 1 does not compete with the IL-2 signaling blockers research-grade basiliximab (A) and daclizumab (B), but it does compete with 7G7B6 (a non-IL-2 blocker) (C). [Figure 61] This figure shows the characterization of antibody 5 compared to an anti-human CD25 Fc silent control antibody for ADCC induction in the Reporter Bioassay. CD25-expressing SR-786 cells were co-cultured with Jurkat T cells genetically engineered to express the NFAT response element (NFAT-RE-luc2) that drives FcγRIIIa and luciferase expression, in the presence of various antibody concentrations (shown in the figure). [Figure 62]This figure shows the functional characterization of antibody 5 compared to human IgG1 isotype controls regarding phagocytosis of differentiated Treg cells in an ADCP assay. Treg cells were co-cultured with MCSF-differentiated macrophages in the presence of various antibody concentrations (shown in the figure). Two-color flow cytometry analysis was performed using CD14+ stained macrophages and eFluor450-labeled Treg cells. Residual target cells were defined as cells that are eFluor450-labeled / CD14-. Double-labeled cells (eFluor450-labeled / CD14+) were considered to represent phagocytosis of the target by macrophages. The phagocytosis of target cells was calculated using the following formula: Phagocytosis (%) = 100 × [(Double Positive Rate) / (Double Positive Rate + Residual Target Rate)]. [Figure 63] This figure shows the characterization of antibodies 6, 7, 8, and 9 bound to CD25 expressed on Karpas 299 cells, compared to human IgG1 isotype controls, at gradually increasing antibody concentrations. [Figure 64] This figure shows a competitive assay in Octet. Immobilized rhCD25 is conjugated with the first Ab (antibody 7), followed by the first Ab again (control), or either the second Ab, daclizumab (A) or basiliximab (B). [Figure 65] This figure shows the characterization of antibody 7 for blocking IL-2 signaling in a STAT5 phosphorylation assay using human-derived PBMCs, compared to human IgG1 isotype control, daclizumab-Hyp, or the absence of the primary antibody. Cells were incubated with 10 μg / ml antibody, followed by 10 U / ml IL-2. Analysis was limited to the percentage of CD3-positive cells that phosphorylated STAT5. [Figure 66] This figure shows the functional characterization of antibody 7 compared to an anti-human CD25 Fc silent control antibody for ADCC induction in the Reporter Bioassay. CD25-expressing SR-786 cells were co-cultured with Jurkat T cells genetically engineered to express the NFAT response element (NFAT-RE-luc2) that drives FcγRIIIa and luciferase expression, in the presence of various antibody concentrations (shown in the figure). [Figure 67] This figure shows the functional characterization of antibody 7 compared to an anti-human CD25 Fc silent control antibody regarding phagocytosis of differentiated Treg cells in an ADCP assay. Treg cells were co-cultured with MCSF-differentiated macrophages in the presence of various antibody concentrations (shown in the figure). Two-color flow cytometry analysis was performed using CD14+ stained macrophages and eFluor450 dye-labeled Treg cells. Residual target cells were defined as cells that are eFluor450 dye+ / CD14-. Double-labeled cells (eFluor450 dye+ / CD14+) were considered to represent phagocytosis of the target by macrophages. The phagocytosis of target cells was calculated using the following formula: Phagocytosis (%) = 100 × [(Double Positive Rate) / (Double Positive Rate + Residual Target Rate)]. [Figure 68] This figure shows the characterization of antibodies 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 bound to CD25 expressed on Karpas 299 cells, compared to a human IgG1 isotype control, at gradually increasing antibody concentrations. [Figure 69] This figure shows a competitive assay in Octet. Immobilized rhCD25 is conjugated with a first Ab (antibody 19), followed by either the first Ab again (control), or a second Ab, either daclizumab (A) or basiliximab (B). [Figure 70] This figure shows the characterization of antibodies 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 for IL-2 signaling blockade in a STAT5 phosphorylation assay using human-derived PBMCs, compared to the absence of a human IgG1 isotype control, daclizumab-Hyp, or primary antibody. Cells were incubated with 10 μg / ml antibody, followed by 10 U / ml IL-2. Analysis was limited to the percentage of CD3-positive cells that phosphorylated STAT5. [Figure 71]This figure shows the functional characterization of antibody 19 compared to an anti-human CD25 Fc silent control antibody for ADCC induction in the Reporter Bioassay. CD25-expressing SR-786 cells were co-cultured with Jurkat T cells genetically engineered to express the NFAT response element (NFAT-RE-luc2) that drives FcγRIIIa and luciferase expression, in the presence of various antibody concentrations (shown in the figure). [Figure 72] This figure shows the functional characterization of antibody 19 compared to an anti-human CD25 Fc silent control antibody regarding phagocytosis of differentiated Treg cells in an ADCP assay. Treg cells were co-cultured with MCSF-differentiated macrophages in the presence of various concentrations of antibody (shown in the figure). Two-color flow cytometry analysis was performed using CD14+ stained macrophages and eFluor450 dye-labeled Treg cells. Residual target cells were defined as cells that are eFluor450 dye+ / CD14-. Double-labeled cells (eFluor450 dye+ / CD14+) were considered to represent phagocytosis of the target by macrophages. The phagocytosis of target cells was calculated using the following formula: Phagocytosis (%) = 100 × [(Double Positive Rate) / (Double Positive Rate + Residual Target Rate)]. [Figure 73] This figure shows the functional characterization of antibodies 19, 12, and 20 compared to an anti-human CD25 Fc silent control antibody regarding the phagocytosis of differentiated Treg cells in an ADCP assay. Treg cells were co-cultured with MCSF-differentiated macrophages in the presence of various antibody concentrations (shown in the figure). Two-color flow cytometry analysis was performed using CD14+ stained macrophages and eFluor450 dye-labeled Treg cells. Residual target cells were defined as cells that are eFluor450 dye+ / CD14-. Double-labeled cells (eFluor450 dye+ / CD14+) were considered to represent phagocytosis of the target by macrophages. The phagocytosis of target cells was calculated using the following formula: Phagocytosis (%) = 100 × [(Double Positive Rate) / (Double Positive Rate + Residual Target Rate)]. [Figure 74]This figure shows the therapeutic activity of 7D4 mouse IgG2a, a non-IL-2 blocking anti-CD25 antibody, in combination with Gvax in a B16Bl6 immunotherapy resistance model. Individual mice were treated with Gvax alone or in combination with 7D4. [Figure 75] This figure shows the therapeutic activity of non-IL-2 blocking anti-CD25 antibodies (7D4 and 2E4) compared to IL-2 blocking antibody (PC61) in a CT26 tumor model using female BALB / c mice. The non-blocking anti-CD25 antibodies 7D4 and 2E4 exhibit potent therapeutic activity against solid tumors. Both 7D4 and 2E4 are potenter than the IL-2 blocking antibody PC61. [Figure 76] This figure shows the evaluation of the therapeutic activity of the non-IL-2 blocking anti-CD25 antibody 7D4 mIgG2a in a MCA205 model with single and repeated injections in combination with anti-mouse PD-L1. * indicates mice that were alive at the end of the experiment. [Modes for carrying out the invention]

[0037] The present invention provides a method for treating or preventing cancer in a subject, preferably when the subject has a solid tumor, comprising the step of administering a CD25-binding antibody to the subject, wherein the anti-CD25 antibody is characterized by a structural element that binds to CD25 without interfering with interleukin-2 binding or CD25-mediated signaling, and efficiently depletes Tregs, particularly within the tumor. The CD25-binding antibody defined in the present invention can be used for the treatment or prevention of cancer, preferably solid tumors. Alternatively, the present invention provides the use of an antibody in the manufacture of a drug for the treatment or prevention of cancer, preferably solid tumors, that binds to CD25 and enables both binding to CD25 without interfering with interleukin-2 binding to CD25 and efficient depletion of Tregs. The present invention also provides the use of an antibody in the treatment or prevention of cancer, preferably solid tumors, that binds to CD25 and enables both binding to CD25 without substantially interfering with interleukin-2 binding to CD25 and depletion of Tregs.

[0038] The inventors have found that CD25 can be targeted using anti-CD25 antibodies that do not inhibit (or substantially do not inhibit) the binding of interleukin-2 to CD25 or IL-2 signaling via CD25, for therapeutic purposes, such as depletion of regulatory T cells in established solid tumors. The inventors have found that non-IL-2 blocking anti-CD25 antibodies having isotypes that enhance binding to activated Fcγ receptors lead to effective depletion of tumor-infiltrating regulatory T cells while enabling an optimal Teff response, and that this is a therapeutic approach that can be combined (in combination with or within a bispecific antibody) with other cancer targeting compounds, such as compounds that target immune checkpoint proteins, tumor-associated antigens, or suppressor Fcγ receptors. These findings also make it possible to combine the use of anti-CD25 with interleukin-2 at doses appropriate for cancer treatment.

[0039] CD25 is the α-chain of the IL-2 receptor and is found on activated T cells, regulatory T cells, activated B cells, some NK T cells, some thymocytes, myeloid progenitor cells, and oligodendrocytes. CD25 associates with CD122 and CD132 to form a heterotrimeric complex that acts as a high-affinity receptor for IL-2. The consensus sequence of human CD25 is shown below as Sequence ID No. 1 (Uniprot accession number P01589; the extracellular domain of mature human CD25 corresponding to amino acids 22-240 is underlined and presented as Sequence ID No. 2): 10 20 30 40 50 MDSYLLMWGL LTFIMVPGCQ A ELCDDDPPE IPHATFKAMA YKEGTMLNCE 60 70 80 90 100 CKRGFRRIKS GSLYMLCTGN SSHSSWDNQC QCTSSATRNT TKQVTPQPEE 110 120 130 140 150 QKERKTTEMQ SPMQPVDQAS LPGHCREPPP WENEATERIY HFVVGQMVYY 160 170 180 190 200 QCVQGYRALH RGPAESVCKM THGKTRWTQP QLICTGEMET SQFPGEEKPQ 210 220 230 240 250 ASPEGRPESE TSCLVTTTDF QIQTEMAATM ETSIFTTEYQ VAVAGCVFLL 260 270 ISVLLLSGLT WQRRQRKSRR TI

[0040] As used herein, “CD25-binding antibody” refers to an antibody capable of binding to the CD25 subunit of the IL-2 receptor. This subunit is also known as the α-subunit of the IL-2 receptor. Such an antibody is also referred to herein as “anti-CD25 antibody.”

[0041] Anti-CD25 antibodies are antibodies capable of specifically binding to the CD25 subunit (antigen) of the IL-2 receptor. "Specific binding" and "specifically binding" refer to the antibody binding to the target antigen at a rate of approximately 10°C. -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10 -13 Dissociation constant less than M (K d This is understood to mean that it has ). In a preferred embodiment, the dissociation constant is 10 -8 Less than M, for example 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10 -13 It is within the range of M.

[0042] As used herein, the term “antibody” refers to both the intact immunoglobulin molecule and its fragments containing antigen-binding sites, as well as chimeric antibodies, humanized antibodies, heteroconjugates and / or multispecific antibodies (e.g., bispecific antibodies, diabodies, tribodies and tetrabodies), and Fab', F(ab')2, Fab, Fv, rIgG, polypeptide-Fc fusions, single-strand variants (scFv fragments, VH The antibody comprises antigen-binding fragments including, but is not limited to, polyclonal, monoclonal, genetically modified, and otherwise modified forms of antibodies, including H, Trans-body (trademark), Affibody (trademark), shark single-domain antibodies, single-chain or tandem diabodies (TandAb (trademark)), VHH, Antiticalin (trademark), Nanobody (trademark), minibodies, BiTE (trademark), bicyclic peptides, and other alternative immunoglobulin protein scaffolds. In some embodiments, the antibody may lack covalent modifications that it may have if naturally produced (e.g., glycan attachment). In some embodiments, the antibody may have covalent modifications (e.g., glycan, detectable moiety, therapeutic moiety, catalytic moiety, or attachment of other chemical groups that improve antibody stability or administration, such as polyethylene glycol). In some embodiments, the antibody may be in the form of a masked antibody (e.g., Probody (trademark)). Mask antibodies may contain a blocking or "masking" peptide that specifically binds to the antigen-binding surface of an antibody and prevents the antibody from binding to the antigen. The mask peptide is linked to the antibody by a cleavable linker (e.g., by a protease). Selective cleavage of the linker in the desired environment, i.e., the tumor environment, dissociates the masking / blocking peptide, allowing antigen binding to occur in the tumor, thereby limiting potential toxicity issues. The term "antibody" may also refer to antibody-like molecules such as camelid antibodies (heavy-chain only antibodies) and anticarin (Skerra (2008) FEBS J 275, 2677-83).In some embodiments, the antibodies are polyclonal or oligoclonal, generating a panel of antibodies where each associates with a single antibody sequence and binds to somewhat different epitopes within the antigen (e.g., different epitopes within the extracellular domain of human CD25 that associate with different reference anti-human CD25 antibodies). Polyclonal or oligoclonal antibodies can be provided in a single preparation for medical use as described in the literature (Kearns JD et al., 2015. Mol Cancer Ther. 14:1625-36).

[0043] In one aspect of the present invention, the antibody is monoclonal. The antibody may optionally be humanized or human. In a further aspect, the antibody is human, or an antibody having a format and characteristics that enable its use and administration in a human subject. In one aspect of the present invention, the antibody may be a humanized variant of affinity-mature 7G7B6 or MA251. The affinity-mature antibody has an affinity at least 10% higher affinity for CD25, and / or the CDR sequence is at least 80% identical, preferably 90% identical (across the entire sequence) to the CDR of the parent sequence. The affinity-mature antibody is an antibody having one or more modified amino acids in one or more CDRs that produces an antibody with improved affinity for CD25 compared to a parent strain without modified amino acids.

[0044] Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins having the same structural characteristics. Immunoglobulins can be of any class, such as IgA, IgD, IgG, IgE, or IgM. Immunoglobulins can be of any subclass, such as IgG1, IgG2, IgG3, or IgG4. In a preferred embodiment of the present invention, the anti-CD25 antibody is of the IgG class, preferably the IgG1 subclass. In one embodiment, the anti-CD25 antibody is of the human IgG1 subclass. Alternatively, in one embodiment, the anti-CD25 antibody is of the human IgG2 subclass.

[0045] The Fc region of IgG antibodies interacts with several cellular Fcγ receptors (FcγRs), stimulating and modulating downstream effector mechanisms. There are five activating receptors: FcγRI (CD64), FcγRIIa (CD32a), FcγRIIc (CD32c), FcγRIIIa (CD16a), and FcγRIIIb (CD16b), as well as one inhibitory receptor, FcγRIIb (CD32b). Communication between IgG antibodies and the immune system is regulated and mediated by FcγRs, which relay information detected and collected by the antibody to the immune system, particularly linking the innate and adaptive immune systems under the context of biological agents (Hayes J et al., 2016. J Inflamm Res 9: 209-219).

[0046] IgG subclasses differ in their ability to bind to FcγR, and this differing binding determines their ability to induce various functional responses. For example, in humans, FcγRIIIa is the primary receptor involved in the activation of antibody-dependent cell-mediated cytotoxicity (ADCC), with IgG3, followed immediately by IgG1, exhibiting the highest affinity for this receptor, reflecting their ability to strongly induce ADCC. While IgG2 has been shown to have weak binding to this receptor, it has also been found that anti-CD25 antibodies containing the human IgG2 isotype efficiently deplete Treg cells.

[0047] In preferred embodiments of the present invention, the antibody binds with high affinity to FcγR, preferably to the activating receptor. The antibody preferably binds with high affinity to FcγRI and / or FcγRIIa and / or FcγRIIIa. In specific embodiments, the antibody binds to at least one activated Fcγ receptor at a rate of about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 They bond with a dissociation constant less than M.

[0048] In one embodiment, the antibody is an IgG1 antibody, preferably a human IgG1 antibody, capable of binding to at least one Fc-activated receptor. For example, the antibody may bind to one or more receptors selected from FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb. In one embodiment, the antibody may bind to FcγRIIIa. In one embodiment, the antibody may bind to FcγRIIIa and FcγRIIa, and optionally to FcγRI. In one embodiment, the antibody has a high affinity for these receptors, for example, about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 It is possible to bond with a dissociation constant less than M.

[0049] In one embodiment, the antibody binds to the inhibitory receptor FcγRIIb with low affinity. In one embodiment, the antibody binds to FcγRIIb with approximately 10 -7 Over M, about 10 -6 M or approximately 10 -5 They bond with a dissociation constant greater than M.

[0050] In preferred embodiments of the present invention, the anti-CD25 antibody is of the IgG1 subclass and preferably has ADCC and / or ADCP activity, particularly against human-derived cells, as discussed herein. As previously described (Nimmerjahn F et al., 2005. Science, 310:1510-2), the mIgG2a isotype (corresponding to the human IgG1 isotype) binds to all FcγR subtypes with a high activator-to-inhibitor ratio (A / I) greater than at least 1. In contrast, other isotypes (such as the rIgG1 isotype) bind to only a single activator FcγR (FcγRIII) and an inhibitory FcγRIIb with similar affinity, resulting in a lower A / I ratio (less than 1). This lower A / I ratio may correlate with lower intratumor Treg depletion and lower antitumor therapeutic activity of this isotype. Despite the known FcγR binding profiles for antibodies of the human IgG2 isotype, significant Treg depletion can also be achieved with human IgG2 isotypes of anti-CD25 antibodies. Therefore, in one embodiment, the anti-CD25 antibody is of the IgG2 subclass.

[0051] In preferred embodiments, the anti-CD25 antibody described herein binds to human CD25, preferably with high affinity. More preferably, the anti-CD25 antibody binds to the extracellular region of human CD25 as shown above. In one embodiment, the present invention provides an anti-CD25 antibody as described herein. In particular, the examples provide experimental data generated using an antibody secreted by a 7D4 hybridoma. As shown in the background art of the present invention, this antibody is specific to mouse CD25, binding to one of three epitopes in mouse CD25 distinct from the IL-2 binding site, as shown by comparing a panel of monoclonal antibodies (including PC61), and not blocking the binding of IL-2 to CD25. For example, 7D4 has been shown to bind to mouse CD25 at an epitope containing amino acids 184-194 (REHHRFLASEE) in [Uniprot sequence P01590]. Assays involving 7D4 and mouse CD25 in the literature (e.g., Non-Patent Literature 11, Non-Patent Literature 23, Teege S et al., 2015, Sci Rep 5: 8959), including recombinant antibodies containing the CD25-binding domain of 7D4, or non-IL-2-blocking anti-human CD25 antibodies referred to as MA-251 and 7G7B6, can be adapted, together with the assays disclosed in the Examples, to characterize human antibodies that recognize human CD25 having the same functional characteristics as 7D4 at the level of interaction with CD25 (particularly without blocking IL-2 binding) and Fcγ receptors (particularly by binding to one or more human activated Fcγ receptors and efficiently depleting Tregs) when appropriate isotypes are associated as described in the Examples.

[0052] In one aspect of the present invention, an antibody competes with antibody 7G7B6 for binding to human CD25 and / or binds to the same epitope(s) recognized by antibody 7G7B6. 7G7B6 is a monoclonal antibody having a mouse IgG2a isotype that recognizes human CD25. 7G7B6 has the following sequence: A variable heavy chain region having EVQLVESGGDLVQPRGSLKLSCAASGFTFSSYGMSWVRQTPDKRLELVATINGYGDTTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYFCARDRDYGNSYYYALDYWGQGTSVTVSS (Sequence ID 3), and sequence: It includes a variable light chain region having QIVLSQSPAILSASPGERVTMTCRASSSVSFMHWLQQKPGSSPKPWIYATSNLASGVSARFSGSGSGTSYSLTITRVEAEDAATYYCQQWSSNPPAFGGGTKLEIK (Sequence ID 4).

[0053] In one embodiment, the antibody comprises heavy chains including the amino acid sequence GFTLDSYGVS (SEQ ID NO: 7) as variable heavy chain CDR1, the amino acid sequence GVTSSGGSAYYADSV (SEQ ID NO: 8) as variable heavy chain CDR2, and the amino acid sequence DRYVYTGGYLYHYGMDL (SEQ ID NO: 9) as variable heavy chain CDR3, and light chains including the amino acid sequence RASQSISDYLA (SEQ ID NO: 11) as variable light chain CDR1, the amino acid sequence YAASTLPF (SEQ ID NO: 12) as variable light chain CDR2, and the amino acid sequence QGTYDSSDWYWA (SEQ ID NO: 13) as variable light chain CDR3. The antibody may compete with 7G7B6 for binding to human CD25. The antibody has the sequence: EVQLVESGGGLIQPGGSLRLSCAAS GFTLDSYGVS WVRQAPGKGLEWV GVTSSGGSAYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRYVYTGGYLYHYGMDL A heavy chain containing a variable heavy chain region including WGQGTLVTVSS (SEQ ID NO: 10), and sequence: DIQMTQSPSSLSASVGDRVTITC RASQSISDYLA WYQQKPGKVPKLLI YAASTLPF GVPSRFSGSGSGTDFLTISSLQPEDVATYYC QGTYDSSDWYWA Preferably, the light chain includes a variable light chain region containing FGGGTKVEI (SEQ ID NO: 14).

