HK40016585B - Fc-optimized anti-cd25 for tumor specific cell depletion - Google Patents

Description

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

Technical Field

This invention belongs to the field of cancer immunotherapy and relates to methods for treating cancer, including methods for treating solid tumors, wherein the methods involve the use of anti-CD25 antibodies.

Background Technology

Cancer immunotherapy involves using the subject's own immune system to treat or prevent cancer. Immunotherapy takes advantage of the fact that cancer cells often have slightly 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. Thus, immunotherapy involves stimulating the immune system to attack tumor cells through these target antigens. However, malignancies, especially solid tumors or hematologic cancers, can evade immune surveillance through a variety of mechanisms inherent to tumor cells and mediated by components of the tumor microenvironment. In the latter, tumor infiltration through an unfavorable balance between regulatory T cells (Treg cells or Tregs), and more specifically effector T cells (Teff) versus Tregs (i.e., a low ratio of Teff to Tregs), has been proposed as a key factor (Smyth Me et al., 2014, Immunol Cell Biol. 92, 473-4).

Since their discovery, Tregs have been found to play a crucial role in mediating immune homeostasis and promoting the establishment and maintenance of peripheral tolerance. However, their role becomes more complex in the context of cancer. The presence of Tregs, which attempt to suppress effector cell responses, can promote tumor progression because cancer cells express both autoantigens and tumor-associated antigens. Therefore, Treg infiltration in established tumors often represents one of the major obstacles to effective antitumor responses and cancer therapy. The inhibitory mechanisms employed by Tregs are thought to have significantly contributed to the limitations or even failures of current therapies, particularly immunotherapies that rely on the induction or enhancement of antitumor responses (Onishi He et al., 2012 Anticanc. Res. 32, 997-1003).

The use of Tregs as a treatment for cancer is supported by studies demonstrating the contribution of Tregs to tumor formation and progression in mouse models. Furthermore, tumor invasion via Tregs has also been associated with poorer prognosis in several human cancers (Shang Be et al., 2015, Sci Rep. 5:15179). Treg cells have been shown to contribute to tumor formation and progression in mouse models, and their absence leads to delayed tumor progression (Elpek et al., 2007 J Immunol. 178(11):6840-8; Golgher et al., 2002; Eur J Immunol. 32(11):3267-75, Jones et al., 2002 Cancer Immun. 22; 2:1; Onizuka et al., 1999 Cancer Res. 59(13):3128-33.; Shimizu et al., 1999, J Immunol. 163(10):5211-8). In humans, high tumor infiltration by Treg cells (more importantly, a low proportion of effector T (Teff) cells versus Treg cells) is associated with poor outcomes in a variety of human cancers (Shang et al., 2015). Conversely, a high Teff/Treg cell ratio is associated with favorable responses to immunotherapy in humans and mice (Hodiet et al., 2008, Proc. Natl. Acad. Sci. USA, 105, 3005-3010; Quezada et al., 2006, J Clin Invest. 116(7): 1935-45). However, Treg depletion in tumors is complex, and findings in this area vary.

CD25 is one of the potential molecular targets for Treg depletion. CD25, also known as the interleukin-2 high-affinity receptor α chain (IL-2Ra), is constitutively expressed at high levels on Treg cells and is absent or expressed at low levels on T effector cells, thus making it a promising target for Treg depletion. IL-2/CD25 interaction has been the focus of several mouse model studies, most of which involve the use of the rat anti-mouse CD25 mouse antibody PC61 (Setiady Y et al., 2010. Eur J Immunol. 40:780-6). The CD25 binding and functional activities of this antibody have been compared with those of a group of monoclonal antibodies produced by different authors (Lowenthal J.W et al., 1985. J. Immunol., 135, 3988-3994; Moreau, J.-L et al., 1987. Eur. J. Immunol. 17, 929-935; Volk HD et al., 1989. Clin. exp. Immunol. 76, 121-5; Dantal J et al., 1991, Transplantation 52:110–5). While initial studies confirmed the prophylactic rather than therapeutic activity of PC61, a recent study showed that this Fc-optimized version of the anti-CD25 antibody leads to the depletion of intratumoral Tregs and provides significant therapeutic benefits in several mouse tumor models (Vargas A et al., 2017, Immunity 48(6), 577-586). Available anti-CD25 antibodies (such as PC61) block or inhibit the binding of IL-2 to CD25, as do many other anti-mouse CD25 antibodies, and most are disclosed as anti-human CD25 antibodies; see, for example, WO2004/045512, WO 2006/108670, WO1993/011238, WO1990/007861 and WO2017/174331. For example, balithimab and dalithimab are anti-human CD25 antibodies that inhibit the binding of IL-2 to CD25 and have been developed to reduce the activation of T effector cells. Baliximab is a chimeric mouse-human CD25 antibody currently approved for graft-versus-host disease, while dalizumab is a humanized CD25 antibody approved for the treatment of multiple sclerosis. However, other anti-CD25 antibodies still allow IL-2 to bind to CD25, such as clone 7D4 (anti-mouse CD25), clone MA251 (anti-human CD25), or 7G7B6 (anti-human CD25) (Rubin et al., 1985, Hybridoma 4(2)91-102, Tanaka et al., 1986, Microbiol. Immunol 30(4), 373-388). 7G7B6 has been used as an investigational antibody and has been suggested as a target for radionuclides targeting CD25-expressing lymphomas (Zhang et al., 2009, Cancer Biother Radiopharm 24(3), 303-309).

For example, 7D4 is a rat IgM anti-mouse CD25 antibody. In the presence of PC61, or after treatment with PC61, or in the presence of antibodies with similar binding properties, 7D4 has been widely used to detect CD25 positive cells (Onizuka Se et al., 1999. Canc Res. 59, 3128–3133). Very few studies have disclosed any functional properties of 7D4-IgM antibody alone or compared to PC61 (Kohm A et al., 2006, J Immunol. 176: 3301-5; Hallett We et al., 2008. Biol Blood Marrow Transplant 14: 1088-1099; Fecci P et al., 2006 Clin Cancer Res. 12: 4294-4305; McNeill A et al., 2007. Scand J Immunol 65: 63–9; Setiady Y et al., 2010. Eur. J Immunol. 40: 780–6; Couper K et al., 2007. J Immunol. 178: 4136–4146). In fact, existing technologies do not offer the possibility of teaching adaptation or modifying the isotype or other structural features of 7D4 in some way to obtain improved antibodies for cancer treatment.

However, the ability of 7D4-IgM antibodies (alone or as engineered antibodies) or any anti-human CD25 antibody (e.g., 7G7B6 or M-A251) designed or characterized to optimally deplete intratumoral Treg cells, either alone or in combination with other antibodies or other anticancer compounds, has been evaluated in detail. As discussed above, the infiltration of Treg cells into tumors, particularly a low proportion of Teff cells compared to Treg cells, can lead to poor clinical outcomes. CD25 has been identified as a Treg marker and is therefore a potential target of interest for therapeutic antibodies designed to deplete Treg cells. Importantly, CD25 is the α subunit of the IL-2 receptor, and IL-2 is a key cytokine in the Teff response. Anti-CD25 antibodies that have been clinically tested to date have blocked IL-2 signaling via CD25 while simultaneously depleting Treg cells. The inventors have now discovered that this blockade of IL-2 signaling restricts the Teff response, and that anti-CD25 antibodies that do not block IL-2 signaling can effectively deplete Treg cells while still allowing IL-2 to stimulate Teff cells, thereby providing antibodies that exhibit strong anti-cancer activity. Therefore, there is a need in the art for a method of treating cancer involving the depletion of Treg cells, particularly a method of treating cancer while still allowing IL-2 to stimulate Teff cells, especially a method of treating cancer using a suitable anti-CD25 antibody.

Summary of the Invention

This invention provides anti-CD25 antibodies and their uses, said anti-CD25 antibodies being characterized by structural elements that allow binding to CD25 without substantially blocking the binding of interleukin-2 (IL-2) to CD25 or the signaling of IL-2 through CD25, and allow for efficient consumption of Tregs, particularly Tregs within tumors. The structural and functional characteristics of 7D4-IgM (as described relative to mouse CD25) have been modified to provide antibodies exhibiting surprisingly improved characteristics in Treg consumption and antitumor efficacy when used alone or in combination with other anticancer drugs. Further structural and functional characteristics of anti-CD25 antibodies that do not block the binding of interleukin-2 to CD25 (and do not block IL-2 signaling through CD25) and efficiently consume Tregs have also been characterized. These findings can be used to define and generate additional anti-human CD25 antibodies that provide comparable antitumor activity in human subjects. Unless the context otherwise requires, references to “anti-CD25 antibody” and the like in this article include its antigen-binding fragment, as well as variants (including affinity-matured variants).

In a key aspect, the present invention provides a method for treating a human subject suffering from cancer, the method comprising the step of administering an anti-CD25 antibody to the subject, wherein the subject suffers from a tumor (preferably a solid tumor), and wherein the antibody does not inhibit the binding of interleukin-2 (IL-2) to CD25.

The terms "non-blocking," "non-blocking," "non-IL-2 blocking," "unblocking," and similar terms used herein (regarding non-blocking of IL-2 binding to CD25 in the presence of anti-CD25 antibody) include embodiments in which the anti-CD25 antibody does not block IL-2 signaling via CD25. That is, compared to IL-2 signaling in the absence of antibody, the anti-CD25 antibody of the present invention inhibits less than 50% of IL-2 signaling via CD25. Preferably, compared to IL-2 signaling in the absence of antibody, the anti-CD25 antibody inhibits less than about 40%, 35%, 30%, and more preferably less than about 25% of IL-2 signaling.

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

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.

In one embodiment, the anti-CD25 antibody specifically binds to an epitope of human CD25, wherein the epitope comprises one or more amino acid residues selected from one or more amino acid segments of SEQ ID NO:1 (YQCVQGYRALHRGP), SEQ ID NO:1 (SVCKMTHGKTRWTQPQLICTG), SEQ ID NO:1 (KEGTMLNCECKRGFR), and SEQ ID NO:1 (NSSHSSWDNQCQCTSSATR). Preferably, the epitope comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 or more amino acid residues selected from one or more amino acid segments selected from the 150th to 163rd amino acid segment of SEQ ID NO: 1 (YQCVQGYRALHRGP), the 166th to 186th amino acid segment of SEQ ID NO: 1 (SVCKMTHGKTRWTQPQLICTG), the 42nd to 56th amino acid segment of SEQ ID NO: 1 (KEGTMLNCECKRGFR), and/or the 70th to 88th amino acid segment of SEQ ID NO: 1 (NSSHSSWDNQCQCTSSATR).