[0054] In another embodiment, the antibody comprises heavy chains including the amino acid sequence SGFSVDIYDMS (SEQ ID NO: 15) as variable heavy chain CDR1, the amino acid sequence YISSSLGATYYADSV (SEQ ID NO: 16) as variable heavy chain CDR2, and the amino acid sequence ERIYSVYTLDYYAMDL (SEQ ID NO: 17) as variable heavy chain CDR3, and light chains including the amino acid sequence QASQGITNNLN (SEQ ID NO: 19) as variable light chain CDR1, the amino acid sequence YAASTLQS (SEQ ID NO: 20) as variable light chain CDR2, and the amino acid sequence QQGYTTSNVDNA (SEQ ID NO: 21) as variable light chain CDR3. The antibody may compete with 7G7B6 for binding to human CD25. The antibody has the sequence: EVQLLESGGGLVQPGGSLRLSCAA SGFSVDIYDMS WVRQAPGKGLEWVA YISSSLGATYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR ERIYSVYTLDYYAMDL A heavy chain containing a variable heavy chain region including WGQGTLVTVSS (SEQ ID NO: 18), and sequence: DIQMTQSPSSLSASVGDRVTITC Q ASQGITNNLN WYQQKPGKVPKLLI YAASTLQS GVPSRFSGSGSGTDFLTISSLQPEDVATYYC QQGYTTSNVDNA Preferably, the light chain includes a variable light chain region containing FGGGTKVEIK (SEQ ID NO: 22).

[0055] In one embodiment, an antibody that can compete with 7G7B6 for binding to human CD25 has the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRDYGNSYYYALDYWGQGTLVTVSS (Sequence ID 23) or, Containing a heavy chain including EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYFCARDRDYGNSYYYALDYWGQGTLVTVSS (SEQ ID NO: 24), and with the amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASSSVSFMHWLQQKPGQAPRPLIYATSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCQQWSSNPPAFGQGTKLEIK (Sequence ID 25) or, Includes a light chain containing QIVLTQSPGTLSLSPGERATLSCRASSSVSFMHWLQQKPGQSPRPLIYATSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCQQWSSNPPAFGQGTKLEIK (SEQ ID NO: 26).

[0056] In one aspect of the present invention, an antibody competes with antibody MA251 for binding to human CD25 and / or binds to the same epitope(s) recognized by antibody MA251. MA251 is a monoclonal antibody having a mouse isotype that recognizes human CD25. MA251 has the following sequence: A variable heavy chain region having QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGIQWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARAYGYDGSWLAYWGQGTLVTVSS (Sequence ID 5), and sequence: It includes a variable light chain region having QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIFATSNLASGVPARFSGSGSGTSYSLTINRVEAEDADTYYCQQWSSNPPTFGGGTKLEIK (Sequence ID 6).

[0057] In one embodiment, an antibody that can compete with MA251 for binding to human CD25 has the following amino acid sequence: QVQLVESGGGVVQPGGSLRLSCAVSGFSLTSYGIQWVRQAPGKGLEWVSVIWAGGSTNYNSALMSRFTISKDNSKSTLYLQMNSLRAEDTAVYYCARAYGYDGSWLAYWGQGTLVTVSS (Sequence ID 27), QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGIQWVRQPPGKGLEWIGVIWAGGSTNYNSALMSRVTISKDNSKSQFSLKLSSVTAADTAVYYCARAYGYDGSWLAYWGQGTLVTVSS (Sequence ID 28), or QVQLVESGGGVVQPGGSLRLSCAVSGFSLTSYGIQWVRQAPGKGLEWVSVIWAGGSTNYNSALMSRFTISKDNSKSTLYLQMNSLRAEDTAVYYCARAYGYDGSWLAYWGQGTLVTVSS (Sequence ID 29), A heavy chain containing a variable heavy chain region and having an amino acid sequence: QIVLTQSPATLSLSPGERATLSCRASSSVSYMHWYQQKPGQAPRPLIFATSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPTFGGGTKLEIK(Sequence ID 30); QIVLTQSPATLSLSPGERATLSCRASSSVSYMHWYQQKPGQAPRPLIFATSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPTFGGGTKLEIK(Sequence ID 31); DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIFATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIK(Sequence ID 32); or, The light chain includes a variable light chain region containing QIQLTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKSPKPLIFATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIK (Sequence ID 33).

[0058] In one embodiment, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 23 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 25. In another embodiment, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 23 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 26. In another embodiment, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 24 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 25. In another embodiment, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 24 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 26. In another embodiment, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 27 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 30. In another embodiment, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 27 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 31. In another embodiment, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 27 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 32. In another embodiment, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 27 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 33. In another embodiment, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 28 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 30. In another embodiment, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 28 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 31. In another embodiment, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 28 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 32. In another embodiment, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 28 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 33. In another embodiment, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 29 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 30. In another embodiment, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 29 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 31. In another embodiment, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 29 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 32.In another embodiment, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 29 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 33.

[0059] In one embodiment, the antibody competes with both antibody 7G7B6 and antibody MA251 for binding to human CD25. In one embodiment, the antibody binds to the same epitope(s) recognized by 7G7B6 and recognized by MA251.

[0060] Competition between the 7G7B6 antibody or MA251 antibody and any additional antibody is discussed, for example, in the examples and can be measured as known in the art. In some embodiments, competition between two antibodies, such as between 7G7B6 or MA251 and any additional antibody, is determined by adding the additional antibody to the assay and measuring the interaction between the 7G7B6 or MA251 antibody and human CD25. One such assay is an Octet-based assay that determines the co-binding of the 7G7B6 or MA251 antibody, any additional antibody, and recombinant human CD25. If binding of the two antibodies to recombinant human CD25 is detected, the antibodies are non-competitive. Alternatively, one such assay is an enzyme-linked immunosorbent assay (ELISA) that detects the binding of the 7G7B6 or MA251 antibody to recombinant human CD25. If the observed signal decreases after the addition of the additional antibody (e.g., by at least 75%), the latter antibody is a competitor to the 7G7B6 or MA251 antibody. The simultaneous binding of 7G7B6 or MA251 antibodies to human CD25-expressing cells with additional antibodies can also be detected using flow cytometry.

[0061] In one embodiment, the present invention provides an anti-CD25 antibody that specifically binds to a human CD25 epitope, where the epitope comprises one or more amino acid residues from an amino acid stretch selected from amino acids 150-163 (YQCVQGYRALHRGP), amino acids 166-186 (SVCKMTHGKTRWTQPQLICTG), amino acids 42-56 (KEGTMLNCECKRGFR), and amino acids 70-88 (NSSHSSWDNQCQCTSSATR) of SEQ ID NO: 1. Preferably, the epitope comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or more residues from the selected amino acid stretch. More preferably, the epitopes include sequences selected from amino acids 150-158 (YQCVQGYRA), 176-180 (RWTQP), 42-56 (KEGTMLNCECKRGFR), and 74-84 (SSWDNQCQCTS) of SEQ ID NO: 1, and combinations thereof. Unlike the IL-2 binding sites in human CD25, antibodies that bind to such epitopes do not block the binding of IL-2 to CD25, as described in the examples.

[0062] In a preferred embodiment, a method for treating a human subject having cancer comprises the step of administering the anti-CD25 antibody of the present invention to the subject, wherein the subject preferably has a solid tumor, and the anti-CD25 antibody is preferably a human IgG1 antibody that does not inhibit the binding of interleukin-2 to CD25, binds with high affinity to at least one activated Fcγ receptor selected from FcγRI(CD64), FcγRIIc(CD32c), and FcγRIIIa(CD16a), and depletes tumor-invasion regulatory T cells. The anti-CD25 antibody has a concentration of 10 to CD25. -7 Less than M, preferably 10 -8 Dissociation constant less than M (K dPreferably, it has (). More preferably, the anti-CD25 antibody binds to human CD25 and brings about effects on IL-2 binding and Treg depletion similar to those of 7D4 on mouse CD25 or 7G7B6 and MA251 on human CD25. In a further embodiment, the anti-CD25 antibody binds to Fcγ receptors with an activating to inhibitory ratio (A / I) greater than 1 and / or binds to FcγRI (CD64), FcγRIIc (CD32c), FcγRIIIa (CD16a) and / or FcγRIIa (CD32a) with a higher affinity than binding to FcγRIIb (CD32b).

[0063] The CD25-binding domain of the 7D4 antibody has been cloned and expressed as a recombinant protein fused to an appropriate constant region. The sequence of the CD25-binding domain of the 7D4 antibody, along with its specificity for different epitopes within the extracellular domain of CD25 and / or other functional activities, can be used for comparison with candidate anti-CD25 antibodies generated and screened by any suitable technique (e.g., producing a panel of hybridomas from rodents immunized with CD25 or generating a library of recombinant antibodies and then screening the antibody repertoire having CD25 fragments for the functional characteristics described herein). The anti-CD25 antibodies identified as a result can also be made as recombinant antibodies, particularly full antibodies, or fragments or variants described herein.

[0064] Natural antibodies and immunoglobulins are typically about 150,000 dalton heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has several constant domains following the variable domain (V H ) at the amino terminus. Each light chain has a variable domain (V L ) at the amino terminus and a constant domain at the carboxy terminus.

[0065] The variable region is capable of interacting with a structurally complementary antigenic target and is characterized by an amino acid sequence different from antibodies of different antigen specificities. The variable region of either the H chain or the L chain contains an amino acid sequence capable of specifically binding to an antigenic target. Within these sequences, there are smaller sequences named "hypervariable" due to the extreme variability among antibodies of different specificities. Such hypervariable regions are also referred to as "complementary determining regions" or "CDR" regions.

[0066] These CDR regions explain the basic specificity of an antibody for a particular antigen determinant structure. CDRs are non - continuous stretches of amino acids within the variable region, and it has been found that, regardless of the species, the positions of these important amino acid sequences within the variable heavy and light chain regions have similar positions within the amino acid sequence of the variable chain. The variable heavy and light chains of all antibodies each have three CDR regions (designated L1, L2, L3, H1, H2, H3) that are non - continuous with each other for their respective light (L) and heavy (H) chains. The recognized CDR regions have been described previously (Kabat et al., 1977. J Biol Chem 252, 660-6616).

[0067] The antibodies of the present invention can function by complement - dependent cytotoxicity (CDC) and / or antibody - dependent cell - mediated cytotoxicity (ADCC) and / or antibody - dependent cell - mediated phagocytosis (ADCP), and any other mechanism that enables targeting, blocking of proliferation and / or depletion of Treg cells.

[0068] "Complement - dependent cytotoxicity" (CDC) refers to the lysis of antigen - expressing cells by the antibodies of the present invention in the presence of complement.

[0069] "Antibody - dependent cell - mediated cytotoxicity" (ADCC) refers to a cell - mediated reaction in which non - specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors recognize the bound antibody on the target cell, thereby resulting in the lysis of the target cell.

[0070] "Antibody-dependent cell-mediated phagocytosis" (ADCP) refers to a cell-mediated reaction in which phagocytic cells (such as macrophages) that express Fc receptors (FcRs) recognize bound antibodies on target cells, thereby inducing phagocytosis of the target cells.

[0071] CDC, ADCC, and ADCP are known in the art and can be measured using available assays (Clynes et al. (1998) Proc Natl Acad Sci USA 95, 652-6) as discussed in the examples. The constant region of the antibody is important for the antibody's ability to immobilize complement and mediate cell-dependent cytotoxicity and phagocytosis. Therefore, as discussed herein, antibody isotypes can be selected based on whether the antibody is desirable for mediating cytotoxicity / phagocytosis.

[0072] As discussed herein, one embodiment of the present invention uses an anti-CD25 antibody that does not inhibit interleukin-2 binding but leads to Treg cell depletion. For example, an anti-CD25 antibody can be used that does not inhibit interleukin-2 binding to CD25 but induces a strong CDC response and / or a strong ADCC and / or a strong ADCP response. Methods for increasing CDC, ADCC and / or ADCP are known in the art. For example, the CDC response can be increased by mutations in antibodies that increase the affinity for C1q binding (ldusogie et al. (2001) J lmmunol 166, 2571-5).

[0073] References in this specification to “not inhibit the binding of interleukin-2 to CD25” can be alternatively expressed as the anti-CD25 antibody being a non-IL-2 blocking antibody or a “non-blocking” antibody (with respect to non-blocking of IL-2 binding to CD25 in the presence of the anti-CD25 antibody), i.e., the antibody does not block the binding of interleukin-2 to CD25 and, in particular, does not inhibit interleukin-2 signaling in CD25-expressing cells. References to “non-blocking,” “non-IL2 blocking,” “not blocking,” or “without blocking” (with respect to non-blocking of IL-2 binding to CD25 in the presence of the anti-CD25 antibody) include embodiments in which the anti-CD25 antibody of the present invention does not block IL-2 signaling via CD25. That is, the anti-CD25 antibody inhibits IL-2 signaling by less than 50% compared to IL-2 signaling in the absence of the antibody. In certain embodiments of the present invention described herein, anti-CD25 antibodies inhibit IL-2 signaling by about 40%, 35%, less than 30%, preferably less than 25%, compared to IL-2 signaling in the absence of the antibody. Anti-CD25 non-IL-2 blocking antibodies allow binding to CD25 without interfering with or substantially interfering with IL-2 binding to CD25. References to non-IL-2 blocking antibodies herein can alternatively be expressed as anti-CD25 antibodies that "do not inhibit the binding of interleukin-2 to CD25" or anti-CD25 antibodies that "do not inhibit IL-2 signaling."

[0074] Some anti-CD25 antibodies can enable IL-2 binding to CD25 but still block signaling via the CD25 receptor. Such anti-CD25 antibodies are not within the scope of this invention. Instead, non-IL-2 blocking anti-CD25 antibodies enable IL-2 binding to CD25 and enhance the level of signaling via the CD25 receptor by at least 50% compared to signaling in the absence of the anti-CD25 antibody.

[0075] CD25-mediated IL-2 signaling is discussed in the examples and can be measured by methods known in the art. Comparisons of IL-2 signaling in the presence and absence of anti-CD25 antibodies can be performed under the same or substantially the same conditions.

[0076] In some embodiments, IL-2 signaling can be determined by measuring the level of phosphorylated STAT5 protein in cells using a standard Stat-5 phosphorylation assay. For example, a Stat-5 phosphorylation assay for measuring IL-2 signaling may involve culturing PMBC cells for 30 minutes in the presence of 10 μg / ml anti-CD25 antibody, followed by the addition of various concentrations of IL-2 (e.g., 10 U / ml, or varying concentrations such as 0.25 U / ml, 0.74 U / ml, 2.22 U / ml, 6.66 U / ml, or 20 U / ml) for 10 minutes. The cells can then be permeabilized, and the level of STAT5 protein can be measured by a fluorescently labeled antibody against the phosphorylated STAT5 peptide, which is then analyzed by flow cytometry. The IL-2 signaling blockade rate can be calculated as follows: Blockade % = 100 × [Stat5 + Cell antibody-free group (%)-Stat5 + cells 10μg / ml antibody group (%)) / (Stat5 + Cell-free Ab group (%)]

[0077] ADCC can be increased by methods such as removing the fucose portion from antibody glycans, for example, by producing antibodies in YB2 / 0 cell lines, or by introducing specific mutations into the Fc portion of human IgG1 (e.g., S298A / E333A / K334A, S239D / I332E / A330L, G236A / S239D / A330L / I332E) (Lazar et al. (2006) Proc Natl Acad Sci USA 103, 2005-2010, Smith et al. (2012) Proc Natl 25 Acad Sci USA 109, 6181-6). ADCP can also be increased by introducing specific mutations into the Fc portion of human IgG1 (Richards et al. (2008) Mol Cancer Ther 7, 2517-27).

[0078] In preferred embodiments of the present invention, the antibody is optimized to induce an ADCC response. That is, the ADCC response is enhanced, increased, or improved compared to other anti-CD25 antibodies, such as unmodified anti-CD25 monoclonal antibodies, which do not inhibit the binding of interleukin-2 to CD25.

[0079] In preferred embodiments of the present invention, the antibody is optimized to induce an ADCP response. That is, the ADCP response is enhanced, increased, or improved compared to other anti-CD25 antibodies, such as unmodified anti-CD25 monoclonal antibodies, which do not inhibit the binding of interleukin-2 to CD25.

[0080] As used herein, "chimeric antibody" refers to an antibody having a variable sequence derived from an immunoglobulin from one species, such as a rat or mouse antibody, and a constant region of an immunoglobulin from another species, such as a human antibody. In some embodiments, the chimeric antibody may have a constant region enhanced for ADCC induction.

[0081] The antibodies according to the present invention may be partially or completely synthetic, in which at least a portion of the polypeptide chain of the antibody is synthesized and possibly optimized for binding to an alloantigen. Such antibodies may be chimeric or humanized antibodies, and may have a completely tetrameric structure or be dimers, comprising only a single heavy chain and a single light chain.

[0082] The antibodies of the present invention may be monoclonal antibodies. As used herein, “monoclonal antibody” is not limited to antibodies produced by hybridoma technology. The term “monoclonal antibody” refers to an antibody derived from a single clone, including any clone of a eukaryote, prokaryote, or phage, and does not refer to a method for producing such an antibody.

[0083] The antibody of the present invention may be a human antibody. As used herein, "human antibody" refers to an antibody having a variable region in which both the framework and the CDR region are derived from a human germline immunoglobulin sequence. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibody of the present invention may contain amino acid residues not encoded by a human germline immunoglobulin sequence (for example, mutations introduced by random or site-directed mutagenesis in vitro, or somatic mutations in vivo).

[0084] A further object of the present invention is an anti-CD25 antibody exhibiting the characteristics described herein. This anti-CD25 antibody can be used as a drug. In further embodiments, the present invention provides a method for treating a disease in a subject, comprising administering an anti-CD25 antibody that does not inhibit the binding of interleukin-2 (IL-2) to CD25 or the signaling of IL-2 via CD25. The disease is preferably cancer, particularly solid tumors.

[0085] In further embodiments, the present invention provides a nucleic acid molecule encoding an anti-CD25 antibody as defined herein. In some embodiments, such provided nucleic acid molecules may contain a codon-optimized nucleic acid sequence and / or may be included in an expression cassette within a suitable nucleic acid vector for expression in host cells such as, for example, bacterial, yeast, insect, fish, mouse, monkey or human cells. In some embodiments, the present invention provides a host cell comprising a heterologous nucleic acid molecule (e.g., a DNA vector) that expresses a desired antibody.

[0086] In some embodiments, the present invention provides a method of making an isolated anti-CD25 antibody as defined above. In some embodiments, such method may comprise culturing a host cell comprising a nucleic acid (e.g., a heterologous nucleic acid that can constitute a host cell and / or can be delivered to a host cell by a vector). The host cell (and / or the heterologous nucleic acid sequence) is preferably arranged and constructed such that an antibody, or an antigen-binding fragment or variant thereof, is secreted from the host cell and isolated from the cell culture supernatant.

[0087] The antibodies of the present invention can be monospecific, bispecific or multispecific. A "multispecific antibody" can be specific for different epitopes of one target antigen or polypeptide or can contain antigen-binding domains specific for two or more target antigens or polypeptides (Kufer et al. (2004) Trends Biotechnol 22, 238-44).

[0088] In one aspect of the present invention, the antibody is a monospecific antibody. In an alternative aspect, as further discussed below, the antibody is a bispecific antibody.

[0089] As used herein, a "bispecific antibody" refers to an antibody that has the ability to bind to two different epitopes, either on a single antigen or polypeptide or on two different antigens or polypeptides.

[0090] The bispecific antibodies of the present invention discussed herein can be produced by biological methods such as somatic cell hybridization; genetic methods such as the expression of a non-natural DNA sequence encoding a desired antibody structure in a cell line or organism; chemical methods (e.g., by chemical coupling to one or more molecular entities such as another antibody or antibody fragment, gene fusion, non-covalent association or other forms); or a combination thereof.

[0091] Technologies and products that enable the production of monospecific or bispecific antibodies are well known in the art, as are extensively outlined in the literature regarding alternative formats, antibody-drug conjugates, antibody design methods, in vitro screening methods, constant regions, post-translational and chemical modifications, and improvements to cancer cell death-inducing features such as Fc engineering (Tiller K and Tessier P, 2015 Annu Rev Biomed Eng. 17: 191-216, Speiss C et al., 2015. Molecular Immunology 67 95-106, Weiner G, 2015. Nat Rev Cancer, 15: 361-370, Fan G et al., 2015. J Hematol Oncol 8:130). Such bispecific antibodies can be supplied in any commercially available format, including Duobody, BiTE DART, CrossMab, Nob-in-Hole, Triomab, or other suitable molecular formats and their fragments.

[0092] As used herein, “epitope” or “antigenic determinant” refers to a site on an antigen to which an antibody binds. As is known in the art, epitopes can be formed from both continuous amino acids (linear epitopes) or discontinuous amino acids juxtaposed by tertiary folding of a protein (conformational epitopes). Epitopes formed from continuous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly at least five or eight to ten, amino acids in their own spatial conformation. Methods for determining the spatial conformation of epitopes are known in the art and include, for example, X-ray crystallography and 2D nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996). For example, the antibody of the present invention may recognize a conformational epitope to which antibody 7G7B6 or MA251 binds. In one embodiment, the conformational epitope includes at least two sequences selected from amino acids 150-158 (YQCVQGYRA), amino acids 176-180 (RWTQP), amino acids 42-56 (KEGTMLNCECKRGFR), and amino acids 74-84 (SSWDNQCQCTS) of SEQ ID NO: 1.

[0093] In some embodiments, the anti-CD25 antibody may be included in an active substance further comprising a conjugate payload, such as a therapeutic or diagnostic agent, particularly for the treatment or diagnosis of cancer. Anti-CD25 antibody conjugates with radionuclides or toxins can be used. Examples of commonly used radionuclides include, for example, 90 Y, 131 I and 67Examples of commonly used toxins are Cu, doxorubicin and calicheamicin. In further embodiments, anti-CD25 antibodies can be modified to have a modified half-life. Methods for achieving a modified half-life are known in the art. In some embodiments, the anti-CD25 antibody is not conjugated with another therapeutic or diagnostic agent. In particular, in some embodiments, the anti-CD25 antibody is not conjugated with a radionuclide. That is, in some embodiments, the anti-CD25 antibody is not radiolabeled.

[0094] In preferred embodiments of the present invention, the subject in any of the embodiments of the present invention described herein is a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cow, hamster, mouse, rat, rabbit, or guinea pig, but most preferably a human. For this reason, it is preferable that the subject is a human in all embodiments of the present invention described herein.

[0095] As used herein, the terms “cancer,” “cancerous,” or “malignant” typically refer to or describe a physiological condition in mammals characterized by uncontrolled cell growth.

[0096] Examples of cancer include, but are not limited to, carcinomas, lymphomas, leukemias, blastomas, and sarcomas. 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 lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), multiple myeloma, gastrointestinal cancers, kidney 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, gastric cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer.

[0097] In one manifestation, cancer is accompanied by a solid tumor. Examples of solid tumors include sarcomas (including cancers arising from mesenchymal-derived transformed cells in tissues such as cavernous bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, hematopoietic tissue, or fibrous connective tissue), carcinomas (including tumors arising from epithelial cells), mesothelioma, neuroblastoma, retinoblastoma, etc. Cancers accompanied by solid tumors are not limited to, but include brain cancer, lung cancer, gastric cancer, duodenal cancer, esophageal cancer, breast cancer, colon and rectal cancer, kidney cancer, bladder cancer, renal cancer, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, oral cancer, sarcoma, eye cancer, thyroid cancer, urethral cancer, vaginal cancer, cervical cancer, lymphoma, etc.

[0098] In one embodiment, cancer includes, but is not limited to, lymphomas such as Hodgkin lymphoma and lymphocytic leukemias such as chronic lymphocytic leukemia (CLL), and is associated with tumors that express CD25.

[0099] In one aspect of the present invention, cancer is identified by the presence of specific tumor-associated markers and antigens such as CD20, HER2, PD-1, PD-L1, SLAM7F, CD47, CD137, CD134, TIM3, CD25, GITR, CD25, EGFR, etc., or is identified as having a biomarker called microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR). Furthermore, antibodies can be used when the identification of specific tumor-associated markers, antigens, or biomarkers is used to determine the precancerous, non-invasive status of the above cancers in patients with conditions such as carcinoma in situ, smoldering myeloma, monoclonal gammaglobulinemia of unknown significance, cervical intraepithelial neoplasia, MALT lymphoma / GALT lymphoma (GALTomes), and various lymphoproliferative disorders. In some embodiments, the subject being treated preferably has a solid tumor.

[0100] In one embodiment of the present invention, the cancer is selected from melanoma, non-small cell lung cancer, renal cancer, ovarian cancer, bladder cancer, sarcoma, and colon cancer. In a preferred embodiment of the present invention, the cancer is selected from melanoma, ovarian cancer, non-small cell lung cancer, and renal cancer. In one embodiment, the cancer is not melanoma, ovarian cancer, or breast cancer. In a preferred embodiment, the cancer is sarcoma, colon cancer, melanoma, or colorectal cancer, or more generally, any human cancer that can be used as a preclinical model to verify the usefulness of the compound in therapeutic management, such as the 4T1, MCA205, B16, CT26, or MC38 cell line.

[0101] As used herein, the term “tumor” in relation to a subject diagnosed with or suspected of having cancer means a malignant or potentially malignant neoplasm or tissue mass of any size, including primary tumors and secondary neoplasms. The terms “cancer,” “malignant disease,” “neoplasm,” “tumor,” and “carcinoma” may also be used herein without distinction to refer to tumors and tumor cells exhibiting an abnormal growth phenotype characterized by relatively abnormal, uncontrolled, and / or autonomous growth and marked uncontrolled cell proliferation. Generally, cells of interest for detection or treatment include precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. The teachings in this disclosure may apply to all cancers.

[0102] As used herein, “solid tumor” refers to an abnormal growth or mass of tissue that does not typically have cysts or fluid areas, particularly tumors and / or metastases (regardless of location) other than leukemia or non-solid lymphoid carcinoma. Solid tumors can be benign or malignant. The various types of solid tumors are named after the type of cells that form the solid tumor and / or the tissue or organ in which the solid tumor is located. Examples of solid tumors include sarcomas (including cancers arising from mesenchymal-derived transformed cells in tissues such as cavernous bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, hematopoietic tissue or fibrous connective tissue), carcinomas (including tumors arising from epithelial cells), melanomas, lymphomas, mesotheliomas, neuroblastomas and retinoblastomas.

[0103] Particularly preferred cancers according to the present invention include those characterized by the presence of solid tumors; that is, the subject does not have non-solid tumors. In all embodiments of the present invention discussed herein, it is preferable that the cancer is a solid tumor, i.e., that the subject has a solid tumor (but does not have a non-solid tumor).

[0104] As used herein, references to "treat" or "treating" cancer define the achievement of at least one positive therapeutic effect, such as, for example, a reduction in the number of cancer cells, a reduction in tumor size, a reduction in the rate of cancer cell invasion into peripheral organs, or a reduction in the rate of tumor metastasis or tumor growth.

[0105] Positive therapeutic effects in cancer can be measured in numerous ways (e.g., Weber (2009) J Nucl Med 50, 1S-10S). For example, regarding tumor growth inhibition, according to the National Cancer Institute (NCI) standard, T / C ≤ 42% is the minimum level of antitumor activity. T / C < 10% is considered a high level of antitumor activity, and T / C (%) = median tumor volume in the treatment group / median tumor volume in the control group × 100. In some embodiments, the treatment achieved by the effective dose is either progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS). PFS, also called "time to tumor progression," indicates the length of time during and after treatment when the cancer does not grow, and includes the time when the patient experiences a complete or partial response, as well as the time when the patient experiences stable disease. DFS indicates the length of time during and after treatment when the patient remains disease-free. OS refers to the extension of average life expectancy compared to an untreated or unenhanced individual or patient.

[0106] As used herein, "prevention" (or preventive measures) refers to delaying or preventing the onset of cancer symptoms. Prevention may be absolute (preventing the disease from occurring) or effective only for some individuals or for a limited time.

[0107] In a preferred embodiment of the present invention, the subject has a settled tumor; that is, the subject already has a tumor classified as, for example, a solid tumor. Thus, the present invention as described herein can be used when the subject already has a tumor such as a solid tumor. Thus, the present invention provides a treatment option that can be used for the treatment of existing tumors. In one embodiment of the present invention, the subject has an existing solid tumor. The present invention can be used preventively or, preferably, as a treatment in a subject who already has a solid tumor. In one embodiment, the present invention is not used as a preventive or prophylaxis method.

[0108] In one embodiment, the present invention as described herein can be used to enhance tumor regression, inhibit or reduce tumor growth, and / or extend survival time, for example, compared with other cancer treatments (e.g., standard treatments for a given cancer).

[0109] In one aspect of the present invention, a method for treating or preventing cancer as described herein further includes the step of identifying a subject having cancer, preferably a subject having a tumor such as a solid tumor. In one embodiment, the method may include identifying a subject having a hematological cancer.

[0110] The dosage regimen of the therapies described herein that are effective in treating cancer patients may vary depending on factors such as the patient's disease state, age and weight, and the ability of the therapy to induce an anti-cancer response in the subject. The selection of an appropriate dosage is within the capabilities of those skilled in the art. For example, these amounts are 0.01 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg. In some embodiments, such amounts are appropriate unit doses (or their whole fractions) for administration according to a dosage regimen (i.e., a therapeutic dosage regimen) that has been determined to correlate with desired or beneficial outcomes when administered to an appropriate population.

[0111] Antibodies according to any aspect of the present invention described herein may be in the form of pharmaceutical compositions further comprising pharmaceutically acceptable carriers, diluents, or excipients. These compositions include, for example, liquid, semi-solid, and solid dosage formulations, such as liquid solutions (e.g., injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, or liposomes. In some embodiments, the preferred form may depend on the intended method of administration and / or therapeutic use. Pharmaceutical compositions containing antibodies can be administered by any suitable method known in the art, including, but not limited to, oral administration, mucosal administration, inhalation administration, topical administration, buccal administration, nasal administration, rectal administration, or parenteral administration (e.g., intravenous, intradrip, intratumoral, intranodal, subcutaneous, intraperitoneal, intramuscular, intradermal, percutaneous, or other types of administration involving physical breaching of the subject's tissue and administration of the pharmaceutical composition through the tissue breach). Such formulations may be in the form of an injectable solution or infusion solution suitable for, for example, intradermal, intratumor, or subcutaneous administration, or intravenous infusion. Administration may include intermittent administration. Alternatively, administration may include continuous administration (e.g., perfusion) for at least a select period of time, concurrent with or between the administration of other compounds.

[0112] In some embodiments, antibodies can be prepared using carriers that prevent rapid release and / or degradation, such as in controlled-release formulations including implanted injectables, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used.

[0113] Those skilled in the art will understand that, for example, the route of delivery (e.g., oral vs. intravenous vs. subcutaneous vs. intratumor) may affect the dose, and / or the required dose may affect the route of delivery. For example, when targeting particularly high concentrations of the active ingredient at a specific site or location (e.g., intratumor), concentrated delivery (e.g., intratumor delivery in this example) may be desired and / or useful. Other factors to consider when optimizing the route and / or administration schedule for a given treatment plan may include, for example, the specific cancer being treated (e.g., type, stage, location, etc.), the subject's clinical condition (e.g., age, overall health, etc.), the presence or absence of concomitant therapy, and other factors known to the physician.

[0114] Pharmaceutical compositions are typically sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other regular structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of antibody, along with one or a combination of the components listed above as needed, into a suitable solvent, followed by filtered sterilization. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and embeddable sustained-release or biodegradable formulations as discussed herein. Sterile injectable formulations can be prepared using non-toxic, parenterally acceptable diluents or solvents. Each pharmaceutical composition used in accordance with the present invention may contain pharmaceutically acceptable dispersants, wetting agents, suspending agents, isotonic agents, coating agents, antibacterial and antifungal agents, carriers, excipients, salts, or stabilizers that are non-toxic to the subject at the doses and concentrations used. Preferably, such composition may further comprise a pharmaceutically acceptable carrier or excipient used in the treatment of cancer for parenteral (e.g., subcutaneous, intradermal, or intravenous), intratumoral, or peritumoral administration, which is suitable for a given method and / or administration site.

[0115] Embodiments of the therapeutic method or composition for use according to the present invention may not be effective in achieving a positive therapeutic effect in all subjects, but appropriate medical practice and Student's t-test, χ² 2 The test shall be conducted using a pharmaceutical composition and administration plan that are suitable for a statistically significant number of subjects, as determined by any statistical test known in the art, such as the Mann-Whitney U test, the Kruskal-Wallis test (H test), the Yonk-Heal-Tapstra test, and the Wilcoxon test.

[0116] Wherever tumor, neoplastic disease, carcinoma or cancer is referred to above and thereafter, it also means, alternatively or additionally, the tumor and / or metastases in the original organ or tissue and / or any other location, regardless of the location of the metastasis.

[0117] As discussed herein, the present invention relates to the depletion of regulatory T cells (Tregs). Therefore, in one aspect of the present invention, an anti-CD25 antibody that does not inhibit the binding of interleukin-2 to CD25 also depletes or reduces tumor-infiltrating regulatory T cells. In one aspect, the depletion is due to ADCC. In another aspect, the depletion is due to ADCP.

[0118] Thus, the present invention provides a method for depleting regulatory T cells in a tumor in a subject, comprising administering to the subject an anti-CD25 antibody that does not inhibit the binding of interleukin-2 to CD25. In a preferred embodiment, Tregs are depleted in a solid tumor. "Depleted" means that the number, ratio, or percentage of Tregs is reduced compared to when an anti-CD25 antibody that does not inhibit the binding of interleukin-2 to CD25 is not administered. In specific embodiments of the present invention described herein, about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 99% of tumor-infiltrating regulatory T cells are depleted.

[0119] As used herein, “regulatory T cells” (“Treg,” “Treg cells,” or “Treg”) refer to a lineage of CD4+ T lymphocytes specialized in controlling autoimmunity, allergies, and infections. They typically modulate the activity of the T cell population, but may also affect certain cell types of the innate immune system. Tregs are usually identified by the expression of the biomarkers CD4, CD25, and Foxp3. Spontaneous Treg cells typically account for about 5%–10% of the peripheral CD4+ T lymphocyte population. However, in the tumor microenvironment (i.e., tumor-infiltrating Treg cells), they can account for as much as 20%–30% of the total CD4+ T lymphocyte population.

[0120] Activated human Treg cells can directly kill target cells such as effector T cells and APCs via perforin-dependent or granzyme B-dependent pathways. Cytotoxic T lymphocyte-associated antigen 4 (CTLA4+) Treg cells induce indoleamine 2,3-dioxygenase (IDO) expression by APCs, which then suppress T cell activation by reducing tryptophan. Treg cells directly inhibit T cell activation by releasing interleukin-10 (IL-10) and transforming growth factor (TGFβ) in vivo, and suppress APC function by inhibiting the expression of MHC molecules, CD80, CD86, and IL-12. Treg cells can also suppress immunosuppression by expressing high levels of CTLA4 that can bind to CD80 and CD86 on antigen-presenting cells, thereby preventing appropriate activation of effector T cells.

[0121] In preferred embodiments of the present invention, the ratio of effector T cells to regulatory T cells in solid tumors is increased. In some embodiments, the ratio of effector T cells to regulatory T cells in solid tumors is increased by 5, 10, 15, 20, 40, or more than 80.

[0122] Immune effector cells refer to immune cells involved in the effector phase of the immune response. Exemplary immune cells include cells of bone marrow or lymphoid origin, such as lymphocytes (e.g., T cells including B cells and cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils.

[0123] Immune effector cells involved in the effector phase of the immune response express specific Fc receptors and perform specific immune functions. Effector cells can induce antibody-dependent cell-mediated cytotoxicity (ADCC). For example, neutrophils can induce ADCC. For instance, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes expressing FcαR are involved in the specific killing of target cells and the presentation of antigens to other components of the immune system, or binding to antigen-presenting cells. Effector cells can also phagocytose target antigens, target cells, or microorganisms. As discussed herein, antibodies according to the present invention can be optimized for their ability to induce ADCC.

[0124] In some embodiments, different agents acting on cancer can be administered in combination with antibodies via the same or different delivery routes and / or according to different schedules. Alternatively or additionally, in some embodiments, one or more doses of the first active agent are administered substantially simultaneously with one or more other active agents, and in some embodiments, via a common route and / or as part of a single composition. Those skilled in the art will further understand that some embodiments of combination therapies provided according to the present invention achieve synergistic effects. In some such embodiments, the doses of one or more agents used in combination may be substantially different (e.g., lower) than those standard, preferred or required when the agent is used in a different treatment plan (e.g., as monotherapy and / or as part of a different combination therapy), and / or may be delivered via alternative routes to the standard, preferred or required routes when the agent is used in a different treatment plan (e.g., as monotherapy and / or as part of a different combination therapy).

[0125] In some embodiments utilizing two or more active substances according to the present invention, such active substances may be administered simultaneously or sequentially. In some embodiments, the administration of one active substance is specifically timed to coincide with the administration of another active substance. For example, in some embodiments, a first active substance is administered so that a specific effect is observed (or is expected to be observed based on population studies showing a correlation between a given administration plan and a specific effect on the subject, for example). In some embodiments, a desired relative administration plan of active substances administered in combination may be assessed or determined empirically, for example, using ex vivo, in vivo, and / or in vitro models. In some embodiments, such assessment or empirical determination is performed in vivo in a patient population (e.g., so that a correlation is established) or alternatively in a specific patient of the subject.

[0126] In another aspect of the present invention, an anti-CD25 antibody that does not inhibit the binding of interleukin-2 to CD25 exhibits improved therapeutic efficacy when combined with an immune checkpoint inhibitor. Combination therapy using an anti-CD25 antibody that does not inhibit the binding of interleukin-2 to CD25 and an immune checkpoint inhibitor may have a synergistic effect in the treatment of established tumors. The data for PD-1 / PD-L1 in this example relate to the interference of PD-1 / PD-L1 interaction. Thus, it is possible to block the interaction between the PD-1 receptor and the PD-L1 ligand, resulting in "PD-1 blockade." In one aspect, the combination may result in enhanced tumor regression, enhanced impairment or reduction of tumor growth, and / or extended survival time using the present invention as described herein, compared to, for example, either an anti-CD25 antibody or PD-1 / PD-L1 blockade alone (directly using an anti-PD1 antibody or indirectly using an anti-PD-L1 antibody). Since anti-CD25 antibodies do not inhibit the binding of interleukin-2 to CD25, combination therapy using anti-CD25 antibodies and immune checkpoint inhibitors may further include the administration of interleukin-2 in doses appropriate for the treatment of cancer.

[0127] As used herein, “immune checkpoint” or “immune checkpoint protein” refers to proteins belonging to suppressive pathways in the immune system, particularly for modulating T cell responses. Under normal physiological conditions, immune checkpoints are crucial for preventing autoimmunity, especially in response to pathogens. Cancer cells can alter the regulation of immune checkpoint protein expression to evade immune surveillance.

[0128] Examples of immune checkpoint proteins include, but are not limited to, PD-1, CTLA-4, BTLA, KIR, LAG3, TIGIT, CD155, B7H3, B7H4, VISTA, and TIM3, as well as OX40, GITR, ICOS, 4-1BB, and HVEM. The term "immune checkpoint protein" can also refer to proteins that bind to other immune checkpoint proteins. Examples of such proteins include PD-L1, PD-L2, CD80, CD86, HVEM, LLT1, and GAL9.

[0129] "Immune checkpoint protein inhibitor" refers to any protein that can interfere with signal transduction and / or protein-protein interactions mediated by immune checkpoint proteins. In one aspect of the present invention, the immune checkpoint protein is PD-1 or PD-L1. In preferred aspects of the present invention described herein, the immune checkpoint inhibitor interferes with PD-1 / PD-L1 interactions via an anti-PD-1 or anti-PD-L1 antibody.

[0130] Thus, the present invention also provides a method for treating cancer, comprising administering to a subject an anti-CD25 antibody that does not inhibit the binding of interleukin-2 to CD25 and a further therapeutic agent, preferably a checkpoint inhibitor. The present invention also provides an anti-CD25 antibody that does not inhibit the binding of interleukin-2 to CD25 and a further therapeutic agent, preferably an immune checkpoint inhibitor, for use in the treatment of cancer.