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

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

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

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

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

In one embodiment, the anti-CD25 antibody specifically binds to the epitopes of the sequence comprising amino acids 150 to 158 (YQCVQGYRA) and 176 to 180 (RWTQP) of SEQ ID NO:1. In another embodiment, the anti-CD25 antibody specifically binds to the epitopes of the sequence comprising amino acids 150 to 158 (YQCVQGYRA) and 176 to 186 (RWTQPQLICTG) of SEQ ID NO:1. In yet another embodiment, the anti-CD25 antibody specifically binds to the epitopes of the sequence comprising amino acids 150 to 163 (YQCVQGYRALHRGP) and 166 to 180 (SVCKMTHGKTRWTQP) of SEQ ID NO:1.

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

The inventors have surprisingly discovered that antibodies binding to specific epitopes of CD25 (including antibodies that compete with 7G7B6 and/or MA251 for CD25 binding) can be used to treat cancer, particularly solid tumors. These antibodies still allow IL-2 signaling via antibody-bound CD25, and the inventors have, for the first time, discovered that, in addition to depleting Treg cells, the antibodies used in this invention allow Teff cells to optimally exert their anti-cancer effects, at least in part by allowing IL-2 to bind to and signal via CD25 expressed on Teff cells.

These antibodies preferably have a dissociation constant (K_{_d} ) for CD25 less than 10 ^{^-7} M and/or a dissociation constant for at least one activating Fcγ receptor less than about 10^{^-6} M. Preferably, the dissociation constant (K_{_d}) for CD25 is in or below the range of 10 ^{^-8} , 10^{^-9}, 10^{^-10} , 10 ^{^-11} , 10^-12, or 10 ^{^-13}. Most preferably, the antibody is a human IgG1 antibody that binds to at least one activating Fcγ receptor with high affinity and depletes tumor-infiltrating regulatory T cells. Most preferably, the anti-CD25 antibody is characterized by other Fcγ receptor-related features, particularly:

(a) Binding to Fcγ receptors with an activation inhibition rate (A/I) greater than 1; and/or

(b) Binds FcγRIIa with a higher affinity than binding FcγRIIb.

Considering the use of anti-CD25 antibodies in treatment methods, these anti-CD25 antibodies may exhibit further preferred characteristics. Anti-CD25 antibodies are preferably monoclonal antibodies, particularly human, chimeric, or humanized antibodies. The antibody may be an affinity-matured variant, optionally a humanized or affinity-matured variant of 7G7B6 or MA251. Furthermore, given that anti-CD25 antibodies interact with immune cells and/or other components of the immune system to exert their activity, compared to existing anti-human CD25 clinical antibodies dalizumab and baliximab, anti-CD25 antibodies may further elicit enhanced CDC, ADCC, and/or ADCP responses, preferably eliciting increased ADCC and/or ADCP responses, more preferably eliciting increased ADCC responses. In some embodiments, compared to existing anti-human CD25 clinical antibodies dalizumab and baliximab, anti-CD25 antibodies may induce a reduced CDC response, more preferably, anti-CD25 antibodies do not elicit a CDC response.

The anti-CD25 antibody of the present invention (as generally defined above and further detailed in specific embodiments) can be used in methods of administering the anti-CD25 antibody to a subject in the treatment of a human subject. In one embodiment, the subject has cancer. Preferably, the subject has a defined solid tumor (preferably in methods that further include the step of identifying a subject with a solid tumor). Such methods may further include administering an additional therapeutic agent to the subject. In one embodiment, the additional agent may be an immune checkpoint inhibitor against the subject, for example in the form of an antibody that binds to and inhibits immune checkpoint proteins. 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 methods of depleting regulatory T cells in a subject's solid tumor, the method including the step of administering the anti-CD25 antibody to the subject.

In another aspect, the anti-CD25 antibody of the present invention can be used to prepare a medicament for treating cancer in human subjects, preferably those suffering from tumors, preferably solid tumors. The antibody can be administered in combination with other therapeutic agents, preferably other cancer therapeutic agents, such as immune checkpoint inhibitors (preferably PD-1/PD-L1 pathway antagonists), cancer vaccines, and/or standard care therapies (e.g., chemotherapy or radiotherapy).

In another aspect, the present invention provides a combination of an anti-CD25 antibody as defined above and an additional anticancer compound (preferably an immune checkpoint inhibitor or other compound as shown in the specific embodiments) for treating cancer in human subjects, preferably wherein the subjects have solid tumors, and the anticancer compound (e.g., an immune checkpoint inhibitor (e.g., a PD-1 antagonist) or cytokine (e.g., interleukin-2)) can be administered simultaneously, separately, or sequentially. Within this scope, the present invention also provides a kit for treating cancer comprising an anti-CD25 antibody as defined above and an anticancer compound (e.g., an immune checkpoint inhibitor (e.g., a PD-1 antagonist)).

In another aspect, the present invention also provides pharmaceutical compositions comprising an anti-CD25 antibody as defined above in a pharmaceutically acceptable medium. Such compositions may also contain anticancer compounds (e.g., immune checkpoint inhibitors (e.g., PD-1 antagonists)).

In another aspect, the present invention also provides a bispecific antibody, said bispecific antibody comprising:

(a) The first antigen-binding moiety that binds to CD25; and

(b) The second antigen-binding portion that binds to another antigen;

The anti-CD25 antibody does not inhibit the binding of interleukin-2 (IL-2) to CD25, and preferably, the bispecific antibody binds with high affinity to at least one activating Fcγ receptor and depletes tumor-infiltrating regulatory T cells. Preferably, this second antigen-binding moiety binds to an antigen selected from immune checkpoint proteins or tumor-associated antigens, or may be based on anti-human activated Fc receptor antibodies (anti-FcgRI, anti-FcgRIIa, anti-FcgRIII) or antagonistic anti-human FcγRIIB antibodies. Therefore, the second antigen-binding moiety can bind to FcRIIb. It can optionally bind to FcgRI, FcgRIIa, and/or FcgRIII, which have antagonistic activity.

Preferably, this bispecific antibody comprises a second antigen-binding moiety that binds to an immune checkpoint protein selected from 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. This immune checkpoint protein is preferably expressed on tumor cells. Preferably, the immune checkpoint protein is selected from PD-1, PD-L1, and CTLA-4. The second antigen-binding moiety that binds to the immune checkpoint protein can be included in a commercially available antibody that is an immune checkpoint inhibitor, 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, anti-CTLA-4 is ipilimumab.

This bispecific antibody can be provided in any commercially available form, including Duobody, BiTE DART, CrossMab, Knobs-in-holes, Triomab, or other suitable bispecific antibodies and their fragments in molecular form.

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

The combination of the antiCD25 antibody of the present invention with another anticancer compound and a bispecific antibody as defined above can be used in a method of treating cancer, the method comprising the step of administering the combination or the bispecific antibody to a subject, particularly when the subject has a solid tumor, and for treating the subject's cancer.

Further objectives of the invention are provided in the specific embodiments and examples, including a further definition of the anti-human CD25 antibody of the invention and its use in methods of treating cancer, in pharmaceutical compositions, in combination with other anticancer compounds, and in bispecific antibodies.

Detailed Implementation

This invention provides a method (preferably when the subject has a solid tumor) for treating or preventing cancer in a subject, the method comprising administering an antibody binding to CD25 to the subject, wherein the anti-CD25 antibody is characterized by a structural element that allows binding to CD25 without interfering with interleukin 2 binding or signaling via CD25 and effectively depletes Tregs, particularly effectively depleting Tregs within the tumor. Antibodies binding to CD25 as defined in this invention can be used to treat or prevent cancer, preferably solid tumors. Alternatively, this invention provides the use of antibodies binding to CD25 and allowing binding to CD25 without interfering with the binding of interleukin 2 to CD25 and effectively depleting Tregs for the preparation of medicaments for treating or preventing cancer (preferably solid tumors). This invention also provides the use of antibodies binding to CD25 and allowing binding to CD25 without substantially interfering with the binding of interleukin 2 to CD25 and depleting Tregs in the treatment or prevention of cancer (preferably solid tumors).

The inventors have discovered that anti-CD25 antibodies targeting CD25 can be used, wherein the anti-CD25 antibodies do not inhibit (or substantially do not inhibit) the binding of interleukin-2 to CD25 or IL-2 signaling via CD25 to deplete regulatory T cells in a therapeutic setting (e.g., in established solid tumors). The inventors have also discovered that isotype-specific, non-IL-2-blocking anti-CD25 antibodies that enhance their binding to activating Fcγ receptors result in the efficient depletion of tumor-infiltrating regulatory T cells while still allowing for optimal Teff responses, i.e., a therapeutic approach that can be associated (in combination with or within bispecific antibodies) with other cancer-targeting compounds (e.g., those targeting immune checkpoint proteins, tumor-associated antigens, or inhibitory Fcγ receptors). These findings also enable the use of anti-CD25 antibodies in combination with interleukin-2 at appropriate doses for the treatment of cancer.

CD25 is the α chain of the IL-2 receptor and is present on activated T cells, regulatory T cells, activated B cells, some NK T cells, some thymocytes, myeloid cell precursors, and oligodendrocytes. CD25 binds to CD122 and CD132 to form a heterotrimeric complex, which acts as a high-affinity receptor for IL-2. The common sequence of human CD25 is shown below in SEQ ID NO:1 (Uniprot accession number P01589; the extracellular domain of mature human CD25 corresponding to amino acids 22 to 240 is underlined and indicated as SEQ ID NO:2):

As used herein, "antibody that binds to CD25" refers to an antibody that can bind to the CD25 subunit of the IL-2 receptor. This subunit is also known as the α subunit of the IL-2 receptor. Such antibodies are also referred to herein as "anti-CD25 antibodies".

Anti-CD25 antibodies are antibodies that specifically bind to the CD25 subunit (antigen) of the IL-2 receptor. "Specific binding" should be understood as meaning that the dissociation constant of the antibody with respect to the antigen of interest is less than about ^10⁻⁶ M, ^10⁻⁷ M, ^10⁻⁸ M, ^10⁻⁹ M, ^10⁻¹⁰ M, ^10⁻¹¹ M, ^10⁻¹² M, or ^10⁻¹³ M. In a preferred embodiment, the dissociation constant is less than ^10⁻⁸ M, for example, in the range of ^10⁻⁹ M, ^10⁻¹⁰ M, ^10⁻¹¹ M, ^10⁻¹² M, or ^10⁻¹³ M.

As used herein, the term "antibody" refers to the complete immunoglobulin molecule and the fragment containing the antigen-binding site, and includes polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugates, and/or multispecific antibodies (e.g., bispecific antibodies, biantibodies, triantibodies, and tetraantibodies) and antigen-binding fragments of antibodies, including, for example, Fab', F(ab') ₂ , Fab, Fv, rlgG, peptide-Fc fusions, single-chain variants (scFv fragments, VHH, shark singledomain antibodies, single-chain or tandem biantibody VHH, microantibodies, bicyclic peptides, and other alternative immunoglobulin scaffolds). In some embodiments, the antibody may lack the covalent modifications (e.g., glycan linkages) that would be present if the antibody were naturally occurring. In some embodiments, the antibody may contain covalent modifications (e.g., glycan linkages, detectable portions, therapeutic portions, catalytic portions, or other chemical groups that provide improved stability or antibody delivery, such as polyethylene glycol). In some embodiments, antibodies may be in the form of masked antibodies (e.g., ). Masked antibodies may comprise blocking or “masking” peptides that specifically bind to the antigen-binding surface of the antibody and interfere with antigen binding. The masking peptide is linked to the antibody via a cleavable linker (e.g., via a protease). Selective cleavage of the linker in the desired environment (i.e., in a tumor environment) allows the masking/blocking peptide to dissociate, enabling antigen binding to occur in the tumor, thereby limiting potential toxicity issues. “Antibody” may also refer to camel antibodies (heavy chain antibodies only) and antibody-like molecules (e.g., anticalciins (Skerra (2008) FEBS J 275, 2677-83)). In some embodiments, antibodies are polyclonal or oligoclonal, produced as a group of antibodies, each associated with a single antibody sequence and binding to more or fewer different epitopes within the antigen (e.g., different epitopes within the extracellular domain of human CD25 associated with different reference anti-human CD25 antibodies). As described in the literature (Kearns JD et al., 2015. Mol Cancer Ther. 14: 1625-36), polyclonal or oligoclonal antibodies can be provided in a single formulation for medical use.

In one aspect of the invention, the antibody is monoclonal. The antibody may additionally or alternatively be humanized or human. In another aspect, the antibody is human, or in any case, an antibody having a form and characteristics that allow its use and administration to human subjects. In one aspect of the invention, the antibody may be an affinity-matured humanized variant of 7G7B6 or MA251. The affinity-matured antibody has at least 10% affinity for CD25 and/or its CDR sequence is at least 80% identical, preferably 90%, to the CDR of the parental sequence (across all sequences). The affinity-matured antibody is an antibody having one or more modified amino acids in one or more CDRs, resulting in improved affinity for CD25 compared to a parental strain without the modified amino acids.

Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins with the same structural characteristics. Immunoglobulins can originate from any class, such as IgA, IgD, IgG, IgE, or IgM. Immunoglobulins can be any subclass such as _IgG1 , _IgG2 , _IgG3 , or _IgG4 . In a preferred aspect of the invention, the anti-CD25 antibody is derived from the IgG class, preferably the IgG1 subclass. In one aspect, the anti-CD25 antibody is derived from the human IgG1 subclass. Alternatively, in one aspect, the anti-CD25 antibody is derived from the human IgG2 subclass.

The Fc region of IgG antibodies interacts with several cellular Fcγ receptors (FcγRs) to stimulate and regulate 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 controlled and mediated by FcγRs, which transmit information sensed and collected by the antibody to the immune system, thus providing a link between the innate and adaptive immune systems, especially in the context of biotherapy (Hayes J et al., 2016. J Inflamm Res 9:209–219).

IgG subclasses exhibit varying abilities to bind to FcγR, and this differential binding determines their capacity to elicit a range of functional responses. For example, in humans, FcγRIIIa is the primary receptor involved in antibody-dependent cell-mediated cytotoxicity (ADCC) activation, with IgG1 following IgG3 exhibiting the highest affinity for this receptor, reflecting their ability to effectively induce ADCC. Although IgG2 has been shown to bind weakly to this receptor, anti-CD25 antibodies containing the human IgG2 isotype have been found to effectively deplete Tregs.

In a preferred embodiment of the invention, the antibody binds to FcγR with high affinity, preferably to the activated receptor. More preferably, the antibody binds to FcγRI and/or FcγRIIA and/or FcγRIIIA with high affinity. In a specific embodiment, the antibody binds to at least one activated Fcγ receptor with a dissociation constant less than about ^10⁻⁶ M, ^10⁻⁷ M, ^10⁻⁸ M, ^10⁻⁹ M, or ^10⁻¹⁰ M.

In one aspect, the antibody is an IgG1 antibody, preferably a human IgG1 antibody, capable of binding to at least one Fc activating receptor. For example, the antibody may bind to one or more receptors selected from FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb. In one aspect, the antibody may bind to FcγRIIIA. In another aspect, the antibody may bind to FcγRIIIA and FcγRIIA and optionally FcγRI. In another aspect, the antibody may bind to these receptors with high affinity, for example, with a dissociation constant less than about ^10⁻⁷ M, ^10⁻⁸ M, ^10⁻⁹ M, or ^10⁻¹⁰ M.

In one respect, the antibody binds to the inhibitory receptor FcγRIIb with low affinity. In another respect, the antibody binds to FcγRIIb with dissociation constants higher than about ^10⁻⁷ M, higher than about ^10⁻⁶ M, or higher than about ^10⁻⁵ M.

In a preferred embodiment of the invention, the anti-CD25 antibody is derived from the IgG1 subclass and preferably possesses ADCC and/or ADCP activity, as discussed herein, particularly for human cells. As previously described (Nimmerjahn F et al., 2005. Science, 310:1510–2), the mIgG2a isotype (which corresponds to the human IgG1 isotype) binds to all FcγR isotypes with a high activation inhibition rate (A/I) (at least greater than 1). In contrast, other isotypes (e.g., the rIgG1 isotype) bind only to a single activating FcγR (FcγRIII) and the inhibitory FcγRIIb with similar affinity, resulting in a low A/I ratio (<1). This lower A/I ratio can be associated with lower intratumoral Treg consumption and lower isotype-based antitumor therapeutic activity. Although the FcγR binding profiles of human IgG2 isotype antibodies are known, significant Treg depletion can also be achieved using human IgG2 isotype antibodies against CD25. Therefore, in one embodiment, the anti-CD25 antibody is derived from the IgG2 subclass.

In a preferred embodiment, the anti-CD25 antibody as described herein binds to human CD25, preferably with high affinity. Still preferably, the anti-CD25 antibody binds to the extracellular region of human CD25, as shown above. In one aspect, the present invention provides an anti-CD25 antibody as described herein. In particular, examples provide experimental data on antibody production secreted by 7D4 hybridomas. As the background of the invention shows, the antibody is specific for mouse CD25, as shown by comparison of monoclonal antibody groups (including PC61), the antibody binds to one of three epitopes in mouse CD25 that are different from the IL-2 binding site, and does not block the binding of IL-2 to CD25. For example, when the corresponding isotype is associated, it has been shown that 7D4 binds to the epitope containing amino acids 184 to 194 (REHHRFLASEE) in [Uniprot sequence P01590] of mouse CD25. Literature (e.g., Setiady Y et al., 2010. Eur. J. Immunol. 40: 780–6; McNeill A et al., 2007. Scand J Immunol. 65: 63-9; Teege S et al., 2015, Sci Rep 5: 8959) relates to the assays of 7D4 and mouse CD25, and the assays disclosed in the examples include recombinant antibodies containing the CD25 binding domain of 7D4 or non-IL-2 blocking anti-human CD25 antibodies named MA-251 and 7G7B6. These assays can be applied to characterize human antibodies that recognize human CD25 and have the same 7D4 functional characteristics at the level of interaction with CD25 (particularly by not blocking IL-2 binding) and with Fcγ receptors (particularly by preferably binding to one or more human activating Fcγ receptors and effectively consuming Tregs), as described in the examples.

In one aspect of the invention, the antibody competes with antibody 7G7B6 for binding to human CD25; and/or binds to the same epitope or epitope recognized by antibody 7G7B6. 7G7B6 is a monoclonal antibody having a mouse IgG2a isotype that recognizes human CD25. 7G7B6 comprises a heavy chain variable region having the following sequence:

EVQLVESGGDLVQPRGSLKLSCAASGFTFSSYGMSWVRQTPDKRLELVATINGYGDTTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYFCARDRDYGNSYYYALDYWGQGTSVTVSS

(SEQ ID NO:3),

And light chain variable regions with the following sequences:

QIVLSQSPAILSASPGERVTMTCRASSSVSFMHWLQQKPGSSPKPWIYATSNLASGVSARFSGSGSGTSYSLTITRVEAEDAATYYCQQWSSNPPAFGGGTKLEIK

(SEQ ID NO:4).

In one embodiment, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises: 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; the light chain comprises: 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 can competitively bind to human CD25 with 7G7B6. Preferably, the antibody comprises a heavy chain and a light chain, wherein the heavy chain includes a heavy chain variable region, and the heavy chain variable region includes the following sequences:

EVQLVESGGGLIQPGGSLRLSCAAS GFTLDSYGVS WVRQAPGKGLEWV GVTSGGSAYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRYVYTGGYLYHYGMDL WGQGTLVTVSS

(SEQ ID NO:10),

The light chain includes a light chain variable region, which contains the following sequence:

DIQMTQSPSSSLSASVGDRVTITC RASQSISDYLA WYQQKPGKVPKLLI YAASTLPF GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QGTYDSSDWYWA FGGGTKVEI

(SEQ ID NO:14).

In another embodiment, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises: 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 the light chain comprises: 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 can competitively bind to human CD25 with 7G7B6. Preferably, the antibody comprises a heavy chain and a light chain, wherein the heavy chain includes a heavy chain variable region, and the heavy chain variable region includes the following sequences:

EVQLLESGGGLVQPGGSLRLSCAA SGFSVDIYDMS WVRQAPGKGLEWVA YISSSLGATYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR ERIYSVYTLDYYAMDL WGQGTLVTVSS

(SEQ ID NO:18),

The light chain includes a light chain variable region, which contains the following sequence:

DIQMTQSPSSSLSASVGDRVTITC QASQGITNNLN WYQQKPGKVPKLLI YAASTLQS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QQGYTTSNVDNA FGGGTKVEIK

(SEQ ID NO:22).