[0131] The present invention additionally provides the use of anti-CD25 antibodies that do not inhibit the binding of interleukin-2 to CD25 and further therapeutic agents, preferably immune checkpoint inhibitors, in the manufacture of drugs for the treatment of cancer. The administration of anti-CD25 antibodies that do not inhibit the binding of interleukin-2 to CD25 and further therapeutic agents such as immune checkpoint inhibitors may be simultaneous, separate, or sequential.

[0132] The present invention provides a combination of an anti-CD25 antibody that does not inhibit the binding of interleukin-2 to CD25 and a further therapeutic agent, preferably an immune checkpoint inhibitor, for use in the treatment of cancer in a subject, wherein the anti-CD25 antibody that does not inhibit the binding of interleukin-2 to CD25 and the further therapeutic agent, such as an immune checkpoint inhibitor, are administered simultaneously, separately, or sequentially. Such an anti-human CD25 antibody that does not inhibit the binding of interleukin-2 to CD25 and exhibits a human IgG1 isotype can be used in combination with an antibody that specifically targets immune checkpoints but lacks sequences that enable ADCC, ADCP, and / or CDC.

[0133] In an alternative embodiment, the present invention provides an anti-CD25 antibody that does not inhibit the binding of interleukin-2 to CD25 for use in the treatment of cancer, wherein the antibody is administered in combination with a further therapeutic agent, preferably an immune checkpoint inhibitor. The present invention also provides the use of anti-CD25 that does not inhibit the binding of interleukin-2 to CD25 in the manufacture of a drug for treating cancer, wherein the drug is administered in combination with a further therapeutic agent, preferably an immune checkpoint inhibitor.

[0134] The present invention provides a pharmaceutical composition comprising an anti-CD25 antibody that does not inhibit the binding of interleukin-2 to CD25 in a pharmaceutically acceptable medium, and optionally a further therapeutic agent, preferably an immune checkpoint inhibitor. As discussed above, the immune checkpoint inhibitor may be a PD-1 inhibitor, i.e., a PD-1 antagonist.

[0135] PD-1 (Programmed Cell Death Protein 1), also known as CD279, is a cell surface receptor expressed on activated T and B cells. Its interaction with its ligands has been shown to weaken the T cell response both in vitro and in vivo. PD-1 binds to two ligands, PD-L1 and PD-L2. PD-1 belongs to the immunoglobulin superfamily. PD-1 signaling requires binding to a PD-1 ligand in close proximity to a peptide antigen presented by the major histocompatibility complex (MHC) (Freeman (2008) Proc Natl Acad Sci USA 105, 10275-6). Therefore, proteins, antibodies, or small molecules that interfere with the co-ligation of PD-1 and the TCR on the T cell membrane are useful PD-1 antagonists.

[0136] In one embodiment, the PD-1 receptor antagonist is an anti-PD-1 antibody or its antigen-binding fragment that specifically binds to PD-1 and blocks the binding of PD-L1 to PD-1. The anti-PD-1 antibody may be a monoclonal antibody. The anti-PD-1 antibody may be human or a humanized antibody. The anti-PD-1 antibody is an antibody capable of specific binding to the PD-1 receptor. Known anti-PD-1 antibodies in the art include nivolumab and pembrolizumab.

[0137] The PD-1 antagonists of the present invention also include compounds or activators that bind to and / or block PD-1 ligands to prevent or inhibit ligand binding to the PD-1 receptor, or that directly bind to and block the PD-1 receptor without inducing inhibitory signaling via the PD-1 receptor. In particular, PD-1 antagonists include small molecule inhibitors of the PD-1 / PD-L1 signaling pathway. Alternatively, PD-1 receptor antagonists can directly bind to the PD-1 receptor without inducing inhibitory signaling, and further bind to ligands of the PD-1 receptor to reduce or inhibit the induction of ligand signaling via the PD-1 receptor. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and induce the transmission of inhibitory signals, fewer cells can be attenuated by the negative signals delivered by PD-1 signaling, thereby achieving a more robust immune response.

[0138] In one embodiment, the PD-1 receptor antagonist is an anti-PD-L1 antibody or its antigen-binding fragment that specifically binds to PD-L1 and blocks the binding of PD-L1 to PD-1. The anti-PD-L1 antibody may be a monoclonal antibody. The anti-PD-L1 antibody may be a human or humanized antibody such as atezolizumab (MPDL3280A).

[0139] The present invention also provides a method for treating cancer, comprising administering to a subject an anti-CD25 antibody that does not inhibit the binding of interleukin-2 to CD25 and an antibody that is an agonist of the T cell activation costimulatory pathway. Examples of antibody agonists of the T cell activation costimulatory pathway include, but are not limited to, agonist antibodies against ICOS, GITR, OX40, CD40, LIGHT, and 4-1BB.

[0140] Further methods for treating cancer include administering anti-CD25 antibodies that do not inhibit the binding of interleukin-2 to CD25, and compounds that reduce, block, inhibit, and / or antagonize FcγRIIb (CD32b). Such FcγRIIb antagonists may be small molecules that interfere with intracellular signaling induced by FcγRIIb, modified antibodies that do not engage with repressive FcγRIIb receptors, or anti-human FcγRIIb (anti-CD32b) antibodies. For example, antagonist anti-human FcγRIIb antibodies have also been characterized for their antitumor properties (Roghanian A et al., 2015, Cancer Cell. 27, 473-488; Rozan C et al., 2013, Mol Cancer Ther. 12:1481-91, International Publication No. 2015173384, International Publication No. 2008002933).

[0141] In a further embodiment, the present invention is (a) A first antigen-binding portion that binds to CD25, (b) A second antigen-binding moiety that binds to an immune checkpoint protein, tumor-associated antigen, anti-human activated Fc receptor antibody (FcgRI, FcgRIIa, FcgRIII), or antagonist anti-human FcγRIIb antibody, The present invention provides a bispecific antibody comprising a bispecific antibody, wherein the anti-CD25 antibody does not inhibit the binding of interleukin-2 (IL-2) to CD25, and preferably binds with high affinity to at least one activated Fcγ receptor, thereby depleting tumor-invading regulatory T cells, and is an IgG1 bispecific antibody. In a preferred embodiment, the second antigen-binding moiety binds to PD-L1.

[0142] As used herein, “tumor-associated antigens” refer to, but are not limited to, antigens expressed on tumor cells that distinguish tumor cells from adjacent non-cancerous cells, and include, however, CD20, CD38, PD-L1, EGFR, EGFRV3, CEA, TYRP1, and HER2. Numerous review articles have been published describing relevant tumor-associated antigens and corresponding therapeutically useful antitumor antibodies (see, for example, Sliwkowski & Mellman (2013) Science 341, 192-8). Examples of such antigens and corresponding antibodies include, but are not limited to, CD22 (blinatumomab), CD20 (rituximab, tositumomab), CD56 (lorbotuzumab), CD66e / CEA (rabetuzumab), CD152 / CTLA-4 (ipilimumab), CD221 / IGF1R (MK-0646), CD326 / Epcam (edrecolomab), CD340 / HER2 (trastuzumab, pertuzumab), and EGFR (cetuximab, panitumumab).

[0143] In one embodiment, the bispecific antibodies according to the present invention described herein result in ADCC, or in one embodiment, enhancement of ADCC.

[0144] A bispecific antibody can bind to a specific epitope on CD25 that does not affect the binding of IL-2 to CD25, and to a specific epitope on an immune checkpoint protein or tumor-associated antigen as defined herein. In a preferred embodiment, the second antigen-binding moiety binds to PD-L1. In a preferred embodiment, the present invention is (a) A first antigen-binding moiety that binds to CD25 and does not affect the binding of IL-2 to CD25, (b) A second antigen-binding moiety that binds to an immune checkpoint protein expressed on tumor cells, This provides a bispecific antibody containing [specific antibody].

[0145] In certain embodiments, the immune checkpoint protein expressed on tumor cells is PD-L1, VISTA, GAL9, B7H3, or B7H4. More preferably, the anti-CD25 antibody is an IgG1 antibody that does not affect the binding of IL-2 to CD25, binds with high affinity to at least one activated Fcγ receptor, and depletes tumor-infiltrating regulatory T cells. Alternatively, the anti-CD25 antibody is a human IgG2 antibody that depletes tumor-infiltrating regulatory T cells. In a particular embodiment, the anti-CD25 antibody is a human IgG2 antibody that binds with high affinity to at least one activated Fcγ receptor, preferably FcγRIIa.

[0146] Those skilled in the art can produce bispecific antibodies using known methods. The bispecific antibodies according to the present invention can be used in any of the embodiments of the present invention described herein. The second antigen-binding moiety in the bispecific antibodies according to the present invention is preferably bound to human PD-1, human PD-L1, or human CTLA-4.

[0147] In one embodiment, a bispecific antibody may bind to CD25 and to immunomodulatory receptors highly expressed on tumor-infiltrating Tregs, such as CTLA4, ICOS, GITR, 4-1BB, or OX40.

[0148] The present invention also provides a kit comprising the anti-CD25 antibody described herein and a further therapeutic agent discussed herein, preferably an immune checkpoint inhibitor, preferably a PD-1 antagonist (directly using an anti-PD1 antibody or indirectly using an anti-PD-L1 antibody). In one embodiment, the immune checkpoint inhibitor is anti-PD-L1. In an alternative embodiment, the kit comprises the anti-CD25 antibody described herein and an antibody that is an agonist of the T cell activation costimulatory pathway. The kit may include instructions for use.

[0149] Any aspect of the present invention described herein can be used in combination with additional therapeutic agents, particularly additional cancer therapies. In particular, the anti-CD25 antibody and optionally an immune checkpoint inhibitor according to the present invention can be administered in combination with a costimulatory antibody, chemotherapy and / or radiotherapy (by irradiating the body from outside the body or by administering a radioconjugate compound), cytokine therapy, targeted therapy, monoclonal antibody therapy, vaccine or adjuvant, or any combination thereof.

[0150] As used herein, "chemotherapeutic entity" refers to a substance that is harmful to cells; that is, a substance that reduces the viability of cells. Chemotherapeutic entities may be cytotoxic drugs. Intended chemotherapeutic agents include, but are not limited to, alkylating agents, anthracyclines, epothyron, nitrosourea, ethyleneimine / methylmelamine, alkyl sulfonates, alkylating agents, antimetabolites, pyrimidine analogs, epipodophylotoxins, enzymes such as L-asparaginase; biological response modifiers such as IFNα, IFN-γ, IL-2, IL-12, G-CSF, and GM-CSF; platinum-coordinated complexes such as cisplatin, oxaliplatin, and carboplatin; substituted ureas such as anthracendione and hydroxyurea; methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine; and adrenal cortical inhibitors such as mitotane (o,p'-DDD) and aminoglutethimide. Examples of suppressants include: adrenal corticosteroid antagonists such as prednisone and equivalents; hormones and antagonists including dexamethasone and aminoglutethimide; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinylestradiol equivalents; antiestrogens such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and nonsteroidal antiandrogens such as flutamide.

[0151] Additional cancer therapies may include the administration of cancer vaccines. As used herein, “cancer vaccine” refers to a therapeutic cancer vaccine that is administered to cancer patients and designed to eradicate cancer cells by enhancing the patient’s own immune response. Examples of cancer vaccines include tumor cell vaccines (autologous and allogeneic), dendritic cell vaccines (ex vivo produced and peptide activated), protein / peptide-based cancer vaccines, and gene vaccines (DNA, RNA, and virus-based vaccines). In principle, therapeutic cancer vaccines can be used to inhibit the further growth of advanced cancers and / or recurrent tumors that have failed to respond to conventional therapies such as surgery, radiation therapy, and chemotherapy. Tumor cell-based vaccines (autologous and allogeneic) include those genetically engineered to secrete single-chain Fv antibodies against soluble immunostimulatory agents such as cytokines (IL-2, IFN-γ, IL-12, GMCSF, FLT3L), and / or ligands for immunostimulatory receptors such as PD-1, CTLA-4, GITR, ICOS, OX40, 4-1BB, and / or OX40 ligands on their membranes. In one embodiment, the cancer vaccine may be a GVAX antitumor vaccine.

[0152] Additional cancer therapies may include other antibodies or small molecule reagents that reduce immunomodulation within the peripheral and tumor microenvironment, such as molecules targeting the TGFβ pathway, IDO (indoleamine dioxygenase), arginase, and / or CSF1R.

[0153] "In combination" may refer to the administration of any additional therapy before, simultaneously with, or after administration of any aspect of the present invention.

[0154] The present invention will now be further illustrated using the following examples, which are intended to assist those skilled in the art in carrying out the invention with reference to the drawings, and are not intended to limit the scope of the invention in any way. [Examples]

[0155] Example 1 - In vitro characterization and preparation of recombinant anti-mouse CD25 Treg depletion antibodies that are either non-IL-2 blockadist or IL-2 blockadist. Materials and methods Antibody origins and their recombinant production The sequences of the variable regions of the heavy and light chains of rat anti-mouse CD25 PC61 were split from a PC-61.5.3 hybridoma (ATCC catalog number TIB-222) by rapid amplification of cDNA ends (RACE), and then cloned into the constant regions of mouse IgG2a and κ chains (or the corresponding mouse IgG1 sequence isolated from a commercially available plasmid (Invivogen)).

[0156] Next, each antibody chain was subcloned into a mouse leukemia virus (MLV) retroviral vector. For preliminary experiments, antibodies were produced using K562 cells transduced with vectors encoding both heavy and light chains. The antibodies were purified from the supernatant using a Protein G HiTrap MabSelect column (GE Healthcare), dialyzed in phosphate-buffered saline (PBS), concentrated, and filtered to sterilize.

[0157] The re-cloned anti-mouse CD25 variable heavy chain DNA sequence derived from the PC-61.5.3 antibody (mouse IgG2a) encodes the following protein sequence: METDTLLLWVLLLWVPGSTGEVQLQQSGAELVRPGTSVKLSCKVSGDTITAYYIHFVKQRPGQGLEWIGRIDPEDDSTEYAEKFKNKATITANTSSNTAHLKYSRLTSEDTATY FCTTDNMGATEFVYWGQGTLVTVSS

[0158] The re-cloned anti-mouse CD25 variable light chain DNA sequence derived from the PC-61.5.3 antibody (mouse IgG2a) encodes the following protein sequence: METDTLLLWVLLLWVPGSTGQVVLTQPKSVSASLESTVKLSCKLNSGNIGSYYMHWYQQREGRSPTNLIYRDDKRPDGAPDRFSGSIDISSNSAFLTINNVQTEDEAMYFCHSYDGRMYIFGGGTKLTV

[0159] 7D4-IgM sequencing was performed on 7D4 hybridomas (ECACC, 88111402). Total RNA or mRNA was extracted and reverse transcribed to obtain cDNA of the antibody heavy and light chains. The variable heavy and light chains were amplified using degenerate forward primers that bind to either the signal peptide or framework region 1, and reverse primers that bind to the antibody constant region. The amplified genes were cloned and sequenced according to a standard approach. cDNA was generated by reverse transcription, and a homopolymeric tail was added to the 3' end of the cDNA. Next, the antibody variable domain gene was amplified using gene-specific primers, followed by a standard cloning and sequencing approach. The DNA was sequenced by conventional Sanger sequencing, and the data was analyzed using DNASTAR Lasergene software. The signal peptide and variable domain sequences were identified by comparison with known sequences in the IMGT database.

[0160] Genes encoding variable heavy and light chain domains were codon-optimized for expression in human cell lines and synthesized using the NheI and AvaI restriction sites at the 5' and 3' positions of the genes. Restriction digestion cloning was performed to insert the 7D4 variable heavy chain domain gene into a separate expression vector containing mouse IgG1 and IgG2a constant domains. Restriction digestion cloning was performed to insert the 7F4 variable light chain domain gene into an expression vector containing mouse κ constant domain. Suspended HEK293 cells cultured in serum-free medium were chemically cotransfected with the heavy and light chain expression vectors and cultured for a further 6 days at 37°C with shaking at 140 rpm in a 5% CO2 environment. The cultures were collected by centrifugation at 4000 rpm and further clarified by filtration through a 0.22 μM filter. The supernatant was loaded onto a Protein A column pre-equilibrated with PBS (pH 7.2), eluted with sodium citrate (pH 3.5), and equilibrated with 10% (v / v) 0.5M Tris (pH 9.0). The neutralizing antibody solution was buffer-changed to PBS (pH 7.2) using a desalting column, and concentrated using a centrifugal concentrator with a molecular weight cutoff of 30 kDa as needed. Protein concentration was determined by measuring absorbance at 280 nm, and purity was determined by SDS-PAGE.

[0161] The re-cloned anti-mouse CD25 heavy chain DNA sequence derived from the 7D4 antibody (mouse IgG1) encodes the following protein sequence: EVQLQQSGAALVKPGASVKMSCKASGYSFPDSWVTWVKQSHGKSLEWIGDIFPNSGATNFNEKFKGKATLTVDKSTSTAYMELSRLTSEDSAIYYCTRLDYGYWGQGVM VTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCK PCICTVPEVSSVFIFPPKPKDVLMISLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPILHQDWLNGKEFKCRVNSAAFPAPIEKTIS KTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0162] The re-cloned anti-mouse CD25 variable heavy chain DNA sequence derived from the 7D4 antibody (mouse IgG2a) encodes the following protein sequence: EVQLQQSGAALVKPGASVKMSCKASGYSFPDSWVTWVKQSHGKSLEWIGDIFPNSGATNFNEKFKGKATLTVDKSTSTAYMELSRLTSEDSAIYYCTRLDYGYWGQGVMVT VSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCP PCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTI SKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0163] The re-cloned anti-mouse CD25κ light chain DNA sequences derived from both 7D4(mIg1) and 7D4(mIg2a) antibodies (mouse IgG2a) encode the following protein sequences: DVVLTQTPPTLSATIGQSVSISCRSSQSLLHSNGNTYLNWLLQRPGQPPQLLIYLASRLESGVPNRFSGSGSGTDFTLKISGVEAEDLGVYYCVQSSHFPNTFGVGTKL EIKRADAAPTVSIFPPSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0164] 2E4 was generated from a 2E4 hybridoma (donated by Dr. Ethan M. Shevach of the National Institutes of Health). Hybridoma sequencing was performed using our proprietary next-generation sequencing (NGS)-based technology. A cDNA library was generated using RNA samples. The library was sequenced on the Illumina platform. The sample transcriptome was reconstructed from raw data using de novo assembly. Variable domain sequences were identified by comparison with known sequences.

[0165] The variable heavy chain domain protein sequence of the anti-mouse CD25 2E4 antibody (mouse IgG1) has the following protein sequence: EVQLVESGGGLVQPGRSLKLSCAASGFTFSDYGMAWVRQAPTKGLEWVASITNGGLNTYYRDSVKGRFTISRDNAKCTLYLQMDSLRSEDTATYYCATGGFSFWGQGTLVTVSS

[0166] The variable light chain domain protein sequence of anti-mouse CD25 2E4(mIg1) has the following protein sequence: DIVMTQSPTSMSISVGDRVTMNCKASQNVDSNVDWYQQKTGQSPKLLIYKASNRYTGVPDRFTGSGSGTDFTFTIRNMQAEDLAVYYCMQSNSYPLTFGSGTKLEIK

[0167] Assessment of the affinity of recombinant antibodies against mouse CD25 ForteBio affinity was generally measured using Octet RED384 as previously described (see, e.g., Estep P et al., 2013. Mabs. 5(2), 270-8). Briefly, ForteBio affinity was measured by loading IgG online onto an AHQ sensor. The sensor was equilibrated offline in assay buffer for 30 minutes, followed by online monitoring for 60 seconds to establish a baseline. The IgG-loaded sensor was exposed to 100 nM antigen for 3 minutes, then transferred to assay buffer for 3 minutes for off-rate measurement. All kinetics were analyzed using a 1:1 binding model.

[0168] result Two mouse hybridomas were selected as reference antibodies to evaluate the CD25 binding and Treg depletion properties of anti-mouse CD25 that were either non-IL-2 blocking or IL-2 blocking (7D4 (mouse IgM isotype) and PC61 (mouse IgG1 isotype), respectively). IL-2 binding-related properties described in the literature were pre-confirmed using the original non-recombinant antibody and recombinant mouse IL-2 (Figure 1A). Recombinant variants of these antibodies were created to test antibodies with more active isotypes and suitable for functional studies (e.g., effects on Treg depletion or other immune cells). Furthermore, in the case of 7D4, isotype changes are necessary because the antibody aggregation properties of the IgM antibody can affect the assay results. Recombinant 7D4 (mIgG1), the original non-recombinant IgM isotype antibody, still allows binding of mouse IL-2 to mouse CD25 (Figure 1B). 7D4(mIgG1), like recombinant PC61(IgG2a), binds to mouse CD25 on the cell surface, but it does not bind to the reference anti-human CD25 antibody (Figure 1C).