In one embodiment, the antibody that can competitively bind to human CD25 with 7G7B6 comprises a heavy chain and a light chain, said heavy chain comprising the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEL VSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RDRDYGNSYYYALDYWGQGTLVTVSS(SEQ ID NO:23), or

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEL VSTINGYGDTTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYFCA RDRDYGNSYYYALDYWGQGTLVTVSS(SEQ ID NO:24);

The light chain comprises the following amino acid sequence:

EIVLTQSPGTLSLSPGERATLSCRASSSVSFMHWLQQKPGQAPRPLIYA TSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCQQWSSNPPAFGQGT KLEIK(SEQ ID NO:25), or

QIVLTQSPGTLSLSPGERATLSCRASSSVSFMHWLQQKPGQSPRPLIYA TSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCQQWSSNPPAFGQGT KLEIK(SEQ ID NO:26).

In one aspect of the invention, the antibody competes with antibody MA251 for binding to human CD25; and/or binds to the same epitope or epitope recognized by antibody MA251. MA251 is a monoclonal antibody having a mouse isotype that recognizes human CD25. MA251 comprises a heavy chain variable region having the following sequence:

QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGIQWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARAYGYDGSWLAYWGQGTLVTVSS

(SEQ ID NO:5)

And light chain variable regions with the following sequences:

QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIFATSNLASGVPARFSGSGSGTSYSLTINRVEAEDADTYYCQQWSSNPPTFGGGTKLEIK

(SEQ ID NO:6).

In one embodiment, the antibody that can competitively bind to human CD25 with MA251 comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region containing the following amino acid sequence:

QVQLVESGGGVVQPGGSLRLSCAVSGFSLTSYGIQWVRQAPGKGLE WVSVIWAGGSTNYNSALMSRFTISKDNSKSTLYLQMNSLRAEDTAVYYC ARAYGYDGSWLAYWGQGTLVTVSS(SEQ ID NO:27);

OR

QVQLVESGGGVVQPGGSLRLSCAVSGFSLTSYGIQWVRQAPGKGLE WVSVIWAGGSTNYNSALMSRFTISKDNSKSTLYLQMNSLRAEDTAVYYC ARAYGYDGSWLAYWGQGTLVTVSS(SEQ ID NO:29);

The light chain includes a light chain variable region, which contains the following amino acid sequence:

QIVLTQSPATLSLSPGERATLSCRASSSVSYMHWYQQKPGQAPRPLIF ATSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPTFGGG TKLEIK(SEQ ID NO:30);

QIVLTQSPATLSLSPGERATLSCRASSSVSYMHWYQQKPGQAPRPLIF ATSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPTFGGG TKLEIK(SEQ ID NO:31);

OR

QIQLTQSPSSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKSPKPLIFA TSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPPTFGGGT KLEIK(SEQ ID NO:33).

In one embodiment, the antibody comprises 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 comprises 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 comprises 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 comprises 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 yet another embodiment, the antibody comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:29 and/or contains SEQ ID NO:33.

In one respect, the antibody competes with both antibody 7G7B6 and antibody MA251 for binding to human CD25. In another respect, the antibody binds to the same epitope that is recognized by both 7G7B6 and MA251.

Competition between 7G7B6 or MA251 antibodies and other antibodies, such as those discussed in the examples and those known in the art, can be measured. In some embodiments, competition between two antibodies (e.g., 7G7B6 or MA251 and another antibody) is determined by adding another antibody to an 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 simultaneous binding of the 7G7B6 or MA251 antibody, the other antibody, and recombinant human CD25. If binding of both 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 (e.g., decreases by at least 75%) after the addition of the other antibody, the latter antibody is a competitor to the 7G7B6 or MA251 antibody. Flow cytometry can also be used to detect the simultaneous binding of 7G7B6 or MA251 antibodies and other antibodies to human CD25-expressing cells.

In one aspect, the present invention provides an anti-CD25 antibody that specifically binds to a human CD25 epitope, wherein the epitope comprises one or more amino acid residues selected from one or more amino acid segments selected from amino acid segments of SEQ ID NO:1 (YQCVQGYRALHRGP), SEQ ID NO:1 (SVCKMTHGKTRWTQPQLICTG), SEQ ID NO:1 (KEGTMLNCECKRGFR), and SEQ ID NO:1 (NSSHSSWDNQCQCTSSATR). Preferably, the epitope comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or more residues from the selected amino acid segment. More preferably, the epitope comprises a sequence selected from the following: amino acids 150 to 158 of SEQ ID NO:1 (YQCVQGYRA), amino acids 176 to 180 of SEQ ID NO:1 (RWTQP), amino acids 42 to 56 of SEQ ID NO:1 (KEGTMLNCECKRGFR), and amino acids 74 to 84 of SEQ ID NO:1 (SSWDNQCQCTS), and combinations thereof. These epitopes differ from the IL-2 binding site in human CD25, and as described in the examples, antibodies binding to such epitopes do not block the binding of IL-2 to CD25.

In a preferred embodiment, a method of treating a human subject suffering from cancer includes administering the anti-CD25 antibody of the present invention to the subject, wherein the subject preferably suffers from a solid tumor, and wherein the anti-CD25 antibody is preferably a human IgG1 antibody that does not inhibit the binding of interleukin 2 to CD25 and binds with high affinity to at least one selected from FcγRI (CD64), FcγRIIc (CD32c), and FcγRIIIa (CD16a) and consumes tumor-infiltrating regulatory T cells. Preferably, the dissociation constant ( _Kd ) of the anti-CD25 antibody to CD25 is less than ^10⁻⁷ M, more preferably less than ^10⁻⁸ M. More preferably, the anti-CD25 antibody binds to human CD25, providing an effect on IL-2 binding and Treg consumption, similar to the effect of 7D4 on mouse CD25 or the effect of 7G7B6 and MA251 on human CD25. In a further embodiment, the anti-CD25 antibody binds to the Fcγ receptor with an activation inhibition rate (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).

The CD25-binding domain of the 7D4 antibody has been cloned and expressed as a recombinant protein fused with an appropriate constant region. The sequence of the CD25-binding domain of the 7D4 antibody, and its specificity and/or other functional activities for different epitopes within the extracellular domain of CD25, can be used to compare candidate anti-CD25 antibodies generated and screened using any suitable technique (either by culturing hybridoma groups from rodents immunized with CD25 or by generating recombinant antibody libraries, followed by screening these libraries with CD25 fragments to be functionally characterized as described herein). Anti-CD25 antibodies thus identified can also be generated as recombinant antibodies, particularly as intact antibodies or as fragments or variants as described herein.

Natural antibodies and immunoglobulins are typically heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has a variable domain (V_{_H} ) at the amino terminus, followed by multiple constant domains. Each light chain has a variable domain (V_{_L} ) at the amino terminus and a constant domain at the carboxyl terminus.

Variable regions are capable of interacting with structurally complementary antigen targets and are characterized by differences in the amino acid sequences of antibodies from different antigen specificities. Variable regions of the H or L chain contain amino acid sequences capable of specifically binding to antigen targets. Among these sequences are smaller sequences referred to as "hypervariates" because they exhibit significant variability between antibodies of different specificities. These hypervariable regions are also known as "complementarity-determining regions" or "CDR" regions.

These CDR regions explain the fundamental specificity of the antibody's specific antigenic determinant structure. CDRs represent discontinuous amino acid segments within the variable region; however, regardless of type, the positional localization of these key amino acid sequences within the variable heavy and light chain regions has been found to have similar localization within the variable chain's amino acid sequence. All antibodies have three CDR regions each for the variable heavy and light chains, and for both the light chain (L) and heavy chain (H), each CDR region is discontinuous (designated L1, L2, L3, H1, H2, H3). Accepted CDR regions have been previously described (Kabat et al., 1977. J Biol Chem 252, 6609-6616).

The antibodies of the present invention can function through complement-dependent cytotoxicity (CDC) and/or antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody-dependent cell-mediated phagocytosis (ADCP), as well as any other mechanisms that allow targeting, blocking proliferation, and/or consuming Treg cells.

"Complement-dependent cytotoxicity" (CDC) refers to the lysis of cells expressing antigens by the antibodies of this invention in the presence of complement.

Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated response in which nonspecific cytotoxic cells expressing Fc receptors (FcRs), such as natural killer (NK) cells, neutrophils, and macrophages, recognize antibodies bound to target cells, leading to target cell lysis.

Antibody-dependent cell-mediated phagocytosis (ADCP) refers to a cell-mediated response in which phagocytes (such as macrophages) expressing Fc receptors (FcRs) recognize antibodies bound to target cells, leading to the phagocytosis of the target cells.

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

As discussed herein, in one embodiment of the invention, an anti-CD25 antibody that does not inhibit interleukin 2 binding and causes Treg cell depletion is used. For example, an anti-CD25 antibody that does not inhibit interleukin 2 binding to CD25 and elicits a strong CDC response and/or a strong ADCC and/or a strong ADCP response can be used. Methods for increasing CDC, ADCC, and/or ADCP are known in the art. For example, the CDC response can be increased by a mutation in the antibody that increases C1q binding affinity (ldusogieet al. (2001) J lmmunol 166, 2571-5).

The phrase "does not inhibit the binding of interleukin-2 to CD25" mentioned herein can also be expressed as the anti-CD25 antibody being a non-IL-2 blocking antibody or a "non-blocking" antibody (regarding the non-blocking of IL-2 binding to CD25 in the presence of the anti-CD25 antibody), meaning the antibody does not block the binding of interleukin-2 to CD25, and particularly does not inhibit interleukin-2 signaling in cells expressing CD25. The references to "non-blocking," "non-IL2 blocking," "non-blocking," or "non-blocking" (regarding the non-blocking of IL-2 binding to CD25 in the presence of the anti-CD25 antibody) include embodiments of the present invention in which the anti-CD25 antibody does not block IL-2 signaling via CD25. That is, compared to IL-2 signaling in the absence of the antibody, the anti-CD25 antibody inhibits less than 50% of IL-2 signaling. In the specific embodiments of the present invention as described herein, compared to IL-2 signaling in the absence of the antibody, the anti-CD25 antibody inhibits IL-2 signaling by less than about 40%, 35%, 30%, preferably less than about 25%. Anti-CD25 non-IL-2 blocking antibodies allow binding to CD25 without interfering with the binding of IL-2 to CD25, or substantially do not interfere with the binding of IL-2 to CD25. The non-IL-2 blocking antibodies mentioned in this article can also 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."

Some anti-CD25 antibodies allow IL-2 to bind to CD25 but still block signal transduction through the CD25 receptor. Such anti-CD25 antibodies are not within the scope of this invention. Conversely, non-IL-2 blocking anti-CD25 antibodies allow IL-2 to bind to CD25 to promote at least 50% of the signal transduction level through the CD25 receptor, compared to signal transduction in the absence of anti-CD25 antibodies.

IL-2 signaling via CD25 can be measured using methods discussed in the examples and known in the art. Comparisons of IL-2 signaling in the presence and absence of anti-CD25 antibody agents can be performed under the same or substantially the same conditions.

In some implementations, 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 include culturing PMBC cells for 30 minutes in the presence of an anti-CD25 antibody at a concentration of 10 μg/ml, followed by the addition of different concentrations of IL-2 (e.g., 10 U/ml or different concentrations of 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 using a fluorescently labeled antibody against the phosphorylated STAT5 peptide, which is analyzed by flow cytometry. The percentage of IL-2 signaling blocked can be calculated as follows: %blocking = 100 × [(%Stat5 ⁺ cells without antibody - %Stat5 ⁺ cells with 10 μg/ml antibody) / (%Stat5 ⁺ cells without antibody)].

ADCC can be increased by removing the fucose moiety from the antibody glycan, for example by generating antibodies in the YB2/0 cell line, or by introducing specific mutations into the Fc region 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 region of human IgG1 (Richards et al. (2008) Mol Cancer Ther 7, 2517-27).

In a preferred embodiment of the invention, the antibody is optimized to elicit an ADCC response, that is, the ADCC response is enhanced, increased, or improved compared to other anti-CD25 antibodies, including those that do not inhibit the binding of interleukin 2 to CD25, such as unmodified anti-CD25 monoclonal antibodies.

In a preferred embodiment of the invention, the antibody is optimized to elicit an ADCP response, that is, the ADCP response is enhanced, increased, or improved compared to other anti-CD25 antibodies, including those that do not inhibit the binding of interleukin 2 to CD25, such as unmodified anti-CD25 monoclonal antibodies.

As used herein, a "chimeric antibody" can refer to an antibody having a variable sequence derived from an immunoglobulin of one species (e.g., a rat or mouse antibody) and a constant region of an immunoglobulin of another species (e.g., a human antibody). In some embodiments, a chimeric antibody may have a constant region enhanced for inducing ADCC.

The antibodies according to the invention can also be partially or wholly synthetic, wherein at least a portion of the polypeptide chain of the antibody is synthetic and may be optimized for binding to its homologous antigen. Such antibodies can be chimeric antibodies or humanized antibodies, and can be a complete tetrameric structure, or can be a dimer containing only a single heavy chain and a single light chain.

The antibodies of this invention can also 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 eukaryotic, prokaryotic, or phage clone, rather than the method of producing it.

The antibodies of this invention can also be human antibodies. As used herein, "human antibody" refers to an antibody having a variable region, wherein both the framework region and the CDR region are derived from human immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human immunoglobulin sequence. The human antibodies of this invention may contain amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo).

Anti-CD25 antibodies possessing the features described herein represent another object of the invention. Anti-CD25 antibodies can be used in medicine. In another embodiment, the invention provides a method of treating a disease of a subject, comprising administering an anti-CD25 antibody that does not inhibit the binding of interleukin-2 (IL-2) to CD25 or IL-2 signaling via CD25. Preferably, the disease is cancer, particularly for the treatment of solid tumors.

In another embodiment, 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 contained in an expression cassette within a suitable nucleic acid vector for expression in host cells (e.g., bacterial, yeast, insect, fish, mouse, monkey, or human cells). In some embodiments, the present invention provides a host cell containing a heterologous nucleic acid molecule (e.g., a DNA vector) expressing the desired antibody.

In some embodiments, the present invention provides a method for preparing isolated anti-CD25 antibodies as defined above. In some embodiments, such a method may include culturing host cells containing nucleic acids (e.g., heterologous nucleic acids contained in and/or delivered to the host cells via a vector). Preferably, host cells (and/or heterologous nucleic acid sequences) are prepared and constructed such that the antibody or its antigen-binding fragment or variant is secreted from the host cells and isolated from the cell culture supernatant.

The antibodies of the present invention can be monospecific, bispecific, or multispecific. A “multispecific antibody” can be specific to different epitopes of a target antigen or polypeptide, or can contain antigen-binding domains specific to more than one target antigen or polypeptide (Kufer et al. (2004) Trends Biotechnol 22, 238-44).

In one aspect of the invention, the antibody is a monospecific antibody. As discussed further below, in another aspect, the antibody is a bispecific antibody.

As used in this article, a "bispecific antibody" is an antibody that has the ability to bind to a single antigen or polypeptide or two different epitopes on two different antigens or polypeptides.

The bispecific antibodies of the present invention, as discussed herein, can be produced by: biological methods, such as somatic cell hybridization; or genetic methods, such as expressing a non-natural DNA sequence encoding the desired antibody structure in a cell line or organism; chemical methods (e.g., by chemical coupling, genetic fusion, non-covalent binding, or otherwise binding to one or more molecular entities, such as another antibody or antibody fragment); or combinations thereof.

Technologies and products that allow for the generation of monospecific or bispecific antibodies are known in the art, as extensively reviewed in the literature, and also involve alternative forms, antibody-drug conjugates, antibody design methods, in vitro screening methods, constant regions, post-translational modifications and chemical modifications, and improved features for triggering cancer cell death, 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). This bispecific antibody can be provided in any commercially available form, including Duobody, BiTE DART, CrossMab, Knobs-in-holes, Triomab, or other suitable molecular forms and fragments thereof.

As used herein, an epitope or antigenic determinant refers to a site on an antigen where an antibody binds. As is well known in the art, epitopes can be formed from consecutive amino acids (linear epitopes) or from non-continuous amino acids arranged side-by-side in a protein's ternary fold (conformational epitopes). Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed by ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more often at least five, or eight to ten amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes are well known in the art, including, 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 antibodies of the present invention can recognize conformational epitopes bound by antibody 7G7B6 or MA251. In one embodiment, the conformational epitope comprises at least two sequences selected from amino acids 150 to 158 of SEQ ID NO:1 (YQCVQGYRA), amino acids 176 to 180 of SEQ ID NO:1 (RWTQP), amino acids 42 to 56 of SEQ ID NO:1 (KEGTMLNCECKRGFR), and amino acids 74 to 84 of SEQ ID NO:1 (SSWDNQCQCTS).

In some embodiments, the anti-CD25 antibody may be contained in a pharmaceutical preparation that also comprises a conjugated payload (e.g., a therapeutic or diagnostic agent), specifically for cancer treatment or diagnosis. Anti-CD25 antibody conjugates having a radionuclide or toxin may be used. Common examples of radionuclides are, for example, ^90γ , ^131I , and ^67Cu , and common examples of toxins are doxorubicin and calcineurin. In further embodiments, the anti-CD25 antibody may be modified to have an altered half-life. Methods for achieving an altered half-life are known in the art. In some embodiments, the anti-CD25 antibody is not conjugated to another therapeutic or diagnostic agent. In particular, in some embodiments, the anti-CD25 antibody is not conjugated to a radionuclide, i.e., in some embodiments, the anti-CD25 antibody is not radiolabeled.

In a preferred embodiment of the invention, the object of any aspect of the 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. Therefore, in all aspects of the invention described herein, the object is preferably a human.

As used in this article, the terms “cancer,” “cancerous,” or “malignant” refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth.

Examples of cancer include, but are not limited to, carcinoma, lymphoma, leukemia, germ cell tumors, and sarcomas. More specific examples of these 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 (intestinal) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer.

In one respect, cancer involves solid tumors. Examples of solid tumors are sarcomas (including cancers arising from transformed cells of mesenchymal origin in tissues such as cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells, or fibrous connective tissue), carcinomas (including tumors arising from epithelial cells), mesotheliomas, neuroblastomas, retinoblastomas, etc. Cancers involving solid tumors include, but are not limited to, brain cancer, lung cancer, stomach cancer, duodenal cancer, esophageal cancer, breast cancer, colon and rectal cancer, kidney cancer, bladder cancer, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, oral cancer, sarcomas, eye cancer, thyroid cancer, urethral cancer, vaginal cancer, cervical cancer, lymphomas, etc.

In one respect, cancer involves tumors that express CD25, including but not limited to lymphomas such as Hodgkin's lymphoma and lymphocytic leukemias such as chronic lymphocytic leukemia (CLL).

In one aspect of the invention, cancer is identified by the presence of specific tumor-associated markers and antigens (e.g., CD20, HER2, PD-1, PD-L1, SLAM7F, CD47, CD137, CD134, TIM3, CD25, GITR, CD25, EGFR, etc.), or by cancer being identified as having biomarkers known as high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR). Furthermore, antibodies may be used when the identification of specific tumor-associated markers, antigens, or biomarkers is used to define a patient's precancerous, non-invasive state of the aforementioned cancers (e.g., carcinoma in situ, smoldering myeloma, monoclonal gammopathy of undetermined significance, cervical intraepithelial neoplasia, MALTomas/GALTomes, and various lymphoproliferative disorders). Preferably, in some embodiments, the treated subject has a solid tumor.

In one aspect of the invention, the cancer is selected from melanoma, non-small cell lung cancer, kidney cancer, ovarian cancer, bladder cancer, sarcoma, and colon cancer. In a preferred aspect of the invention, the cancer is selected from melanoma, ovarian cancer, non-small cell lung cancer, and kidney cancer. In one embodiment, the cancer is not melanoma, ovarian cancer, or breast cancer. In a preferred aspect, the cancer is sarcoma, colon cancer, melanoma, or colorectal cancer, or more generally, any human cancer in which the 4T1, MCA205, B16, CT26, or MC38 cell lines can represent a preclinical model for validating compounds that can be used for their therapeutic administration.

As used herein, the term “tumor” applies to any object diagnosed or suspected of having a tumor, and cancer refers to any malignant or potentially malignant growth or mass of tissue of any size, including primary tumors and secondary growths. The terms “cancer,” “malignant tumor,” “growth,” “tumor,” and “cancer” are also used interchangeably herein to refer to tumors and tumor cells that exhibit relatively abnormal, uncontrolled, and/or autonomous growth, thus displaying an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. Typically, target cells used for detection or treatment include precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells.

As used in this article, a "solid tumor" is an abnormal growth or mass of tissue that typically does not contain cysts or fluid-filled areas, particularly tumors and/or metastases (wherever they may be) other than leukemia or non-solid lymphoma. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them and/or the tissue or organ in which they are located. Examples of solid tumors are sarcomas (including cancers arising from transformed cells of mesenchymal origin in tissues such as cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells, or fibrous connective tissue), carcinomas (including tumors originating from epithelial cells), melanomas, lymphomas, mesotheliomas, neuroblastomas, and retinoblastomas.