[0169] Furthermore, the DNA sequences encoding the variable domains of the 7D4 heavy chain (and PC61) were cloned into a vector that enables the expression of a mouse CD25-binding domain possessing the mouse IgG2a isotype (functionally corresponding to human IgG1). In this way, it is possible to compare two recombinant anti-mouse CD25 antibodies that can efficiently deplete tumor Tregs but have optimized ADCC activity exhibiting specific characteristics regarding mouse IL-2 binding to mouse CD25. The resulting recombinant anti-mouse CD25 antibodies were tested for their CD25 affinity. Different isotypes (mouse IgG2a or mouse IgG1) do not affect this characteristic, as Kd is similar between the 7D4-based recombinant antibodies (approximately 1 nM) and equivalent to one of the PC61s (mIgG2a) (measured at 4.6 nM).

[0170] The functional properties of these recombinant antibodies were also compared in in vitro assays to determine their effects on granzyme B production in response to anti-CD3 and anti-CD28 stimulation (Figure 2). Granzyme B (GnzB) is a serine proteinase expressed by memory T cells, NK cells, and activated CD4 and CD8 T cells, which strongly express and secrete GnzB during immune responses. This enzyme is an important mediator of cell death, histopathology, and disease. In vitro stimulation and proliferation of T cells by anti-CD3 and anti-CD28 antibodies (more than 80% of CD4 T cells proliferate and express GnzB) can be influenced by cytokines and antibodies. When this stimulation was combined with a neutralizing anti-IL-2 antibody, granzyme B production, rather than proliferation, was inhibited. The frequency of proliferating and GnzB-producing cells decreased from more than 80% to less than 1%, while the frequency of proliferating cells remained above 90%. This indicates that granzyme B production, rather than cell proliferation, depends on IL-2 signaling. A similar decrease in granzyme B-producing T cells is observed when PC61(mIgG1) is added to stimulated T cells. However, 7D4(mIgG1) primarily preserves the ability of CD4 T cells to respond to anti-CD3 and anti-CD28 stimulation by producing GnzB (over 65% of cells still produce GnzB and proliferate). These results suggest that PC61-based antibodies block IL-2 signaling, while 7D4 has only a minimal effect on this signaling, thus confirming that anti-human CD25 antibodies that do not affect IL-2 signaling can be used as surrogate antibodies to evaluate the therapeutic potential of such antibodies, particularly in relation to Treg depletion and tumor-specific characteristics.

[0171] Example 2 - Treg depletion and anticancer properties of recombinant anti-mouse CD25 Treg depletion antibodies that are either non-IL-2 blockadist or IL-2 blockadistist. Materials and methods mouse In vivo studies were conducted by Charles River Discovery Services North Carolina (CR Discovery Services). Female BALB / c mice (BALB / c AnNcr1, Charles River) and female C57BL / 6 mice (C57BL / 6Ncr1, Charles River) were 7 to 9 weeks old at the start of the study. CR Discovery Services adheres particularly closely to the recommendations of the Guide for Care and Use of Laboratory Animals regarding restraint, rearing, surgical procedures, feed and fluid regulation, and veterinary care. CR Discovery Services' animal care and use programs are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, which ensures compliance with recognized standards for laboratory animal care and use.

[0172] Cell lines and tissue cultures MCA205 tumor cells (3-methylcholanthrene-induced weakly immunogenic fibrosarcoma cells; by G. Kroemer of the Gustave Roussy Cancer Institute) were cultured in Dulbecco's modified Eagle medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FCS, Sigma), 100 U / mL penicillin, 100 μg / mL streptomycin, and 2 mM L-glutamine (all by Gibco). MC38 mouse colon cancer cells (CR discovery services) were grown to the mid-log phase in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum, 2 mM glutamine, 100 units / mL penicillin G, 100 μg / mL streptomycin sulfate, and 25 μg / mL gentamicin. CT26 mouse colon cancer cells (CR discovery services) were grown in RPMI-1640 medium containing 10% fetal bovine serum, 2 mM glutamine, 100 units / mL penicillin G sodium, 100 μg / mL streptomycin sulfate, and 25 μg / mL gentamicin. All tumor cells were cultured in tissue culture flasks in a humidified incubator at 37°C under an atmosphere of 5% CO2 and 95% air. K562 cells used for antibody production were cultured in phenol red-free Iscove modified Dulbecco medium (IMDM) supplemented with 10% IgG-depleted FCS (Life Technologies).

[0173] in vivo tumor experiments Cultured tumor cells were either trypsin-treated (MCA205) or not (MC38 and CT26), washed, resuspended in PBS, and subcutaneously injected into the flank (5 × 10 for the MCA205 and MC38 models of C57BL / 6 mice). 5 Cells; 3 × 10 for the CT26 model of BALB / c mice 5(15 cells). Antibodies were injected intraperitoneally (ip) at the time indicated in the diagram's caption. For functional experiments, tumors and draining lymph nodes were collected 12 days after tumor transplantation and processed for flow cytometry analysis as described (Simpson et al. (2013) J Exp Med 210, 1695-710). For therapeutic experiments, tumors were measured twice a week, and their volume was calculated as the product of three orthogonal diameters.

[0174] Flow cytometry Acquisition was performed using BD LSR II Fortessa (BD Biosciences). The following antibodies were used: anti-CD3 (clone 145-2C11, eBioscience, 25003182), anti-CD4 (clone RM4-5, BD Biosciences, 560782), anti-CD8 (clone 53-6.7, Biolegend, 100750), anti-granzyme B (clone GB11, Invitrogen), anti-FoxP3 (clone FJK-16s, eBiosciences), and Ki67 (clone SolA15, eBiosciences, 48569882). Lymph nodes (inguinal, axillary, and upper arm) and tumors of mice were dissected in serum-free RPMI. Lymph nodes were dispersed through a 70 μm filter, while tumors were mechanically destroyed using gentleMACS (Miltenyl Biotech) and digested at 37°C for 30 minutes with a mixture of 0.33 mg / ml DNase (Sigma-Aldrich) and 0.27 mg / ml Liberase TL (Roche) in serum-free RPMI. The tumors were filtered through a 70 μm filter, and the resulting tumor single-cell suspension was enriched with leukocytes by applying a Ficoll-paque (GE Healthcare) gradient. The tumors and LNs were washed in complete RPMI, resuspended in FACS buffer (500 mL PBS, 2% FCS, 2 mM EDTA), and placed in a round-bottom 96-well plate. Master mixes of surface antibodies were prepared at the dilutions recommended by the manufacturers: anti-CD3 (clone 145-2C11, ebioscience, 25003182), anti-CD4 (clone RM4-5, BD biosciences, 560782), and anti-CD8 (clone 53-6.7, Biolegend, 100750). A fixable viability dye (eFlour780, eBioscience) was also included in the surface master mix.After permeabilization for 20 minutes using the intracellular fixation and permeabilization buffer set (eBioscience), an intracellular staining panel consisting of the following antibodies, used at the manufacturer's recommended dilutions, was applied: anti-granzyme B (clone GB11, Invitrogen), anti-FoxP3 (clone FJK-16s, eBiosciences), and Ki67 (clone SolA15, eBiosciences, 48569882).

[0175] result The MCA205 sarcoma mouse model allows for the rapid evaluation of immunological responses and overall efficacy against solid tumors using a panel of immunomodulatory compounds. In particular, recombinant mouse IgG2a-based anti-mouse CD25 antibodies were tested to assess changes in T cell subpopulations present as tumor-infiltrating lymphocytes or within peripheral lymph nodes, as well as tumor growth and viability in mice exposed to MCA205. An additional antibody (anti-mouse PD1) was included in the study as a negative control for immunological effects against Tregs.

[0176] Immunological analysis showed that the 7D4 antibody, when cloned into the mouse IgG2a backbone, exhibited similar ability to PC61 (mouse IgG2a) in depleting Tregs and subsequently increasing the Teff-to-Treg ratio in both tumors and periphery, while anti-PD1, either alone or in combination, was ineffective (Figure 3). Therefore, any further effects measured using 7D4 (mIgG2a) as a surrogate antibody against non-IL-2 blocking anti-human CD25 antibodies do not appear to be related to changes in Treg depletion characteristics.

[0177] Furthermore, MCA205 model mice treated with 7D4 showed a higher percentage of GnzB-positive cells, such as proliferative CD4-positive and CD8-positive T cells, not only in response to anti-PD1 treatment but also to Il-2-blocking PC61(mIg2a). In mice treated in this way, 7D4(mIg2a) not only did not affect Teff cells in the same way as PC61(mIg2a), but also increased the frequency of Teff cells compared to PC61(mIg2a), suggesting even higher antitumor activity of anti-human CD25 antibodies that do not block IL-2 / CD25 interaction (Figure 4).

[0178] The use of 7D4(mIg2a) and functionally equivalent anti-human CD25 antibodies for cancer immunotherapy, particularly for solid tumors, can be tested not only in the MCA205 mouse model but also in other models such as CT26 and MC38 (colon cancer) or B16 (melanoma) models. Both IgG2a anti-mouse CD25 antibodies show therapeutic activity against engrafted CT26 tumors when administered in combination with an anti-PD1 antibody. Interestingly, when used as monotherapy, the non-IL-2 blocking 7D4(mIg2a) antibody shows significantly higher therapeutic activity than PC61-based antibodies of the same isotype. At the end of the experiment, only mice treated with 7D4(mIg2a) were all 50 mm. 3 The control of tumor growth down to a volume of less than 50 mm was demonstrated, and all mice treated with PC61(mIg2a) reached 50 mm. 3 They did not show smaller tumors, and furthermore, 8 out of 10 mice had tumors of 2000 mm. 3 The endpoint is reached. This is also demonstrated by the difference in survival, with all mice treated with 7D4(mIg2a) still surviving at day 50, compared to only 2 out of 10 mice treated with PC61(mIg2a). In fact, even if the efficacy results of PC61(mIg2a) are significantly improved by the combination with anti-PD1, the efficacy of 7D4(mIg2a) is not further improved, at least when this antibody is used at this concentration.

[0179] Since these 7D4 and PC61-based antibodies exhibit similar ability to deplete Tregs (see Figure 3), such differences in efficacy can be explained, at least in part, by the less significant impact of 7D4(mIg2a) on the interaction between IL-2 and its receptor. This suggests that the lack of IL-2 / IL-2 receptor blocking activity is not only not detrimental to therapeutic activity but may even provide therapeutic benefits. Therefore, this data supports the selection of non-IL-2 / IL-2 receptor blocking CD25-targeted antibodies for use in cancer therapy. These favorable properties of 7D4(mIg2a) antibodies were also confirmed when anti-mouse PD-L1 was used in the same CT26 mouse model (Figure 6) or in the MC38 mouse model with the same antibody combination (Figures 7 and 8).

[0180] These data suggest that, based on 7D4 properties, antibodies with appropriate isotypes, Treg depletion properties, and CD25 binding properties can be combined with other anticancer compounds, such as antibodies targeting immune checkpoint proteins (e.g., against PD-1 and anti-PD-L1) or other cancer-related targets. This approach can be pursued by constructing and administering two products as a novel mixture of monospecific antibodies or a novel bispecific antibody. This approach, involving the construction of bispecific antibodies combining two antigen-binding properties with therapeutically relevant isotypes (e.g., human IgG1), can be validated using Duobody technology, which enables efficient association of single heavy and light chains from two different monospecific antibodies, each separately constructed and possessing a single matching point mutation in the CH3 domain, and allows for Fab exchange in a single heteromer protein (Labrijn AF et al., Nat Protoc. 2014, 9:2450-63). The functional properties of such 7D4-based duobody products (e.g., anti-PD1 or anti-PD-L1) can be evaluated using the cell interaction and depletion models used to validate the 7D4-based antibodies and antibody combinations described above.

[0181] These results also demonstrate that the 7D4 binding properties of mouse CD25 can be utilized in anti-human CD25 antibodies, provided that the isotype is selected to suit this mechanism of action (e.g., human IgG1) without interfering with the interaction of IL-2 with its receptor and IL-2 signaling in CD25-expressing cells. In fact, several other properties can be considered for screening anti-human CD25 antibody candidates that have further improved properties with respect to preparation, use, and / or administration for the treatment of cancer, particularly solid tumors.

[0182] These characteristics all have a nanomolar range Kd relative to human CD25, but can also be defined for known anti-human CD25 features such as Humax-TAC, basiliximab, or daclizumab, which all block the binding of human IL-2 to human CD25 (clone M-A251 is used as a potential reference non-IL-2 blocking anti-human CD25 to be added to the selection of anti-human CD25s of the present invention).

[0183] These characteristics may be one or more of the following: K is lower than 25 nM, preferably less than 10 nM, and more preferably less than 1 nM. D Affinity for recombinant isolated monomer human CD25 (established using techniques such as Octet, Kinexa, ELISA, or other technologies); K is lower than 75 nM, preferably less than 30 nM, and even more preferably less than 3 nM. D Cross-reactivity of recombinant isolated monomer cynomolgous monkey CD25 (established using techniques such as Octet, Kinexa, ELISA, or other methods); K is lower than 100 nM, preferably less than 10 nM, and more preferably less than 1 nM. D Affinity for recombinant human CD25 on the surface of CHO or MJ cells (established using techniques such as flow cytometry, cell-based ELISA, or other techniques); K is lower than 300 nM, preferably less than 30 nM, and even more preferably less than 3 nM.D Affinity for recombinant rhesus monkey CD25 on the surface of CHO cells (established using techniques such as flow cytometry, cell-based ELISA, or other techniques); K is lower than 100 nM, preferably less than 10 nM, and more preferably less than 1 nM. D Human Treg cell binding (established using techniques such as flow cytometry, cell-based ELISA, or other technologies); K is lower than 300 nM, preferably less than 30 nM, and even more preferably less than 3 nM. D Binding of cynomolgus monkey Treg cells (established using techniques such as flow cytometry, cell-based ELISA, or other technologies); Absence of inhibition of the interaction between human recombinant IL-2 and human recombinant CD25 in biochemical assays (less than 25% of IL-2 binding to CD25 is blocked in the screening described in Example 1); Absence of IL-2-induced signaling in cell-based assays such as STAT5 phosphorylation in activated CD8-positive or CD4-positive T cells or CD25-expressing cell lines, or upregulation of granzyme B after activation in CD4-positive T cell assays (inhibition of less than 25% of baseline signaling, as described in Example 1); and / or Evaluation of relevant efficacy in cell-based assays such as ADCC, ADCP, and / or CDC assays in human CD25-expressing cell lines or primary Treg cells (EC50 less than 10 nM, preferably less than 1 nM, and more preferably less than 0.1 nM).

[0184] Example 3 - Further in vivo mouse model experiments using non-IL2 blocking anti-mouse CD25 antibody Materials and methods Therapeutic activity of non-IL-2 blocking antibodies: In female BALB / c mice obtained from Charles River, 3 × 10¹⁶ units in 0% Matrigel were administered. 5CT26 tumor cells were subcutaneously injected into the flank (n=15 per group). Animals were randomized to treatment groups based on body weight on day 1. Treatment was initiated on day 6, with mice treated with a single injection of 200 μg / animal of each antibody (mouse IgG2a isotype, IL-2 neutralizing antibody, PC61 mIgG1, an anti-mouse CD25 that blocks IL-2 signaling of the mouse IgG1 isotype, and 7D4 mIgG2a, an anti-mouse CD25 that does not block IL-2 signaling of the mouse IgG2a isotype). Animals were treated with monotherapy with one antibody per group, or in combination with 7D4 mIgG2a and IL-2 neutralizing antibody, or 7D4 mIgG2a and PC61 mIgG1 antibody. Mice were treated when the tumor volume reached 2000 mm³. 3 They were slaughtered when they reached a certain age or after 50 days, whichever came first.

[0185] Therapeutic activity of non-IL-2 blocking antibodies compared to blocking antibodies 3 x 10 5 Individual CT26 cells were subcutaneously transplanted into the flank. Pair-matching was performed on tumors measuring 30 mm. 3 ~60mm 3 The procedure was initiated on day 0, when the target was reached. Treatment was administered via ip at 10 mg / kg on day 1 and every two weeks thereafter. The group was either treated with IL-2 neutralizing antibody PC61-m2a, non-IL-2 blocking antibody 7D4, or non-IL-2 blocking antibody 2E4, or left untreated.

[0186] Therapeutic activity of non-IL-2 blocking antibodies in combination with aPDL1 therapy As specified in the diagram, 50,000 MCA205 tumor cells were subcutaneously injected into mice in groups of n=10 or n=5. The animals were randomized to a treatment group. The animals were treated with either 7D4 mIgG2a or aPD-L1 (clone 10F.9G2) as monotherapy, in combination with 7D4 mIgG2a and PD-L1 (clone 10F.9G2), or untreated. The groups were given one of the following: a7D4 mIgG2a alone - day 10 (200 μg), aPD-L1 rIgG2b (10F.9G2) - days 6, 9, and 12 (200 μg), aPD-L1 + a7D4 combination (aPD-L1 on days 6, 9, and 12, a7D4 on day 10), or aPD-L1 + a7D4 combination (aPD-L1 on days 6, 9, and 12, a7D4 on day 10) - an additional shot of a7D4 on day 15 + aPD-L1 on day 18 (only 5 mice).

[0187] result The anti-CD25 depleted non-IL-2 blocking antibody 7D4 mIgG2a induced tumor rejection in treated mice, and other antibodies were ineffective as monotherapy compared to isotype control mouse IgG2a. Combination with either PC61 mIgG1 or IL2 nAb IL2 blocking antibodies suppressed the therapeutic activity of the non-IL-2 blocking antibody 7D4 mIgG2a (Figure 13). This demonstrates that the non-IL-2 blocking characteristic of 7D4 mIgG2a is important for its therapeutic activity. It is also suggested that the therapeutic activity of this antibody relies on an anti-tumor immune response mediated by T effector cells that are IL-2 signaling-dependent for optimal activity. These results indicate that the lack of IL-2 / CD25 blocking activity is required for the optimal therapeutic activity of CD25-targeted antibodies, supporting the use of the anti-CD25 non-IL-2 blocking antibodies described herein in cancer therapy.

[0188] These results further demonstrate that the lack of IL-2 / CD25 blocking activity is not detrimental to antibody therapeutic activity, and support the use of the anti-CD25 non-IL-2 blocking antibodies described herein in cancer therapy.

[0189] These results further demonstrate that the non-IL-2 blocking antibodies 7D4 and 2E4 are more potent than the IL-2 blocking antibody PC61. The anti-CD25 non-blocking antibodies 7D4 and 2E4 exhibit potent therapeutic activity against solid tumors (Figure 75).

[0190] The results showed that single or repeated injections of the non-IL2 blocking aCD25 antibody 7D4 after initiation of aPDL1 therapy enhanced the antitumor response. Teff cells activated by aPDL1 treatment were preserved and enhanced by the aCD25 antibody (Figure 76).

[0191] Example 4 - Epitope characterization of anti-CD25 non-IL-2 blocking antibody Epitope Binning Antibody epitope binning was performed using a standard sandwich format binning assay on a Forte Bio Octet Red384 system (Pall Forte Bio Corporation, Menlo Park, CA). Anti-mouse CD25 PC61 antibody was loaded onto an AMC sensor, and unoccupied Fc binding sites on the sensor were blocked with unrelated mouse IgG1 antibody. The sensor was then exposed to a 15 nM target antigen, followed by 7D4 antibody. Data were processed using ForteBio's Data Analysis Software 7.0. Additional binding by a second antibody after antigen association indicates unoccupied epitopes (non-competitive substance), while absence of binding indicates epitope blockade (competitive substance).

[0192] Epitope mapping of anti-CD25 non-IL-2 blocking antibodies A set of peptides exhibiting the human CD25 sequence (Uniprot reference number P01589), including linear, single-loop, β-turn mimics, disulfide-bridged mimics, discontinuous disulfide-bridged mimics, and discontinuous epitope mimics, was synthesized using solid-phase Fmoc synthesis (Pepscan BV, The Netherlands; Timmermann P et al., 2007 J. Mol. Recognit., 20, 283-99, Langedijk JP et al., 2011, Analytical Biochemistry. 417:149-155). Antibody binding to each synthetic peptide was tested using ELISA (Pepscan, The Netherlands). The peptide array was incubated with primary antibody solution (overnight at 4°C). After washing, the peptide array was incubated with a 1000-fold dilution of appropriate antibody peroxidase conjugate (2010-05; Southern Biotech) at 25°C for 1 hour. After washing, the peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 μl / ml of 3% H2O2 were added. Color development was measured after 1 hour. The color development was quantified using a charge-coupled device (CCD) camera and image processing system. The values ​​obtained by the CCD camera were in the range of 0 mAU to 3000 mAU, similar to those obtained with a standard 96-well plate ELISA reader. To validate the quality of the synthesized peptides, separate sets of positive and negative control peptides were synthesized in parallel and screened using unrelated control antibodies.

[0193] result Epitope binning was performed to determine whether the antibody bound to epitopes that overlapped with those of the commercially available mouse anti-human non-IL-2 blocking CD25 antibody, 7G7B6. The antibody was further characterized to determine its epitopes against the non-IL-2 blocking antibody. The epitopes of the anti-mouse CD25 blocking antibody PC61 were determined for comparison. The results of epitope mapping are shown in Table 1 for the anti-human CD25 antibody and in Table 2 for the anti-mouse CD25 antibody.

[0194] [Table 1]

[0195] The amino acid (aa) sequence numbering is based on human CD25 from the sequence published under Uniprot accession number P01589.

[0196] [Table 2]

[0197] The amino acid (aa) sequence numbering is based on mouse CD25 from the sequence published under Uniprot accession number P01590.

[0198] Epitope mapping studies using Pepscan technology show that anti-human antibodies bind to human CD25 at epitopes that do not overlap with the IL-2 binding site on CD25. Anti-human antibodies bind to epitopes different from those of basiliximab and daclizumab. The epitopes for basiliximab and daclizumab contain residues in the region of amino acids 137-143 (SEQ ID NO: 1) that overlap with the interaction site between CD25 and IL-2 (Binder M et al, Cancer Res 2007 vol 54 67(8): 3518-23). ​​Anti-mouse CD25 non-blocking antibodies 2E4 and 7D4 recognize epitopes different from those of PC61.

[0199] Example 5: Characterization of mouse anti-CD25 antibody Antibody binding to mouse CD25-expressing CHO cells. Binding to CD25-expressing CHO cells was tested by staining with test samples (anti-CD25 primary antibodies, 7D1, PC61, and 2E4) at a concentration of 30 mg / ml, followed by a semi-logarithmic dilution series (7 points) on ice for 30 minutes. Subsequently, staining was performed on ice for 30 minutes with a secondary antibody (Alexa Fluor 647-AffiniPure Fab fragment goat anti-human IgG (H+L) (Jackson ImmunoResearch)) at a concentration of 1 mg / ml. All samples were stained in double series. Live cells were gated using FSC vs. SSC parameters by flow cytometry at the time of sample acquisition. The mean fluorescence intensity (MFI) of stained cells was plotted on an XY chart, the MFI was graphed against the logarithm of the concentration, and the data was fitted to a nonlinear regression curve to calculate EC50. The results shown in Figure 11 confirm that anti-mouse CD25 antibodies bind to mouse CD25-expressing CHO cells.

[0200] Affinity measurement of anti-mCD25 antibodies The affinity for the anti-mouse CD25 antibodies 7D4, PC61, and 2E4 was measured using SPR with a CM-5 sensor chip in a Biacore 2000 at an ambient experimental temperature of 25°C, and their K levels were measured accordingly. DThis was determined by measuring the following: Anti-mouse antibody was first immobilized over the entire flow cell in analytical buffer (pH 7.4, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20) to 16,000–18,000 RU over 10 minutes. The ligand (antibody test) was then loaded to a capture level of 119–163 RU. Next, the analyte (his-tagged recombinant mouse CD25) was associated in analytical buffer at a 2-fold dilution starting from 800 nM down to a minimum concentration of 3.13 nM over 6 minutes. Dissociation was performed in analytical buffer over 10 minutes. Regeneration between sample concentrations was performed in 10 mM glycine (pH 1.7) for 10 minutes. A flow rate of 25 μl / min was maintained throughout the process. The kinetic data were fitted by reference subtraction using the global model bivalent analyte analysis software provided by Biacore. SPR-based analysis is shown in Figure 12. The Kd values ​​for the anti-mouse CD25 antibodies established in this assay are as follows: 2.6 × 10 for 7D4. -9 For M and 2E4, the numbers are 114 x 10. -9 For M and PC61, 3.6 × 10 -9 M (Results will not be shown).

[0201] Anti-mouse antibody competition in Octet Antibody competition was performed using a standard sandwich binning assay with a Forte Bio Octet Red96 system (Pall Forte Bio Corp., USA). A 10 nM anti-mouse CD25 antibody was loaded onto an AMC sensor for 900 seconds, and unoccupied Fc binding sites on the sensor were blocked with an unrelated mouse IgG2a antibody. The sensor was exposed to a 15 nM target antigen (his-tagged mouse CD25) for 600 seconds, followed by exposure to a second anti-CD25 antibody (similarly 10 nM). Data were processed using Forte Bio Data Analysis Software 9.0. Additional binding by the second antibody after antigen association indicates an unoccupied epitope, while absence of binding indicates epitope blockade.

[0202] Competitive binding to mCD25 is observed between 7D4 and 2E4 (Figure 13(A)), but not between 7D4 and PC61 (Figure 13(B)).

[0203] In vitro IL-2 signaling by STAT5 phosphorylation assay: Pan T cells were isolated from splenocytes using Invitrogen's Dynabeads® FlowComp® Mouse Pan T (CD90.2) kit (catalog number: 11465D). 200,000 cells were plated and allowed to stand at 37°C for 2 hours. Antibody was added at 50 μg / ml and incubated with the cells at 37°C for 30 minutes, followed by stimulation of the cells with IL2 (50 U / ml) at 37°C for 10 minutes.

[0204] Cells were fixed and permeabilized with eBioscience® Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen), and IL-2-induced STAT5 phosphorylation was stopped by treatment with BD Phosflow Perm Buffer III (BD Biosciences). Next, cells were simultaneously stained with surface and intracellular fluorescent dye-labeled antibodies (STAT5-Alexa Fluor 647 clone 47 / stat5 / pY694 (BD Bioscience), CD3-PerCP-Cy5.5 clone 17A2 (Biolegend), CD4-PE clone RM4-5 (Biolegend), FoxP3-AF488 clone FJK-16s (Ebioscience)). Samples were acquired using a Fortessa LSR X20 flow cytometer (BD Bioscience) and analyzed using BD FACSDIVA software. Doublets were excluded using FCS-H vs. FCS-A, and lymphocytes were defined using SSC-A vs. FCS-A parameters. CD3 + T cells were defined using a plot of CD3 PerCP-Cy5.5-A versus FCS-A, and the gate was plotted on a histogram showing count versus STAT5 Alexa Fluor 647-A.+ CD3 + The T cell population was determined. The IL-2 signaling blockade rate was calculated as follows: Blockade (%) = 100 × [(Stat5 + Cell Ab-free group (%) - Stat5 + cells 50μg / ml Ab group (%)) / (Stat5 + Cell-free Ab group (%)). Different T cell subsets (CD4 + CD8 + CD4 + Further analysis of STAT5 phosphorylation by FoxP3-) was also performed, with each subset evaluated by gating, and analyzed as described above. Graphing and statistical analysis were performed using GraphPad Prism v7 (results are not shown). The results are shown in Figure 14.

[0205] result: The anti-mouse antibodies 7D4 and 2E4 were further evaluated for their ability to bind to CD25 and their ability to avoid interfering with IL-2 signaling in CD25-expressing target cells. While the non-IL-2 blockers 7D4 and 2E4 compete for binding to CD25, PC61 (an IL-2 signaling blocker) does not compete with 2E4 or 7D4 for binding to CD25 (Figure 12).

[0206] The STAT5 assay confirmed that 7D4 and 2E4 did not block IL-2 signaling, while IL-2 signaling was blocked by the "blocking" antibody PC61 (Figure 14).

[0207] Example 6: In vivo depletion of Treg cells 1 × 10⁶ units in 200 μl of RPMI 1640 medium 5 Four T1 cells were transplanted into the second thoracic adipose tissue of Balb / c mice. The tumor was 50 mm. 3 ~100mm 3At this stage, mice were randomized and administered a single, fixed intraperitoneal dose of mouse anti-mouse CD25(7D4) antibody at a dose of either 2 μg, 20 μg, or 200 μg per mouse. Tumor tissue and whole blood were isolated on days 3 and 9 for immunophenotyping.

[0208] result: Based on immunophenotyping analysis on days 3 and 9 after administration, antibody 7D4 showed Treg depletion activity in both whole blood and tumor tissue (Figure 15).

[0209] Example 7: Characterization of anti-CD25 antibody 7G76B Binding of anti-CD25 antibodies to human CD25-expressing cells: 7G76B was evaluated by binding to the lymphoma human cell lines Karpas 299, SU-DHL-1, and SR-786, as well as to in vitro differentiated Treg cells. Binding to CD25-expressing human cell lines (SU-DHL-1 and SR-786) was tested by first blocking the cells with Trustine (Biolegend), then incubating them at 4°C for 30 minutes with an anti-CD25 antibody titrated in a semi-logarithmic dilution series starting from the highest concentration of 20 μg / ml, washing, and incubating with a PE-conjugated anti-human IgG Fc antibody (Biolegend). The cells were washed again, resuspended in FACS buffer containing DAPI, and acquired using Intellicyt iQue. Live cells were gated using FSC vs. SSC parameters by flow cytometry at the time of sample acquisition. The geometric mean intensity of stained cells was plotted on an XY chart, the geometric mean intensity was graphed against the logarithm of the concentration, and the data was fitted to a nonlinear regression curve to calculate the EC50.

[0210] The binding of the test substance (anti-CD25 primary antibody) to CD25-expressing Karpas 299 cells and in vitro differentiated Treg cells was tested by staining with a 30 mg / ml antibody followed by a semi-logarithmic dilution series (7 points) on ice for 30 minutes. Subsequently, the cells were stained with a 1 mg / ml secondary antibody (Alexa Fluor 647-AffiniPure Fab fragment goat anti-human IgG (H+L) (Jackson ImmunoResearch)) on ice for 30 minutes. All samples were stained in double series. Live cells were gated using FSC vs. SSC parameters by flow cytometry at the time of sample acquisition. The mean fluorescence intensity (MFI) of stained cells was plotted on an XY chart, the MFI was graphed against the logarithm of the concentration, and the data was fitted to a nonlinear regression curve to calculate the EC50. The results shown in Figures 16 and 21 confirm that the anti-CD25 antibody binds to CD25-expressing cells.

[0211] In vitro IL-2 signaling by STAT5 phosphorylation assay: IL-2 blockade was characterized using a STAT5 phosphorylation assay, and IL-2 signaling was tested. Pre-frozen PBMCs (Stemcell Technologies) were cultured in a U-bottom 96-well plate for 30 minutes in the presence of 10 μg / ml anti-CD25 antibody. Then, IL-2 (Peprotech) at various concentrations of 0.1 U / ml, 1 U / ml, or 10 U / ml was added for 10 minutes in RPMI 1640 (Life Technologies) containing 10% FBS (Sigma), 2 mM L-glutamine (Life Technologies), and 10000 U / ml Pen-Strep (Sigma). Cells were fixed and permeabilized with eBioscience® Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen), and IL-2-induced STAT5 phosphorylation was stopped by treatment with BD Phosflow Perm Buffer III (BD Biosciences). Next, cells were simultaneously stained with surface and intracellular fluorescent dye-labeled antibodies (STAT5-Alexa Fluor 647 clone 47 / stat5 / pY694 (BD Bioscience), CD3-PerCP-Cy5.5 clone UCHT1 (Biolegend), CD4-BV510 clone SK3 (BD ​​Bioscience), CD8-Alexa Fluor 700 clone RPA-T8 (Invitrogen), CD45RA-PE-Cy7 clone HI100 (Invitrogen), FoxP3-Alexa Fluor 488 clone 236A / E7 (Invitrogen)). Samples were acquired using a Fortessa LSR X20 flow cytometer (BD Bioscience) and analyzed using BD FACSDIVA software. Doublets were excluded using FCS-H vs. FCS-A, and lymphocytes were identified using SSC-A vs. FCS-A parameters. CD3 + T cells were defined using a plot of CD3 PerCP-Cy5.5-A versus FCS-A, and the gate was plotted on a histogram showing count versus STAT5 Alexa Fluor 647-A. + CD3 +The population of T cells was determined. The IL-2 signaling blockade rate was calculated as follows: Blockade (%) = 100×[(Stat5 + cells without Ab group (%) - Stat5 + cells 10 μg / ml Ab group (%)) / (Stat5 + cells without Ab group (%))]. Further analysis of STAT5 phosphorylation by different T cell subsets (CD4 + , CD8 + , CD4 + FoxP3 + , naive and memory T cells) was also evaluated by gating on each subset and analyzed as described above. Graphing and statistical analysis were performed using GraphPad Prism v7 (results not shown). The results are shown in Figures 17 and 22.

[0212] In vitro T cell activation assay: The effect of IL-2 signaling on Teff responses was characterized in a T cell activation assay that tests for upregulation of intracellular granzyme B (GrB) and proliferation. Cryopreserved primary human pan T cells (Stemcell Technologies) were labeled with eFluor450 cell proliferation dye (Invitrogen) according to the manufacturer's recommendations and seeded at 1×10 5Cells were added in wells. Subsequently, cells were treated with 10 μg / ml anti-CD25 antibody or control antibody, followed by Human T-Activator CD3 / CD28 (20:1 cell-to-bead ratio; Gibco), and incubated for 72 hours in a 5% CO2 humidified incubator at 37°C. To assess T cell activation, cells were stained with eBioscience Fixable Viability Dye efluor780 (Invitrogen), followed by fluorescent dye-labeled antibodies against surface T cell markers (CD3-PerCP-Cy5.5 clone UCHT1 (Biolegend), CD4-BV510 clone SK3 (BD ​​Bioscience), CD8-Alexa Fluor 700 clone RPA-T8 (Invitrogen), CD45RA-PE-Cy7 clone HI100 (Invitrogen), CD25-BUV737 clone 2A3 (BD ​​Bioscience)). The cells were then fixed and permeabilized with eBioscience® Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen), and subsequently stained for intracellular GrB and nuclear FoxP3 (granzyme B-PE clone GB11 (BD Bioscience), FoxP3-APC clone 236A / E7). Samples were acquired using a Fortessa LSR X20 flow cytometer (BD Bioscience) and analyzed using BD FACSDIVA software. Doublets were excluded using FCS-H vs. FCS-A, and lymphocytes were identified using SSC-A vs. FCS-A parameters. Live CD3 + CD4 gated from lymphocytes + and CD8 + T cell subsets were evaluated using a plot of GrB-PE-A versus proliferating eFluor450-A. Results were obtained for all CD4 + The percentage of proliferative GrB-positive cells from the T cell population is presented. Graphing and statistical analysis were performed using GraphPad Prism v7. The results are shown in Figure 18.

[0213] In vitro ADCC assay: Antibody-dependent cell-mediated cytotoxicity assays (ADCC assays) were performed using SU-DHL-1 or SR-786 (CD25-positive) human cell lines as target cells to characterize anti-human CD25 antibodies, and human NK cells as the source of effector cells. NK cells were isolated from healthy donor PBMCs using an NK cell-negative isolation kit (Stemcell Technologies). NK cells were cultured overnight in the presence of 2 ng / mL IL-2 (Peprotech). SU-DHL-1 or SR-786 target cells were loaded with calcein AM (Thermofisher) and plated in the presence of anti-CD25 or isotype antibodies in the presence of anti-CD25 or isotype antibodies for 30 minutes at 37°C and 5% CO2 in four replicate test groups per condition. After incubation, NK cells were added to wells in a 1:10 target:effector (T:E) ratio (10,000 target cells and 100,000 effector cells) and incubated at 37°C and 5% CO2 for 4 hours. Calcein fluorescence in the supernatant was read out using a BMG Fluostar plate reader. Specific lysis rates were calculated for target cells alone (0% lysis) and target cells treated with 0.1% saponin (100% lysis). Raw data graphs were created using Graphpad Prism v7 to generate dose-response curves. Target cell lysis rates were plotted on an XY chart, normalized calcein AM release rates were graphed against the logarithm of concentration, and the data were fitted to a nonlinear regression curve to calculate EC50. The results are shown in Figure 19.

[0214] in vitro ADCP assay: An antibody-dependent cell-mediated phagocytosis (ADCP) assay was performed using in vitro differentiated Tregs as target cells and monocyte-derived macrophages as effector cells. PBMCs were isolated from leucocyte cones by Ficoll gradient centrifugation. Monocytes (CD14+ cells) were isolated using CD14 microbeads (Miltenyi Biotec). Monocytes were cultured for 5 days in RPMI 1640 (Life Technologies) containing 10% FBS (Sigma), 2 mM L-glutamine (Life Technologies), and 10,000 U / ml Pen-Strep (Sigma) in the presence of 50 ng / ml M-CSF, and fresh medium containing M-CSF was added after 3 days. Regulatory T cells (Tregs) were isolated using the Human Treg Cell Differentiation Kit (R&D Systems). These cells were incubated for 5 days in a 5% CO2 humidified incubator at 37 °C and labeled with the eFluor450 dye (Invitrogen) according to the manufacturer's recommendations. On day 5, macrophages and eFluor450 dye-labeled Tregs were co-cultured for 4 hours at an effector-to-target ratio of 10:1 in the presence of an anti-CD25 antibody or a control as follows. For the 10:1 effector-to-target ratio, target cells (Tregs) were added at 1 × 10 4 cells / well, and effector cells (macrophages) were added at 1 × 10 5 cells / well. Then, the anti-CD25 antibody was added at a maximum concentration of 1 μg / ml, followed by a two-fold serial dilution (7 points). The cells and antibody were incubated at 37 °C, 5% CO2 for 4 hours. To assess ADCP, the cells were placed on ice, stained with the cell surface marker CD14 (CD14-PerCP-Cy5.5 clone MfP9 (BD Biosciences)), and fixed with eBioscience fixation buffer. Two-color flow cytometry analysis was performed using a Fortessa LSR X20. Residual target cells were defined as cells that were + eFluor450 dye - / CD14 +This was defined as follows. Double-labeled cells (eFluor450 dye+ / CD14+) were considered to represent phagocytosis of targets by macrophages. The phagocytosis of target cells was calculated using the following formula: Phagocytosis (%) = 100 × [(Double-positive rate) / (Double-positive rate + Residual target rate)]. The results are shown in Figure 20.

[0215] statistics: Curve fitting was performed using Prism software (GraphPad) to determine the EC50 value and maximum activity.

[0216] Human antibodies do not block the IL2-CD25 interaction. Interference with IL-2 ligand binding to CD25 was investigated using a standard sandwich binning assay on a Forte Bio Octet Red384 system (Pall Forte Bio Corp., USA). MA251 antibody was loaded onto an AHQ sensor, and unoccupied Fc binding sites on the sensor were blocked with unrelated human IgG1 antibody. The sensor was exposed to 100 nM human CD25, followed by 100 nM human IL-2. Data were processed using Forte Bio Data Analysis Software 7.0. Additional binding by human IL-2 after antigen association indicated an unoccupied epitope (non-competitive substance), while the absence of binding indicated epitope blockade (competitive substance).

[0217] result: The 7G7B6 antibody was further evaluated for its ability to not interfere with IL-2 signaling and to kill CD25-expressing target cells. In the STAT5 assay, 7G7B6 did not block IL-2 signaling regardless of the tested IL-2 concentration, while IL-2 signaling was completely blocked by the reference antibody daclizumab (Figure 17). Daclizumab, which has been shown to block the interaction between CD25 and IL-2 via the so-called "Tac" epitope (Queen C et al, 1989, PNAS. 86(24):10029-10033 and Bielekova B, 2013, Neurotherapeutics, 10(1):55-67), binds to a different epitope than 7G7B6 (Figure 10 and Figure 24B), which can explain why daclizumab blocks IL-2 signaling in the STAT5 phosphorylation assay, while 7G7B6 does not (Figure 17). In addition, daclizumab reduces the effector response of activated T cells, possibly due to the blockade of IL-2 signaling, while 7G7B6, which does not block IL-2 signaling, does not adversely affect the T cell response (Figure 18). Finally, the 7G7B6 chimeric antibody kills CD25-expressing cells, tumor cells, or regulatory T cells by ADCC (Figure 19) and ADCP (Figure 20) compared to the IgG1 isotype antibody.

[0218] In conclusion, characterization of 7G7B6 as a chimeric antibody revealed that it exhibits potent killing of CD25-positive cells (Treg or cancer cell lines) without interfering with IL-2 signaling, and consequently without inhibiting the T effector response. Therefore, 7G7B6 is a Treg-depleting antibody that can be applied to the treatment of cancer, either as monotherapy or in combination.