Particularly preferred cancers according to the invention include those characterized by the presence of a solid tumor, i.e., the subject does not have a non-solid tumor. In all aspects of the invention as discussed herein, the preferred cancer is a solid tumor, i.e., the subject has a solid tumor (and does not have a non-solid tumor).

The definition of “treat/treating” cancer used in this article is limited to achieving at least one positive therapeutic effect, such as a reduction in the number of cancer cells, a reduction in tumor size, a slowdown in the rate at which cancer cells infiltrate into peripheral organs, or a slowdown in the rate of tumor metastasis or tumor growth.

The effectiveness of active cancer treatment can be measured in many ways (e.g., Weber (2009) J Nucl Med 50, 1S-10S). For example, in terms of tumor growth inhibition, a T/C ≤ 42% is the lowest level of antitumor activity according to the National Cancer Institute (NCI) criteria. A T/C < 10% is considered a high level of antitumor activity, where T/C (%) = median tumor volume in the treatment group / median tumor volume in the control group × 100. In some implementations, the effective amount of treatment achieved is any of progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS). PFS, also known as “time to tumor progression,” represents the length of time during and after treatment when cancer does not grow, including the amount of time a patient experiences a complete or partial response, and the amount of time a patient experiences stable disease. DFS refers to the length of time a patient remains disease-free during and after treatment. OS refers to the extension of life expectancy compared to an untreated or pristine individual or patient.

The term "prevention" (or preventive measures) used in this article refers to delaying or preventing the onset of cancer symptoms. Prevention may be absolute (thus preventing the disease from occurring) or effective only in certain individuals or for a limited period of time.

In a preferred aspect of the invention, the subject suffers from an established tumor, i.e., a subject already has a tumor (e.g., a tumor classified as a solid tumor). Therefore, when a subject already has a tumor (e.g., a solid tumor), the invention as described herein can be used. Thus, the invention provides a treatment option that can be used to treat an existing tumor. In one aspect of the invention, the subject has an existing solid tumor. The invention can be used as a prevention for a subject already suffering from a solid tumor, or preferably as a treatment for a subject already suffering from a solid tumor. In one aspect, the invention is not used as a prevention or preventative measure.

In one aspect, using the invention as described herein, for example, compared with other cancer treatments (e.g., standard care for a given cancer), tumor regression may be enhanced, tumor growth may be impaired or reduced, and/or survival time may be increased.

In one aspect of the invention, the method for treating or preventing cancer as described herein further includes the step of identifying a subject suffering from cancer, preferably a subject suffering from a tumor (e.g., a solid tumor). In one embodiment, the method may include identifying a subject suffering from a hematologic cancer.

The dosage regimens for the effective treatment of cancer patients described herein can vary depending on factors such as disease state, age, and patient weight, as well as the ability of the treatment to elicit an anticancer response in the subject. The selection of an appropriate dose is within the capabilities of those skilled in the art. Examples include 0.01, 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 mg/kg. In some embodiments, such amounts are unit doses (or portions thereof) suitable for administration according to a dosing regimen (i.e., a treatment dosing regimen) that has been determined to be associated with the desired or beneficial outcome when administered to the relevant population.

Antibodies according to any aspect of the invention as described herein may be in the form of pharmaceutical compositions that additionally include pharmaceutically acceptable carriers, diluents, or excipients. These compositions include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), dispersions or suspensions, tablets, pills, or liposomes. In some embodiments, preferred forms may depend on the intended route of administration and/or therapeutic application. Antibody-containing pharmaceutical compositions may be administered by any suitable method known in the art, including but not limited to oral, mucosal, inhalation, topical, oral, nasal, rectal, or parenteral administration (e.g., intravenous, infusion, intratumoral, intranodal, subcutaneous, intraperitoneal, intramuscular, intradermal, transdermal, or other types of administration involving physical breakthrough of target tissue and administration of pharmaceutical compositions via tissue breakthrough). Such formulations may be, for example, injectable or infusionable solutions suitable for intradermal, intratumoral, or subcutaneous administration or for intravenous infusion. Administration may involve intermittent administration. Alternatively, administration may involve continuous administration (e.g., infusion) for at least a selected period of time, simultaneously with or between administration of other compounds.

In some embodiments, antibodies can be prepared using carriers that protect them from rapid release and/or degradation, such as controlled-release formulations, like implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used.

For example, those skilled in the art will understand that the route of delivery (e.g., oral vs. intravenous vs. subcutaneous vs. intratumoral, etc.) can affect the dose and/or the desired dose can affect the route of delivery. For example, focused delivery may be necessary and/or useful in cases where a particular high concentration of the agent is desired at a specific site or location (e.g., intratumoral) (e.g., intratumoral delivery in this instance). Other factors to consider when optimizing the route and/or dosing regimen for a given treatment may include, for example, the specific cancer to be treated (e.g., type, stage, location, etc.), the patient's clinical condition (e.g., age, overall health, etc.), the availability of combination therapies, and other factors known to the physician.

Pharmaceutical compositions should generally be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the desired amount of antibody with one or a combination of the components listed above into a suitable solvent, followed by filtration sterilization as needed. Formulations for parenteral administration include, but are not limited to, the suspensions, solutions, emulsions in oily or aqueous carriers, pastes, and implantable sustained-release or biodegradable formulations described herein. Sterile injectable formulations can be prepared using non-toxic, parenteral-acceptable diluents or solvents. Each pharmaceutical composition used according to the invention may include pharmaceutically acceptable dispersants, wetting agents, suspending agents, isotonic agents, coating agents, antibacterial agents, and antifungal agents; the carrier, excipients, salts, or stabilizers are non-toxic to the target at the dose and concentration used. Preferably, such a composition may further comprise a pharmaceutically acceptable carrier or excipient for treating cancer, said carrier or excipient being compatible with a given method and/or site of administration, such as for parenteral (e.g., subcutaneous, intradermal, or intravenous), intratumoral, or peritumoral administration.

While embodiments of the treatment methods or compositions used according to the present invention may not be effective in achieving positive therapeutic effects in every subject, they should achieve positive therapeutic effects using pharmaceutical compositions and dosing regimens that are consistent with good medical practice and a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, ^χ² -test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

In the context of the above and subsequent references to tumor, neoplasm, carcinoma, or cancer, regardless of the location of the tumor and/or metastasis, metastasis in the original organ or tissue and/or any other location may be implied.

As discussed herein, the present invention relates to the depletion of regulatory T cells (Tregs). Therefore, in one aspect of the invention, anti-CD25 antibodies that do not inhibit the binding of interleukin-2 to CD25 also deplete or reduce tumor-infiltrating regulatory T cells. In one aspect, depletion is achieved through ADCC. In another aspect, depletion is achieved through ADCP.

Thus, the present invention provides a method for consuming regulatory T cells in a target tumor, comprising administering an anti-CD25 antibody that does not inhibit the binding of interleukin 2 to CD25 to the target. In a preferred embodiment, Tregs consume solid tumors. “Consumption” means a reduction in the proportion or percentage of Tregs relative to when no anti-CD25 antibody that does not inhibit the binding of interleukin 2 to CD25 is administered. In specific embodiments of the invention as described herein, more than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of tumor-infiltrating regulatory T cells are consumed.

As used in this article, “regulatory T cells” (“Treg/Treg cells/Treg”) refers to a lineage of CD4+ T lymphocytes that specifically controls autoimmunity, allergic reactions, and infection. They typically regulate the activity of the T cell population, but they can also influence certain cell types of the innate immune system. Tregs are usually identified by the expression of the biomarkers CD4, CD25, and Foxp3. Naturally occurring Treg cells typically comprise about 5 to 10% of peripheral CD4+ T lymphocytes. However, within the tumor microenvironment (i.e., tumor-infiltrating Treg cells), they can account for 20 to 30% of the total CD4+ T lymphocyte population.

Activated human Treg cells can directly kill target cells, such as effector T cells and APCs, via perforation factor or granzyme B-dependent pathways. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4+) Treg cells induce indoleamine 2,3-dioxygenase (IDO) expression via APCs, which in turn inhibits T cell activation by reducing tryptophan. Treg cells can release interleukin-10 (IL-10) and transforming growth factor (TGFβ) in vivo, thereby directly inhibiting T cell activation and suppressing APC function by inhibiting the expression of MHC molecules, CD80, CD86, and IL-12. Treg cells can also suppress immunity by expressing high levels of CTLA4, which can bind to CD80 and CD86 on antigen-presenting cells and prevent proper activation of effector T cells.

In a preferred embodiment of the 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 to more than 5, 10, 15, 20, 40, or 80.

Immune effector cells are immune cells that participate in the effector phase of the immune response. Exemplary immune cells include myeloid or lymphoid cells, such as lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils.

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), such as neutrophils capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes expressing FcαR participate in the specific killing of target cells and present antigens to other components of the immune system or bind to antigen-presenting cells. Effector cells can also phagocytose target antigens, target cells, or microorganisms. As discussed herein, antibodies according to the invention can be optimized for their ability to induce ADCC.

In some embodiments, different anticancer agents may be administered via the same or different delivery routes and/or in combination with antibodies according to different regimens. Alternatively or additionally, in some embodiments, one or more doses of a first active agent are administered substantially simultaneously with one or more other active agents, and in some embodiments, they are administered substantially simultaneously with one or more other active agents via a common route and/or as part of a single composition. Those skilled in the art will further understand that some embodiments of the combination therapies provided according to the invention achieve synergistic effects; in some such embodiments, the doses of one or more agents used in the combination may be substantially different (e.g., lower) and/or when the agents are used in different treatment regimens (e.g., as a monotherapy and/or as part of different combination therapies), they may be delivered via alternative rather than standard, preferred, or necessary routes.

In some embodiments, when two or more active agents are used according to the invention, these active agents may be administered simultaneously or sequentially. In some embodiments, administration of one agent relative to administration of another agent is particularly timed. For example, in some embodiments, a first agent is administered to observe a specific effect (or to anticipate observing a specific effect, e.g., based on a population study showing a correlation between a given dosing regimen and a specific effect of interest). In some embodiments, the desired relative dosing regimen of the combined agents may be empirically assessed or determined, 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., to establish a correlation), or in specific patients of interest.