[0219] The MA-251 antibody was further evaluated for its ability to not interfere with IL-2 signaling. The MA251 antibody was assessed in an IL2-CD25 ocet competitive assay. Simultaneous IL2 binding and MA251 binding to CD25 were observed (Figure 23), indicating that MA251 binds non-competitively. In the STAT5 assay, MA-251 did not block IL-2 signaling regardless of the tested IL-2 concentration, while IL-2 signaling was completely blocked by the reference antibody daclizumab (Figure 22). Daclizumab, which has been shown to block the interaction between CD25 and IL-2 via the so-called "Tac" epitope (Queen C et al, 1989 and Bielekova B, 2013), binds to a different epitope than MA-251 (Figure 10 and Figure 24(E)), which can explain why daclizumab blocks IL-2 signaling in the STAT5 phosphorylation assay, while MA-251 does not (Figure 22).

[0220] Example 8: Anti-human CD25 Ab competitive assay Antibody competition was performed using a standard sequential binding assay on a Forte Bio Octet Red96 system (Pall Forte Bio Corp., USA). 26.8 nM his-tagged recombinant human CD25 was loaded onto a Ni-NTA biosensor for 200 seconds. After a baseline step with kinetic buffer, the sensor was exposed to a 66.6 nM primary antibody for either 600 or 1800 seconds, followed by a secondary anti-CD25 antibody (similarly 66.6 nM for either 600 or 1800 seconds). Data were processed using Forte Bio Data Analysis Software 9.0. Additional binding by the secondary antibody indicated an unoccupied epitope (no competition for the epitope), while the absence of binding indicated epitope blockade (competition for the epitope).

[0221] result Non-blocking agents for IL-2 signaling mAbs (antibody 1 and antibody 3) compete with each other or with 7G7B6 and MA251, but not with research-grade daclizumab or research-grade basiliximab (Examples (A) to (N) in Figure 24). IL-2 signaling blockers (i.e., TSK031) compete with research-grade daclizumab and research-grade basiliximab, but not with 7G7B6 (Examples (O) to (Q) in Figure 24).

[0222] Example 9: Therapeutic analysis of non-blocking antibodies On day 0, 1 × 10⁶ units in 200 μl of RPMI 1640 7 100 SU-DHL-1 cells were transplanted into the right flank. On day 12, mice with palpable tumors were randomized to either vehicle treatment or treatment with antibody 1 at 2 mg / kg twice weekly. On day 15, the tumor size was 100 mm. 3 ~200mm 3 Mice were randomized and administered either a vehicle and either antibody 1 at 2 mg / kg twice weekly or a single dose of antibody 1 at 10 mg / kg.

[0223] result Antibody 1 inhibited tumor growth in 9 out of 10 mice with palpable tumors that were administered 2 mg / kg twice weekly (Figure 25(A) and (B)). Tumor size was 100 mm. 3 ~200mm 3 In mice, antibody 1 inhibited tumor growth at doses of 2 mg / kg twice weekly and a single dose of 10 mg / kg (Figure 25(C)-(E)).

[0224] Example 10: Affinity measurement of anti-human CD25 antibody The affinity for anti-human CD25 antibodies was measured using SPR with a CM-5 sensor chip in a Biacore 2000 at an ambient experimental temperature of 25°C, and their K DThis was determined by measuring the following: Anti-human antibodies were first immobilized over the entire flow cell in analytical buffer (pH 7.4, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20) to 12,000–14,000 RU over 10 minutes. The ligand (antibody test) was then loaded to a capture level of 145–190 RU. Next, the analyte (his-tagged recombinant mouse CD25) was associated in analytical buffer at a 2-fold dilution starting from 400 nM and going down to a minimum concentration of 3.13 nM over 6 minutes. Dissociation was performed in analytical buffer over 10 minutes. Regeneration between sample concentrations was performed in 10 mM glycine (pH 1.7) for 10 minutes. A flow rate of 25 μl / min was maintained throughout the process. For Figures 26(C) and 26(D), the dynamic data was fitted using reference subtraction with the global two-state reaction conformational change analysis software provided by Biacore. The 1:1 Langmuir model obtained by reference subtraction was used in Figures 26(A), 26(B), and 26(E).

[0225] Affinity measurements for ForteBio were generally performed using Octet RED384, as previously described (see, for example, Estep P et al., 2013. Mabs. 5(2), 270-8).

[0226] Alternatively, affinity for anti-human CD25 antibodies can be determined by biolayer interferometry using the Octet Red 96 system (Pall Forte Bio Corp., USA) to obtain their K DThis was determined by measuring [the relevant parameters]. The sensor was equilibrated offline in dynamic buffer for 10 minutes, and then monitored online for 60 seconds to establish a baseline. 13.32 nM antibody was loaded onto the AHC biosensor for 200 seconds, followed by various concentrations of his-tagged rhCD25 (3-fold serial dilutions, 50 nM to 0.54 nM) for 600 seconds, and then dissociated in dynamic buffer for 400 seconds. Dynamic data were fitted by reference subtraction using global 1:1 analysis software provided by Pall Forte Bio. The results are shown in Figure 26(F).

[0227] result: The results are shown in Figure 26. The Kd values ​​for the anti-CD25 antibodies established in this assay are as follows: 3.2 × 10⁻¹⁰ for antibody 1. -9 For M, antibody 3 is 3.8 × 10 -9 M, for daclizumab: 0.61 × 10 -9 M.

[0228] Example 11: Characterization of anti-CD25 antibodies (Antibody 1 to Antibody 21) Binding of anti-CD25 antibodies to CD25-expressing cells: Candidate hits are evaluated by binding to human lymphoma cell lines such as Karpas 299, SU-DHL-1, and SR-786 cells, in vitro differentiated Treg cells, activated human or cyno-PBMCs, HSC-F cynomolgus monkey T cell lines, and CHO cells.

[0229] Binding to CD25-expressing human cell lines (SU-DHL-1 and SR-786) was tested by first blocking the cells with Trustine (Biolegend), then incubating them at 4°C for 30 minutes with an anti-CD25 antibody titrated in a semi-logarithmic dilution series starting from the highest concentration of 20 μg / ml, washing, and incubating with a PE-conjugated anti-human IgG Fc antibody (Biolegend). The cells were washed again, resuspended in FACS buffer containing DAPI, and acquired using Intellicyt iQue. Live cells were gated using FSC vs. SSC parameters by flow cytometry at the time of sample acquisition. The geometric mean intensity of stained cells was plotted on an XY chart, the geometric mean intensity was graphed against the logarithm of concentration, and the data was fitted to a nonlinear regression curve to calculate EC50.

[0230] The binding of CD25 to CD25-expressing Karpas 299 cells and in vitro differentiated Treg cells was tested by staining with a test substance (anti-CD25 primary antibody) at a concentration of 30 mg / ml, followed by a semi-logarithmic dilution series (7 points) on ice for 30 minutes. Subsequently, staining was performed on ice for 30 minutes with a secondary antibody at a concentration of 1 mg / ml (Alexa Fluor 647-AffiniPure Fab fragment goat anti-human IgG (H+L) or Alexa Fluor 647-AffiniPure F(ab')2 fragment rabbit anti-human IgG Fcγ fragment (Jackson ImmunoResearch)). All samples were stained in double series. Live cells were gated using FSC vs. SSC parameters by flow cytometry at the time of sample acquisition. The mean fluorescence intensity (MFI) of stained cells was plotted on an XY chart, the MFI was graphed against the logarithm of the concentration, and the data was fitted to a nonlinear regression curve to calculate EC50.

[0231] The binding of CD25-expressing activated human and cynomolgus monkey PMBCs was tested by staining the test material (anti-CD25 primary antibody) with 20 mg / ml antibody followed by a semi-logarithmic dilution series (7 points) on ice for 30 minutes. Subsequently, staining was performed on ice for 30 minutes with a secondary antibody (rabbit anti-human Fcg F(ab')2 (Jackson ImmunoResearch)) at a concentration of 5 mg / ml. All samples were stained in triplicate. To minimize cross-linking-induced cell death mediated by secondary antibody binding, cell lines were tested simultaneously in four test material staining cohorts. Live lymphocytes were gated using FSC vs. SSC parameters by flow cytometry at the time of sample acquisition. Gated CD4 + and CD8 + The mean fluorescence intensity (MFI) of T cell subsets was plotted on an XY chart, the MFI was graphed against the logarithm of the concentration, and the data was fitted to a nonlinear regression curve to calculate the EC50.

[0232] The binding of CD25-expressing HSC-F cynomolgus monkey T cell lines was tested by staining with a test substance (anti-CD25 primary antibody) at a concentration of 20 mg / ml, followed by a semi-logarithmic dilution series (7 points) on ice for 30 minutes. Subsequently, the cells were stained with a secondary antibody (rabbit anti-human Fcg F(ab')2 (Jackson ImmunoResearch)) at a concentration of 5 mg / ml on ice for 30 minutes. All samples were stained in triplicate. To minimize cross-linking-induced cell death mediated by secondary antibody binding, the cell lines were tested simultaneously in four test substance staining cohorts. Live lymphocytes were gated using FSC vs. SSC parameters by flow cytometry at the time of sample acquisition. The mean fluorescence intensity (MFI) of live cells was plotted on an XY chart, the MFI was graphed against the logarithm of the concentration, and the data was fitted to a nonlinear regression curve to calculate the EC50.

[0233] Binding to CD25-expressing CHO cells was also tested. Approximately 100,000 cells overexpressing the antigen were washed with washing buffer and incubated with 100 μl of 100 nM IgG at room temperature for 15 minutes. The cells were then washed twice with washing buffer and incubated with 100 μl of 1:100 human-PE on ice for 15 minutes. The cells were then washed twice with washing buffer and analyzed using a FACS Canto II analyzer (BD Biosciences). Unmodified CHO cell lines were also used as a negative control.

[0234] In vitro IL-2 signaling by STAT5 phosphorylation assay: IL-2 blockade was characterized using a STAT5 phosphorylation assay, and IL-2 signaling was tested. Pre-frozen PBMCs (Stemcell Technologies) were cultured in a U-bottom 96-well plate for 30 minutes in the presence of 10 μg / ml anti-CD25 antibody. Then, IL-2 (Peprotech) at various concentrations of 10 U / ml, 0.25 U / ml, 0.74 U / ml, 2.22 U / ml, 6.66 U / ml, or 20 U / ml was added for 10 minutes in RPMI 1640 (Life Technologies) containing 10% FBS (Sigma), 2 mM L-glutamine (Life Technologies), and 10000 U / ml Pen-Strep (Sigma). Cells were fixed and permeabilized with eBioscience® Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen), and IL-2-induced STAT5 phosphorylation was stopped by treatment with BD Phosflow Perm Buffer III (BD Biosciences). Next, cells were simultaneously stained with surface and intracellular fluorescent dye-labeled antibodies (STAT5-Alexa Fluor 647 clone 47 / stat5 / pY694 (BD Bioscience), CD3-PerCP-Cy5.5 clone UCHT1 (Biolegend), CD4-BV510 clone SK3 (BD ​​Bioscience), CD8-Alexa Fluor 700 clone RPA-T8 (Invitrogen), CD45RA-PE-Cy7 clone HI100 (Invitrogen), FoxP3-Alexa Fluor 488 clone 236A / E7 (Invitrogen)). Samples were acquired using a Fortessa LSR X20 flow cytometer (BD Bioscience) and analyzed using BD FACSDIVA software. Doublets were excluded using FCS-H vs. FCS-A, and lymphocytes were identified using SSC-A vs. FCS-A parameters. CD3+ T cells were defined using a plot of CD3 PerCP-Cy5.5-A versus FCS-A, and the STAT5+CD3+ T cell population was determined by plotting the gate on a histogram showing count versus STAT5 Alexa Fluor 647-A.The rate of IL-2 signaling blockade was calculated as follows: blockade (%) = 100 × [(Stat5+ cell group without Ab (%) - Stat5+ cell group with 10 μg / ml Ab (%)) / (Stat5+ cell group without Ab (%))]. Further analysis of STAT5 phosphorylation by different T cell subsets (CD4+, CD8+, CD4+FoxP3+, naive, and memory T cells) was also assessed by gating each subset and analyzed as described above. Graphing and statistical analysis were performed using GraphPad Prism v7.

[0235] In vitro T cell activation assay: The effect of IL-2 signaling on the Teff response was characterized in a T cell activation assay that tests the upregulation and proliferation of intracellular granzyme B (GrB). Pre-frozen primary human pan-T cells (Stemcell Technologies) were labeled with eFluor450 cell proliferation dye (Invitrogen) according to the manufacturer's recommendations and placed in a U-bottom 96-well plate containing RPMI 1640 (Life Technologies) with 10% FBS (Sigma), 2 mM L-glutamine (Life Technologies), and 10000 U / ml Pen-Strep (Sigma) at a rate of 1 × 10⁶ cells. 5Cells were added in wells. Subsequently, cells were treated with 10 μg / ml anti-CD25 antibody or control antibody, followed by Human T-Activator CD3 / CD28 (20:1 cell-to-bead ratio; Gibco), and incubated for 72 hours in a 5% CO2 humidified incubator at 37°C. To assess T cell activation, cells were stained with eBioscience Fixable Viability Dye efluor780 (Invitrogen), followed by fluorescent dye-labeled antibodies against surface T cell markers (CD3-PerCP-Cy5.5 clone UCHT1 (Biolegend), CD4-BV510 clone SK3 (BD ​​Bioscience), CD8-Alexa Fluor 700 clone RPA-T8 (Invitrogen), CD45RA-PE-Cy7 clone HI100 (Invitrogen), CD25-BUV737 clone 2A3 (BD ​​Bioscience)). The cells were then fixed and permeabilized with eBioscience® Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen), and subsequently stained for intracellular GrB and nuclear FoxP3 (granzyme B-PE clone GB11 (BD Bioscience), FoxP3-APC clone 236A / E7). Samples were acquired using a Fortessa LSR X20 flow cytometer (BD Bioscience) and analyzed using BD FACSDIVA software. Doublets were excluded using FCS-H vs. FCS-A, and lymphocytes were identified using the SSC-A vs. FCS-A parameter. Gated CD4+ and CD8+ T cell subsets from live CD3+ lymphocytes were assessed using a GrB-PE-A vs. proliferating eFluor450-A plot. Results are presented as the percentage of proliferating GrB-positive cells from the total CD4+ or CD8+ T cell population. Graphing and statistical analysis were performed using GraphPad Prism v7.

[0236] In vitro ADCC assay: Antibody-dependent cell-mediated cytotoxicity assays (ADCC assays) were performed using SU-DHL-1 or SR-786 (CD25-positive) human cell lines as target cells to characterize anti-human CD25 antibodies, and human NK cells as the source of effector cells. NK cells were isolated from healthy donor PBMCs using an NK cell-negative isolation kit (Stemcell Technologies). NK cells were cultured overnight in the presence of 2 ng / mL IL-2 (Peprotech). SU-DHL-1 or SR-786 target cells were loaded with calcein AM (Thermofisher) and plated in the presence of anti-CD25 or isotype antibodies in the presence of anti-CD25 or isotype antibodies for 30 minutes at 37°C and 5% CO2 in four replicate test groups per condition. After incubation, NK cells were added to wells in a 1:10 target:effector (T:E) ratio (10,000 target cells and 100,000 effector cells) and incubated at 37°C and 5% CO2 for 4 hours. Calcein fluorescence in the supernatant was read out using a BMG Fluostar plate reader. Specific lysis rates were calculated for target cells alone (0% lysis) and target cells treated with 0.1% saponin (100% lysis). Raw data graphs were created using Graphpad Prism v7 to generate dose-response curves. Target cell lysis rates were plotted on an XY chart, normalized calcein AM release rates were graphed against the logarithm of concentration, and the data were fitted to a nonlinear regression curve to calculate EC50.

[0237] ADDC was also determined using a luciferase reporter system assay. CD25-expressing SR786 cells, referred to herein as target (T) cells, were incubated at 37°C for 20 minutes in low-IgG FBS medium (4% FBS in RPMI) with different concentrations of mAbs (or control IgG) for CD25. ADCC effector (E) cells were then added to the cell-mAb mixture in a 1:1 E:T ratio. The effector cells were Jurkat cells (Promega) that stably transfect with a luciferase reporter system and overexpress CD16 / FcγRIIIA. After overnight incubation at 37°C, the cells were lysed, and luciferase activity was measured using luminescence emission by hydrolysis of a specific luciferase substrate, according to the manufacturer's instructions (Promega Bio-Glow protocol).

[0238] In vitro ADCP assay using differentiated macrophages and Treg cells: Antibody-dependent cell-mediated phagocytosis (ADCP) assays were performed using in vitro differentiated Treg cells as target cells and monocyte-derived macrophages as effector cells. PBMCs were isolated from leukocyte cones by Ficol gradient centrifugation. Monocytes (CD14+ cells) were isolated using CD14 microbeads (Miltenyi Biotec). Monocytes were cultured for 5 days in RPMI 1640 (Life Technologies) containing 10% FBS (Sigma), 2 mM L-glutamine (Life Technologies), and 10000 U / ml Pen-Strep (Sigma) in the presence of 50 ng / ml M-CSF, and fresh medium containing M-CSF was added after 3 days. Regulatory T cells (Tregs) were isolated using the Human Treg Cell Differentiation Kit (R&D Systems). These cells were incubated in a 37°C 5% CO2 humidified incubator for 5 days and labeled with eFluor450 dye (Invitrogen) according to the manufacturer's recommendations. On day 5, macrophages and eFluor450-labeled Tregs were co-cultured for 4 hours in the presence of anti-CD25 antibody or control at a 10:1 effector-to-target ratio, as described below. For a 10:1 effector-to-target ratio, target cells (Tregs) were cultured at a ratio of 1 × 10⁶. 4 Add cells / well and add effector cells (macrophages) 1 × 10⁶ 5Cells were added per well. Anti-CD25 antibody was then added in a logarithmic series (7 points) at the highest concentration of 1 μg / ml. Cells and antibody were incubated at 37°C in 5% CO2 for 4 hours. To assess ADCP, cells were placed on ice, stained with the cell surface marker CD14 (CD14-PerCP-Cy5.5 clone MfP9 (BD Biosciences)), and fixed with eBioscience fixation buffer. Two-color flow cytometry analysis was performed using a Fortessa LSR X20. Residual target cells were defined as cells labeled eFluor450 dye+ / CD14-. Macrophages were defined as CD14+. Double-labeled cells (eFluor450 dye+ / CD14+) were considered to represent phagocytosis of the target by macrophages. The phagocytic activity of target cells was calculated using the following formula: Phagocytosis (%) = 100 × [(Double Positive Rate) / (Double Positive Rate + Residual Target Rate)].

[0239] In vitro ADCP assay using FcγRIIa-H reporter assay ADCP Bioassay Effector Cells (FcγRIIa-H) were obtained from Promega (catalog number G9881 / 5; lot number 0000261099). 5000 SUDHL-1 target cells per well were plated in a 96-well white polystyrene plate (Costar; catalog number 3917) (25 μl / well). The test antibody was serially diluted using a 3-fold dilution, and 25 μl was added to the cells. 50,000 effector cells were added to each well in a 25 μl volume, resulting in a 10:1 ratio of effector to target cells. All target cells, antibodies, and effector cells were plated in cell culture medium. The plate was incubated at 37°C for 18 hours. The plate was then removed from the incubator and kept at room temperature for 20 minutes. 60 μl of Bio-Glo luciferase assay substrate buffer was added to each well, followed by a 30-minute incubation. Luminescence was then measured using the GloMax Multi Detection System (Promega).

[0240] statistics: Curve fitting was performed using Prism software (GraphPad) to determine the EC50 value and maximum activity.

[0241] result Antibody 1 was evaluated for its ability to not interfere with IL-2 signaling and to kill CD25-expressing target cells. The results of binding to rhCD25 and competitive IL-2 binding analysis are shown in Figures 27 and 28. Ligand binding assays using Octet showed that antibody 1 did not affect IL-2 binding to CD25 (Figure 29). This was confirmed in the STAT5 assay, where antibody 1 did not block IL-2 signaling regardless of the tested IL-2 concentration, and IL-2 signaling was completely blocked by the reference antibody daclizumab (Figure 31). Daclizumab, which has been shown to block the interaction between CD25 and IL-2 via the so-called "Tac" epitope (Queen C et al, 1989 and Bielekova B, 2013), binds to a different epitope than antibody 1 (Figure 30), which can explain why daclizumab blocks IL-2 signaling in the STAT5 phosphorylation assay, while antibody 1 does not (Figure 31). In addition, daclizumab reduces the effector response of activated T cells, possibly due to the blockade of IL-2 signaling, while antibody 1, which does not block IL-2 signaling, does not adversely affect the T cell response (Figure 32). Finally, antibody 1 kills CD25-expressing cells, tumor cells, or regulatory T cells by ADCC (Figure 33) and ADCP (Figure 34) compared to IgG1 isotype antibodies.

[0242] In conclusion, antibody 1 was characterized to exhibit potent killing of CD25-positive cells (Treg or cancer cell lines) without interfering with IL-2 signaling and consequently without inhibiting the T effector response. Therefore, antibody 1 is a Treg-depleting antibody that can be applied to the treatment of cancer as monotherapy or in combination.

[0243] Antibody 3 was evaluated for its ability to not interfere with IL-2 signaling and to kill CD25-expressing target cells. The results of binding to rhCD25 and competitive IL-2 binding analysis are shown in Figures 35 and 36. Ligand binding assays using Octet showed that antibody 3 did not affect IL-2 binding to CD25 (Figure 37). This was confirmed in the STAT5 assay, where antibody 3 did not block IL-2 signaling regardless of the tested IL-2 concentration, while IL-2 signaling was completely blocked by the reference antibody daclizumab (Figure 39). Daclizumab, which has been shown to block the interaction between CD25 and IL-2 via the so-called "Tac" epitope, binds to a different epitope than antibody 3 (Figure 38), which can explain why daclizumab blocked IL-2 signaling in the STAT5 phosphorylation assay while antibody 3 did not (Figure 39). In addition, daclizumab reduces the effector response of activated T cells, likely due to the blockade of IL-2 signaling, while antibody 3, which does not block IL-2 signaling, has minimal, if any, effect on the T cell response compared to conditions without the antibody or with an isotype control (Figure 40). Finally, antibody 3 kills CD25-expressing cells, tumor cells, or regulatory T cells by ADCC (Figure 41) and ADCP (Figure 42) compared to IgG1 isotype antibodies.

[0244] In conclusion, antibody 3 was characterized to exhibit potent killing of CD25-positive cells (Treg or cancer cell lines) without interfering with IL-2 signaling and consequently without inhibiting the T effector response. Therefore, antibody 3 is a Treg-depleting antibody that can be applied to the treatment of cancer as monotherapy or in combination.

[0245] Antibody 4 was evaluated for its ability to not interfere with IL-2 signaling and to kill CD25-expressing target cells. The results of binding to rhCD25 and competitive IL-2 binding analysis are shown in Figures 43 and 44. Ligand binding assays using Octet showed that antibody 4 did not affect IL-2 binding to CD25 (Figure 45). This was confirmed in the STAT5 assay, where antibody 4 did not block IL-2 signaling regardless of the tested IL-2 concentration, and IL-2 signaling was completely blocked by the reference antibody daclizumab (Figure 47). Daclizumab, which has been shown to block the interaction between CD25 and IL-2 via the so-called "Tac" epitope, binds to a different epitope than antibody 4 (Figure 46), which can explain why daclizumab blocked IL-2 signaling in the STAT5 phosphorylation assay while antibody 4 did not (Figure 47). In addition, daclizumab reduces the effector response of activated T cells, possibly due to the blockade of IL-2 signaling, while antibody 4, which does not block IL-2 signaling, does not adversely affect the T cell response (Figure 48). Finally, antibody 4 kills CD25-expressing cells, tumor cells, or regulatory T cells by ADCC (Figure 49) and ADCP (Figure 50) compared to IgG1 isotype antibodies.

[0246] In conclusion, antibody 4 was characterized to exhibit potent killing of CD25-positive cells (Treg or cancer cell lines) without interfering with IL-2 signaling and consequently without inhibiting the T effector response. Therefore, antibody 4 is a Treg-depleting antibody that can be applied to the treatment of cancer as monotherapy or in combination.

[0247] Antibody 2 was evaluated for its ability to not interfere with IL-2 signaling and to kill CD25-expressing target cells. The results of binding to rhCD25 and competitive IL-2 binding analysis are shown in Figures 51 and 52. Ligand binding assays using Octet showed that antibody 2 did not affect IL-2 binding to CD25 (Figure 53). This was confirmed in the STAT5 assay, where antibody 2 did not block IL-2 signaling regardless of the tested IL-2 concentration, while IL-2 signaling was completely blocked by the reference antibody daclizumab (Figure 55). Daclizumab, which has been shown to block the interaction between CD25 and IL-2 via the so-called "Tac" epitope, binds to a different epitope than antibody 2 (Figure 54), which can explain why daclizumab blocked IL-2 signaling in the STAT5 phosphorylation assay while antibody 2 did not (Figure 55). Finally, antibody 2 kills CD25-expressing cells, tumor cells, or regulatory T cells by ADCC (Figure 56) and ADCP (Figure 57) compared to IgG1 isotype antibodies.

[0248] In conclusion, antibody 2 was characterized to exhibit potent killing of CD25-positive cells (Treg or cancer cell lines) without interfering with IL-2 signaling, and consequently without inhibiting the T effector response. Therefore, antibody 2 is a Treg-depleting antibody that can be applied to the treatment of cancer as monotherapy or in combination.

[0249] Antibody 5: The sequence is: EVQLVESGGGLIQPGGSLRLSCAAS GFTLDSYGVS WVRQAPGKGLEWV GVTSSGGSAYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRYVYTGGYLYHYGMDL The heavy chain variable region containing WGQGTLVTVSS (SEQ ID NO: 10) and sequence: DIQMTQSPSSLSASVGDRVTITC RASQSISDYLA WYQQKPGKVPKLLI YAASTLPF GVPSRFSGSGSGTDFLTISSLQPEDVATYYC QGTYDSSDWYWAIt is characterized as containing a variable light chain including FGGGTKVEIK (SEQ ID NO: 14).

[0250] The sequences of the complementarity determination regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs) specified above were defined according to the Kabat numbering scheme.

[0251] Antibody 5 was evaluated for its ability to not interfere with IL-2 signaling and to kill CD25-expressing target cells. The results of binding to rhCD25 are shown in Figure 58. The STAT5 assay showed that antibody 5 did not block the tested IL-2 signaling, while IL-2 signaling was completely blocked by antibody daclizumab (Figure 59). The competition assay showed that antibody 5 did not compete with the IL-2 signaling blockers daclizumab or basiliximab (Figures 60(A) and (B)), but competed with 7G7B6 (a non-IL-2 blocker) (Figure 60(C)). Finally, antibody 5 killed CD25-expressing cells, tumor cells, or regulatory T cells by ADCC (Figure 61) and ADCP (Figure 61) compared to an anti-human CD25 Fc silent control antibody.

[0252] In conclusion, antibody 5 was characterized to exhibit potent killing of CD25-positive cells (Treg or cancer cell lines) without interfering with IL-2 signaling and consequently without inhibiting the T effector response. Therefore, antibody 5 is a Treg-depleting antibody that can be applied to the treatment of cancer as monotherapy or in combination.

[0253] Antibodies 6, 7, 8, and 9 are characterized as containing the following sequences:

[0254] Antibody 6 has the following sequence: EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYGMS WVRQAPGKGLELVS TINGYGDTTYYPDSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYGNSYYYALDY The heavy chain variable region containing WGQGTLVTVSS (SEQ ID NO: 23) and sequence: EIVLTQSPGTLSLSPGERATLSC RASSSVSFMH WLQQKPGQAPRPLI YATSNLAS GIPDRFSGSGSGTDYTLTISRLEPEDFAVYYC QQWSSNPPA It is characterized as containing a variable light chain including FGQGTKLEIK (SEQ ID NO: 25).

[0255] Antibody 7 has the following sequence: EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYGMS WVRQAPGKGLELVS TINGYGDTTYYPDSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYGNSYYYALDY The heavy chain variable region containing WGQGTLVTVSS (SEQ ID NO: 23) and sequence: QIVLTQSPGTLSLSPGERATLSC RASSSVSFMH WLQQKPGQSPRPLI YATSNLAS GIPDRFSGSGSGTDYTLTISRLEPEDFAVYYC QQWSSNPPA It is characterized as containing a variable light chain including FGQGTKLEIK (SEQ ID NO: 26).

[0256] Antibody 8 has the following sequence: EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYGMS WVRQAPGKGLELVS TINGYGDTTYYPDSVK GRFTISRDNAKNTLYLQMNSLRAEDTAVYFCAR DRDYGNSYYYALDY The heavy chain variable region containing WGQGTLVTVSS (SEQ ID NO: 24) and sequence: EIVLTQSPGTLSLSPGERATLSC RASSSVSFMH WLQQKPGQAPRPLI YATSNLAS GIPDRFSGSGSGTDYTLTISRLEPEDFAVYYC QQWSSNPPA It is characterized as containing a variable light chain including FGQGTKLEIK (SEQ ID NO: 25).

[0257] Antibody 9 has the following sequence: EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYGMS WVRQAPGKGLELVS TINGYGDTTYYPDSVK GRFTISRDNAKNTLYLQMNSLRAEDTAVYFCAR DRDYGNSYYYALDY The heavy chain variable region containing WGQGTLVTVSS (SEQ ID NO: 24) and sequence: QIVLTQSPGTLSLSPGERATLSC RASSSVSFMH WLQQKPGQSPRPLI YATSNLAS GIPDRFSGSGSGTDYTLTISRLEPEDFAVYYC QQWSSNPPA It is characterized as containing a variable light chain including FGQGTKLEIK (SEQ ID NO: 26).

[0258] The sequences of the complementarity determination regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs) specified above were defined according to the Kabat numbering scheme.

[0259] Epitope mapping results showed that antibodies 6, 7, 8, and 9 bound to human CD25 in the amino acid regions 150-163 (YQCVQGYRALHRGP) and 166-180 (SVCKMTHGKTRWTQP) of SEQ ID NO: 1, and 10 times the human CD25 extracellular protein sequence. -8 M~10 -10 It was shown that the Kd values ​​within the range of M can be joined.

[0260] Antibodies 6, 7, 8, and 9 were evaluated for their ability to not interfere with IL-2 signaling and to kill CD25-expressing target cells. The results of binding to rhCD25 are shown in Figure 63. The STAT5 assay showed that the antibodies did not block the tested IL-2 signaling, while IL-2 signaling was completely blocked by the antibody daclizumab (Figure 65). The competition assay showed that antibody 7 did not compete with the IL-2 signaling blockers daclizumab or basiliximab (Figures 64(A) and (B)). Finally, antibody 7 killed CD25-expressing cells, tumor cells, or regulatory T cells by ADCC (Figure 66) and ADCP (Figure 67) compared to an anti-human CD25 Fc silent control antibody.

[0261] In conclusion, antibodies 6, 7, 8, and 9 were characterized to exhibit potent killing of CD25-positive cells (Treg or cancer cell lines) without interfering with IL-2 signaling and consequently without inhibiting the T effector response. Therefore, these antibodies are Treg-depleting antibodies that can be applied to the treatment of cancer as monotherapy or in combination.

[0262] Antibodies 10, 11, 12, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 are characterized as containing a heavy chain variable region with the following sequence:

[0263] [Table 3] TIFF0007895705000004.tif90169

[0264] The sequences of the complementarity determination regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs) specified above were defined according to the Kabat numbering scheme.

[0265] Epitope mapping results showed that antibodies 10, 11, 12, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 bound to human CD25 in the amino acid regions 150-163 (YQCVQGYRALHRGP) and 166-180 (SVCKMTHGKTRWTQP) of SEQ ID NO: 1, and 10 of the human CD25 extracellular protein sequence were found to be linked to the human CD25 extracellular protein sequence. -8 M~10 -10 It was shown that the Kd values ​​within the range of M can be joined.

[0266] The antibodies were evaluated for their ability to not interfere with IL-2 signaling and to kill CD25-expressing target cells. The results of binding to rhCD25 are shown in Figure 68. The STAT5 assay showed that the antibodies did not block the tested IL-2 signaling, while IL-2 signaling was completely blocked by the antibody daclizumab (Figure 70). A competition assay showed that antibody 19 did not compete with the IL-2 signaling blockers daclizumab or basiliximab (Figures 69(A) and (B)). Finally, antibodies 12, 19, and 20 killed CD25-expressing cells, tumor cells, or regulatory T cells by ADCC (Figure 71) and ADCP (Figures 72 and 73) compared to an anti-human CD25 Fc silent control antibody.

[0267] In conclusion, antibodies 10, 11, 12, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 were characterized and showed potent killing of CD25-positive cells (Treg or cancer cell lines) without interfering with IL-2 signaling, and consequently without inhibiting the T effector response. Therefore, these antibodies are Treg-depleting antibodies that can be applied to the treatment of cancer as monotherapy or in combination.

[0268] Example 12: Therapeutic analysis in combination with cancer vaccine The therapeutic activity of 7D4 mouse IgG2a, a non-IL-2 blocking anti-CD25 antibody, in combination with GVAX in a B16Bl6 immunotherapy-resistant mouse model was determined. On day 0, 50 × 10⁶ 3 1 x 10¹ B16Bl6 cells were transplanted using id. On day 5, 200 μg of non-IL-2 blocking anti-CD25 antibody was administered via ip or not. On days 6, 9, and 12, mice were immunoadjuvanted with GM-CSF (GVAX). 6 Individual B16Bl6 cells were either treated with irradiation (150 Gy) or left untreated. Tumor growth and mouse survival were monitored until day 33. The results are shown in Figure 74.

[0269] A synergistic effect was observed in the B16Bl6 model with the combination of GVAX and the 7D4 non-blocking anti-CD25 antibody. Therefore, administration of 7D4 in combination with a cancer vaccine enhanced the vaccine-induced antitumor response. These results suggest that non-IL2 blocking anti-CD25 depletion antibodies can be used in combination with cancer vaccines for the treatment of cancer in humans. Furthermore, these data suggest that non-IL2 blocking depletion antibodies can enhance the vaccine-induced immune response and potentially have a broader scope of application than cancer.

[0270] All references cited in the above specification constitute part of this specification by reference. Various modifications and variations of the methods and systems of the present invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in relation to certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the embodiments of the described invention that will be apparent to those skilled in the art in molecular biology, cellular immunology or related fields are intended to be included in the appended claims.

Claims

1. A pharmaceutical composition for treating cancer in a human subject having a solid tumor, comprising a human IgG1 anti-CD25 antibody, wherein the antibody has ADCC activity and does not inhibit the binding of interleukin-2 (IL-2) to CD25 (excluding pharmaceutical compositions in which the human IgG1 anti-CD25 antibody is administered to the subject in a combination of systemic and intracranial administration).

2. The aforementioned anti-CD25 antibody competes with antibody 7G7B6 for binding to human CD25, or The aforementioned anti-CD25 antibody competes with antibody MA251 for binding to human CD25. The pharmaceutical composition according to claim 1.

3. The anti-CD25 antibody specifically binds to the human CD25 epitope, and the epitope, At least one sequence selected from amino acids 150-158 of SEQ ID NO: 1 (YQCVQGYRA), amino acids 176-180 of SEQ ID NO: 1 (RWTQP), amino acids 42-56 of SEQ ID NO: 1 (KEGTMLNCECKRGFR), amino acids 74-84 of SEQ ID NO: 1 (SSWDNQCQCTS), amino acids 166-180 of SEQ ID NO: 1 (SVCKMTHGKTRWTQP), amino acids 70-84 of SEQ ID NO: 1 (NSSHSSWDNQCQCTS), and amino acids 150-158 of SEQ ID NO: 1 (YQCVQGYRA); The sequences of amino acids 42-56 (KEGTMLNCECKRGFR) and 150-160 (YQCVQGYRALH) of SEQ ID NO: 1; The sequences of amino acids 42-56 (KEGTMLNCECKRGFR) and 74-84 (SSWDNQCQCTS) of SEQ ID NO: 1; The sequences of amino acids 150-163 (YQCVQGYRALHRGP), 166-180 (SVCKMTHGKTRWTQP), 42-56 (KEGTMLNCECKRGFR), and 74-84 (SSWDNQCQCTSSATR) of SEQ ID NO: 1; The sequences of amino acids 150-158 (YQCVQGYRA) and 176-180 (RWTQP) of SEQ ID NO: 1; The sequence of amino acids 150-158 (YQCVQGYRA) and amino acids 176-186 (RWTQPQLICTG) of SEQ ID NO: 1; or The sequences of amino acids 150-163 (YQCVQGYRALHRGP) and amino acids 166-180 (SVCKMTHGKTRWTQP) of SEQ ID NO: 1; A pharmaceutical composition according to claim 1 or 2, comprising:

4. The aforementioned anti-CD25 antibody, (a) An IgG1 antibody that binds with high affinity to at least one activated Fcγ receptor selected from FcγRI, FcγRIIc and / or FcγRIIIIa, and depletes tumor infiltration regulatory T cells, (b) Binds to the Fcγ receptor in an activating-to-inhibiting ratio (A / I) greater than 1, or (c) Binds to FcγRI, FcγRIIc and / or FcγRIIIIa with a higher affinity than it binds to FcγRIIb. A pharmaceutical composition according to any one of claims 1 to 3.

5. The aforementioned antibody (a) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 3 and a light chain containing the amino acid sequence of SEQ ID NO: 4, (b) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 5 and a light chain containing the amino acid sequence of SEQ ID NO: 6 (c) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 10 and a light chain containing the amino acid sequence of SEQ ID NO:

14. (d) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 18 and a light chain containing the amino acid sequence of SEQ ID NO: 22, (e) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 23 and a light chain containing the amino acid sequence of SEQ ID NO: 25, (f) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 23 and a light chain containing the amino acid sequence of SEQ ID NO: 26, (g) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 24 and a light chain containing the amino acid sequence of SEQ ID NO:

25. (h) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 24 and a light chain containing the amino acid sequence of SEQ ID NO: 26, (i) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 27 and a light chain containing the amino acid sequence of SEQ ID NO: 30, (j) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 27 and a light chain containing the amino acid sequence of SEQ ID NO: 31, (k) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 27 and a light chain containing the amino acid sequence of SEQ ID NO:

32. (l) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 27 and a light chain containing the amino acid sequence of SEQ ID NO: 33, (m) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 28 and a light chain containing the amino acid sequence of SEQ ID NO:

30. (n) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 28 and a light chain containing the amino acid sequence of SEQ ID NO: 31, (o) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 28 and a light chain containing the amino acid sequence of SEQ ID NO:

32. (p) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 28 and a light chain containing the amino acid sequence of SEQ ID NO:

33. (q) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 29 and a light chain containing the amino acid sequence of SEQ ID NO: 30, (r) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 29 and a light chain containing the amino acid sequence of SEQ ID NO: 31, (s) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 29 and a light chain containing the amino acid sequence of SEQ ID NO: 32, and (t) An antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 29 and a light chain containing the amino acid sequence of SEQ ID NO:

33. A pharmaceutical composition according to any one of claims 1 to 4, selected from the group consisting of the following.

6. The aforementioned anti-CD25 antibody, 10 for CD25 －７ Having a dissociation constant (Kd) less than M, It inhibits IL-2 signaling by less than 50%. It is a monoclonal antibody. Human antibodies, chimeric antibodies, or humanized antibodies Its affinity maturation mutant, and / or Enhancement of CDC and / or ADCP response, preferably an increase in ADCP response, A pharmaceutical composition according to any one of claims 1 to 5.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the anti-CD25 antibody inhibits CD25-mediated IL-2 signaling by less than 50% compared to IL-2 signaling in the absence of the antibody.

8. The pharmaceutical composition according to claim 7, wherein the antibody inhibits IL-2 signaling by about 40%, 35%, or less than 25% compared to IL-2 signaling in the absence of the antibody.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the antibody is a humanized and / or affinity-matured variant of 7G7B6 or MA251.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the anti-CD25 antibody is used for administration to a subject having a colonized tumor.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the antibody is used for administration in combination with a further therapeutic agent.

12. The aforementioned further therapeutic agent: Immune checkpoint inhibitors; or Cancer vaccine; The pharmaceutical composition according to claim 11.

13. Use of an anti-CD25 antibody as defined in any one of claims 1 to 9 for the manufacture of a drug for the treatment of cancer in a human subject, wherein the subject has a solid tumor.

14. A combination drug for the treatment of cancer in a human subject, comprising an anti-CD25 antibody as described in any one of claims 1 to 9, wherein the subject has a solid tumor, and the combination drug is administered simultaneously, separately, or sequentially with a further therapeutic agent.

15. The aforementioned further therapeutic agent: Immune checkpoint inhibitors; or Cancer vaccine; The combination drug according to claim 14.

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