In another aspect of the invention, anti-CD25 antibodies that do not inhibit the binding of interleukin 2 to CD25 have improved therapeutic effects when combined with immune checkpoint inhibitors. Combination therapy using anti-CD25 antibodies that do not inhibit the binding of interleukin 2 to CD25 and immune checkpoint inhibitors can have a synergistic effect in treating established tumors. Data regarding PD-1/PD-L1 in this embodiment pertains to interference with PD-1/PD-L1 interactions. Therefore, the interaction between the PD-1 receptor and the PD-L1 ligand may be blocked, resulting in "PD-1 blockade." In one aspect, for example, compared to anti-CD25 antibodies or PD-1/PD-L1 blockade (direct use of anti-PD1 antibodies, or indirect use of anti-PD-L1 antibodies), using the invention described herein can lead to enhanced tumor regression, impaired or reduced tumor growth, and/or prolonged survival. When the anti-CD25 antibody does not inhibit the binding of interleukin-2 to CD25, the combination therapy with the anti-CD25 antibody and the immune checkpoint inhibitor may further include administration of interleukin-2 at a dose appropriate for the treatment of cancer.

As used herein, "immune checkpoint" or "immune checkpoint protein" refers to proteins belonging to the inhibitory pathways of the immune system, particularly those regulating T-cell responses. Under normal physiological conditions, immune checkpoints are crucial for preventing autoimmunity, especially during responses to pathogens. Cancer cells can alter the regulation of immune checkpoint protein expression to evade immune surveillance.

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. Immune checkpoint proteins can also refer to proteins that bind to other immune checkpoint proteins. These proteins include PD-L1, PD-L2, CD80, CD86, HVEM, LLT1, and GAL9.

"Immune checkpoint protein inhibitor" refers to any protein that can interfere with immune checkpoint protein-mediated signal transduction and/or protein-protein interactions. In one aspect of the invention, the immune checkpoint protein is PD-1 or PD-L1. In a preferred aspect of the invention as described herein, the immune checkpoint inhibitor interferes with PD-1/PD-L1 interactions via anti-PD-1 or anti-PD-L1 antibodies.

Therefore, the present invention also provides a method for treating cancer, comprising administering an anti-CD25 antibody that does not inhibit the binding of interleukin 2 to CD25 and a further therapeutic agent to a subject, said further therapeutic agent preferably being 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 for treating cancer, said further therapeutic agent preferably being an immune checkpoint inhibitor.

Furthermore, the present invention provides the use of an anti-CD25 antibody that does not inhibit the binding of interleukin 2 to CD25 and other therapeutic agents in a medicament for the treatment of cancer, wherein the other therapeutic agents are preferably immune checkpoint inhibitors. The administration of the anti-CD25 antibody that does not inhibit the binding of interleukin 2 to CD25 and other therapeutic agents (e.g., immune checkpoint inhibitors) can be simultaneous, separate, or sequential.

This invention provides a combination of an anti-CD25 antibody that does not inhibit the binding of interleukin 2 to CD25 and an additional therapeutic agent for treating cancer, wherein the additional therapeutic agent is preferably an immune checkpoint inhibitor, wherein the anti-CD25 antibody that does not inhibit the binding of interleukin 2 to CD25 and the additional therapeutic agent (e.g., an immune checkpoint inhibitor) are administered simultaneously, alone, or sequentially. Such an anti-CD25 antibody that does not inhibit the binding of interleukin 2 to CD25 and presents a human IgG1 isotype can be particularly combined with antibodies that target immunosuppressive sites but lack sequences that allow ADCC, ADCP, and/or CDC.

In another aspect, the present invention provides an anti-CD25 antibody that does not inhibit the binding of interleukin-2 to CD25 for the treatment of cancer, wherein the antibody is for administration in combination with another therapeutic agent, preferably an immune checkpoint inhibitor. The present invention also provides the use of the anti-CD25 antibody that does not inhibit the binding of interleukin-2 to CD25 in the preparation of a medicament for the treatment of cancer, wherein the medicament is for administration in combination with another therapeutic agent, preferably an immune checkpoint inhibitor.

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

PD-1 (programmed cell death protein 1), also known as CD279, is a cell surface receptor expressed on activated T cells and B cells. Interactions with its ligands have been shown to attenuate T cell responses 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 proximity to peptide antigens presented by the major histocompatibility complex (MHC) for binding to PD-1 ligands (Freeman (2008) Proc Natl Acad Sci USA 105, 10275-6). Therefore, proteins, antibodies, or small molecules that prevent the co-linking of PD-1 and TCR on the T cell membrane are useful PD-1 antagonists.

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 can be a monoclonal antibody. The anti-PD-1 antibody can be a human antibody or a humanized antibody. An anti-PD-1 antibody is an antibody capable of specifically binding to the PD-1 receptor. Anti-PD-1 antibodies known in the art include nivolumab and pembrolizumab.

The PD-1 antagonists of the present invention also include compounds or agents that bind to and/or block PD-1 ligands to interfere with or inhibit ligand binding to the PD-1 receptor or directly bind to and block the PD-1 receptor without inducing inhibitory signal transduction via the PD-1 receptor. In particular, the PD-1 antagonists include small molecule inhibitors of the PD-1/PD-L1 signaling pathway. Alternatively, the PD-1 receptor antagonist may bind directly to the PD-1 receptor without inducing inhibitory signal transduction, and may also bind to ligands of the PD-1 receptor to reduce or inhibit ligand-triggered signal transduction via the PD-1 receptor. By reducing the number and/or amount of ligands that bind to the PD-1 receptor and trigger inhibitory signal transduction, fewer cells are attenuated by the negative signal delivered via PD-1 signal transduction, and a stronger immune response can be achieved.

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

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 acts as an agonist of the T-cell activation co-stimulatory pathway. The antibody agonists of the T-cell activation co-stimulatory pathway include, but are not limited to, agonist antibodies against ICOS, GITR, OX40, CD40, LIGHT, and 4-1BB.

Other approaches to cancer treatment 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). These FcγRIIb antagonists can be small molecules that interfere with FcγRIIb-induced intracellular signaling, modified antibodies that do not bind to inhibitory FcγRIIb receptors, or anti-human FcγRIIb (anti-CD32b antibodies). For example, antagonistic 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; WO2015173384; WO2008002933).

In another aspect, the present invention provides a bispecific antibody comprising:

(a) The first antigen-binding moiety that binds to CD25; and

(b) A second antigen-binding moiety that binds to immune checkpoint proteins, tumor-associated antigens, anti-human activated Fc receptor antibodies (FcgRI, FcgRIIa, FcgRIII) or antagonistic anti-human FcγRIIb antibodies;

The anti-CD25 antibody does not inhibit the binding of interleukin-2 (IL-2) to CD25, and is preferably a bispecific IgG1 antibody that binds to at least one activating Fcγ receptor with high affinity and depletes tumor-infiltrating regulatory T cells. In a preferred embodiment, the second antigen-binding portion binds to PD-L1.

As used herein, “tumor-associated antigen” refers to antigens expressed on tumor cells that distinguish them from neighboring non-cancer cells, and includes, but is not limited to, CD20, CD38, PD-L1, EGFR, EGFRV3, CEA, TYRP1, and HER2. Various review articles describing relevant tumor-associated antigens and corresponding therapeutically useful antitumor antibody drugs have been published (see, for example, Sliwkowski & Mellman (2013) Science 341, 192-8). These antigens and corresponding antibodies include, but are not limited to, CD22 (bonatumab), CD20 (rituximab, tosimob), CD56 (lorvotuzumab), CD66e/CEA (labetuzumab), CD152/CTLA-4 (ipilimumab), CD221/IGF1R (MK-0646), CD326/Epcam (edrecolomab), CD340/HER2 (trastuzumab, pertuzumab), and EGFR (cetuximab, panitumumab).

In one aspect, the bispecific antibody according to the invention, as described herein, causes ADCC, or in another aspect, causes enhanced ADCC.

Bispecific antibodies can bind to specific epitopes on CD25 that do not affect the binding of IL-2 to CD25, as well as specific epitopes on immune checkpoint proteins or tumor-associated antigens as defined herein. In a preferred embodiment, the second antigen-binding portion binds to PD-L1. In a preferred aspect, the present invention provides a bispecific antibody comprising:

(a) The first antigen-binding moiety that binds to CD25 and does not affect IL-2 binding to CD25; and

(b) The second antigen-binding portion that binds to immune checkpoint proteins expressed on tumor cells.

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

Those skilled in the art will be able to generate bispecific antibodies using known methods. The bispecific antibody according to the invention can be used in any aspect of the invention described herein. Preferably, the second antigen-binding portion within the bispecific antibody according to the invention binds to human PD-1, human PD-L1, or human CTLA-4.

In one respect, bispecific antibodies can bind to CD25 as well as to immunomodulatory receptors (such as CTLA4, ICOS, GITR, 4-1BB, or OX40) that are highly expressed on tumor-infiltrating Tregs.

The present invention also provides a kit comprising an anti-CD25 antibody as described herein and an additional therapeutic agent, wherein the additional therapeutic agent is preferably an immune checkpoint inhibitor, preferably a PD-1 antagonist as discussed herein (using an anti-PD1 antibody directly or an anti-PD-L1 antibody indirectly). In one aspect, the immune checkpoint inhibitor is anti-PD-L1. In another embodiment, the kit comprises an anti-CD25 antibody as described herein and an antibody that acts as an agonist of the T-cell activation co-stimulatory pathway. The kit may include instructions for use.

Any aspect of the invention as described herein can be combined with other therapeutic agents, particularly other cancer therapies. In particular, the anti-CD25 antibody and optionally an immune checkpoint inhibitor according to the invention can be administered in combination with co-stimulatory antibodies, chemotherapy and/or radiotherapy (by applying radiation in vitro or by administering a radioconjugated compound), cytokine-based therapies, targeted therapies, monoclonal antibody therapies, vaccines or adjuvants, or any combination thereof.

As used herein, a chemotherapeutic entity refers to an entity that is destructive to cells, i.e., that which reduces cell viability. A chemotherapeutic entity can be a cytotoxic drug. Anticipated chemotherapeutic agents include, but are not limited to, alkylating agents, anthracyclines, epoetomycin, nitrosoureas, ethyleneimine/methylmelamine, alkyl sulfonates, alkylating agents, antimetabolites, pyrimidine analogs, epipodophyllotoxin, enzymes (e.g., L-asparaginase); biological response modifiers, such as IFNα, IFN-γ, IL-2, IL-12, G-CSF, and GM-CSF; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; anthraquinones; substituted ureas (e.g., hydroxyurea); methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine; and adrenal glands. Corticosteroid inhibitors (e.g., mitotane (o,p'-DDD) and aminoacetylimine); hormones and antagonists, including corticosteroid antagonists such as prednisone and its equivalents, dexamethasone and aminoacetylimine; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens such as tamoxifen; androgens including testosterone propionate and flumethasone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprorelin; and nonsteroidal antiandrogens such as flutamide.

Other cancer therapies may include administered cancer vaccines. As used herein, “cancer vaccine” refers to a therapeutic cancer vaccine administered to a cancer patient and designed to eradicate cancer cells by enhancing the patient’s own immune response. Cancer vaccines include tumor cell vaccines (autologous and allogeneic), dendritic cell vaccines (ex vivo generated and peptide-activated), protein/peptide-based cancer vaccines, and gene vaccines (DNA, RNA, and virus-based vaccines). Therefore, in principle, therapeutic cancer vaccines can be used to inhibit the further growth of advanced cancers and/or recurrent tumors that are difficult to treat with conventional therapies such as surgery, radiation therapy, and chemotherapy. Tumor cell-based vaccines (autologous and allogeneic) include genetically modified soluble immunostimulants such as cytokines (IL-2, IFN-γ, IL-12, GMCSF, FLT3L), single-chain Fv antibodies against immunomodulatory receptors (PD-1, CTLA-4, GITR, ICOS, OX40, 4-1BB), and/or ligands expressing immunostimulatory receptors on their tumor cell membranes (e.g., ICOS ligand, 4-1BB ligand, GITR ligand, and/or OX40 ligand). In one embodiment, the cancer vaccine may be a GVAX antitumor vaccine.

Other cancer therapies could include other antibodies or small molecule agents that reduce immune regulation in the peripheral and tumor microenvironment, such as molecules targeting the TGFβ pathway, IDO (indoleamine deoxygenase), arginase, and/or CSF1R.

"Combination" can refer to administering another therapy before, simultaneously with, or after administering any aspect of the invention.

The invention will now be further described with reference to the accompanying drawings and the following embodiments, which are intended to help those skilled in the art to implement the invention and are not intended to limit the scope of the invention in any way.

Attached Figure Description

Figure 1 illustrates the characterization of the 7D4 and PC61 anti-mouse CD25 antibodies used in the examples. The effect on IL-2 binding was assessed using a tandem format cross-blocking assay. Biotinylated mouse CD25 was added to the SA sensor. The sensor was then exposed to 100 nM mouse IL-2, followed by exposure to the anti-mouse CD25 antibody at 150 seconds. Additional antibody binding after IL-2 association indicated that the anti-mouse CD25 did not block IL-2 binding, while the lack of further binding indicated ligand blockage. PC-61mIgG2a showed interference with the mouse IL-2/mouse CD25 interaction compared to 7D4(A). The mouse IL-2/mouse CD25 interaction in the presence of recombinant anti-mouse CD25 7D4(mIgG1) was assessed using a standard sandwich cross-blocking assay. 7D4(mIgG1) was added to the AHQ sensor, and unoccupied Fc binding sites on the sensor were blocked with an unrelated human IgG1 antibody. The sensor was then exposed to 100 nM recombinant mouse CD25 (R&D Systems; catalog 2438-RM-050), followed by recombinant mouse IL-2 (Peprotech; catalog 212-12). Additional binding of mouse IL-2 to 7D4 (mIgG1) mouse CD25 after association indicated an unoccupied epitope, where neither 7D4 (mIgG1) nor mouse IL-2 competed for the epitope in mouse CD25 due to co-binding with mouse CD25. (B) Binding to mouse CD25 was determined using CHO cells expressing mouse CD25 against the anti-human CD25 binding antibody dalizumab (DAC), PC61 (mIgG2a) antibody (original anti-CD25 obtained from clone PC-61 with mouse IgG2a and κ constant regions associated with ADCC), and a7D4 (mIgG1) antibody (anti-D25 obtained from clone 7D4 with mouse IgG1 and κ constant regions). Anti-mouse CD25 IgG binds to cell-expressed mCD25. CHO-mCD25 cells were aliquoted into 96-well assay plates (50,000 cells/well) and incubated for 15 min at 25°C with a solution containing 0.1 mL of antibody (100 nM antibody in PBS + 0.1% bovine serum albumin). Cells were washed three times with ice-cold PBS + 0.1% bovine serum albumin, then labeled with goat anti-human IgG (γ-chain specific) R-PE (Southern Biotech, catalog number 2040-09) and analyzed by flow cytometry (propidium iodide was used to distinguish dead cells). No binding to DAC was detected, while PC61 (mIgG2a) and 7D4 (mIgG1) showed significant binding to these cells (C).

Figure 2 illustrates the effect of anti-mouse CD25 antibody on CD4 T cell-induced granzyme B expression following stimulation with anti-CD3 and anti-CD28. Total CD4-positive T cells were isolated from mouse lymph nodes and spleen using CD4 MicroBeads and labeled with CellTrace ^™ purple dye (ThermoFisher) for measuring cell proliferation. Labeled T cells (10⁵) were seeded in 96-well plates with feeder cells (10⁵; CD90.2 negative fraction, using a mouse pan T Dynabeads kit). Anti-CD3 (clone 145-2C11, BioXcell catalog BE0001-1; 1 μg/ml) and anti-CD28 (clone 37.51, BioXcell catalog BE0015-1; 0.5 μg/ml) were added to the wells (in addition to a control sample containing unstimulated labeled T cells) to activate CD4 T cells and induce proliferation and granzyme B production. The following antibodies (at 25 μg/mL) were then further added 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 served as negative controls): PC61 (mIgG2a), 7D4 (mIgG1), or neutralizing anti-mouse IL-2 antibody (clone Jes6-1A12, BioXcell6032988564) served as positive controls to demonstrate the effect of blocking the interaction between IL-2 and its receptor on T cell activation. The labeled T cell samples were then incubated for approximately 84 hours. The cells were then fixed and permeabilized, and stained with anti-mouse granzyme B antibody (clone GB11, Invitrogen). Granzyme B expression in the cells was then analyzed by flow cytometry using CellTrace purple dye dilution (BV450). The percentage of T cells that proliferate and express granzyme B (GnzB) is shown in the upper left quadrant of each treatment-specific plot (Q9), while the percentage of T cells that proliferate but do not express GnzB is shown in the lower left quadrant of each treatment-specific plot (Q12).

Figure 3 illustrates the in vivo effects of an isoform of anti-mouse CD25 antibody (mouse IgG2a) on immune cells, with or without blocking the interaction between CD25 and IL-2, in the presence or absence of anti-mouse PD1 (aPD1; clone RMP1-14). On day 0, six groups of mice were subcutaneously injected with MCA205 tumor cells (5 × ^10⁵¹⁵ ) and treated as shown in the figure. In four groups, anti-mouse CD25 antibody (aPC61 mIgG2a or a7D4 mIgG2a; 200 μg) was injected intraperitoneally on day 5. In three groups, APD1 (100 μg) was injected intraperitoneally on days 6 and 9 with aPD1 (100 μg). Tumors and lymph nodes were harvested on day 12 and then processed according to the desired type for cell staining and analysis by flow cytometry using the following antibodies, as indicated in each figure: 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 FoxP3 transcription factor staining buffer (eBioscience). The percentages of CD4-positive/Foxp3-positive regulatory T cells and CD4-positive/FoxP3-negative effector CD4 T cells (CD4 Teff) in LN and TIL are shown, as well as the ratios of effector CD8-positive T cells/Treg cells and CD4 Teff/Treg cells. Data analysis was performed in Flowjo version 10.0.8 (Tree Star Inc.). Statistical analysis was performed in Prism 6 (GraphPad Software, Inc.); p-values were calculated using Kruskall-Wallis ANOVA and Dunn's post-hoc test (ns = p >0.05; **** = p < 0.0001).

Figure 4 illustrates the effects of an isotype of anti-mouse CD25 antibody (IgG2a) that blocks or does not block the interaction between CD25 and IL-2 in vivo on granzyme B production by proliferating T cells, in the presence or absence of anti-mouse PD1 (aPD1; clone RMP1-14). Cell samples were generated in six treatment groups as shown in Figure 3 using an MCA205-based model. Cells were then stained according to the desired type and analyzed by flow cytometry using the following antibodies as indicated in each figure: anti-CD3 (PeCy7, clone 145-2C11, eBioscience, 25003182), anti-CD4 (V500, clone RM4-5, BDbiosciences, 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). Ki67 and granzyme B were stained in the nucleus using the FoxP3 transcription factor staining buffer kit (eBioscience, 00-5523-00). The percentage of GnzB positive cells was compared with the total number of GnzB positive proliferators (as indicated by Ki67 positivity) and the total number of CD4 positive or CD8 positive T cells. Statistical analysis was performed as shown in Figure 3 (ns = p > 0.05; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001).

Figure 5 illustrates the effect of anti-mouse CD25 (IgG2a isotype) administered in combination with or without anti-PD-1 (clone RMP1-14) on eradicating established tumors in a CT26 mouse model. Both 7D4m2a and PC61m2a are anti-mouse CD25 and Treg-eliminating antibodies, but one is non-IL-2 blocking (7D4m2a) while the other is IL-2 blocking (PC61m2). Growth curves for individual mice over time were generated for each treatment group. Each figure indicates the number of tumor-free survivors after 50 days. CT26 cells for implantation were harvested during the logarithmic growth phase and resuspended in cold PBS. On day 1 (D1), 3 × ^10⁵ cells (0.1 mL cell suspension) were subcutaneously injected into the right abdomen of each mouse. On day 6, anti-mouse CD25 (10 mg/kg) was injected intraperitoneally (when palpable tumors were detected). Anti-PD1 was administered intraperitoneally on days 7, 10, 14, and 15. Tumor growth was measured twice weekly in two dimensions using calipers. Tumor size ( ^mm³ ) was calculated using the formula: Tumor volume = (w² × 1)/2, where w = width of the tumor and l = length of the tumor (mm). The study endpoint was a tumor volume of 4000 ^mm³ or 50 days, whichever came first (data points that stopped at earlier dates were due to mouse mortality; the number of surviving animals at the end of the experiment is indicated in each graph).

Figure 6 shows the CT26 tumor growth curves of individual mice that were untreated (PBS, solvent only) and treated with anti-mouse CD25 IgG2a antibodies that both consumed Tregs, but one was non-IL-2 blocking (7D4m2a) and the other was IL-2 blocking (PC61m2), or further treated with or without anti-mouse PD-L1 clone 10F.9G2 (aPDL1; clone 10F.9G2). The model, protocol, and data analysis were the same as in Figure 5.

Figure 7 illustrates the effect of anti-mouse CD25 (IgG2a isotype) administered in combination with or without anti-PD-1 (clone RMP1-14) on eradicating established tumors in an MC38 mouse model. The antibodies tested are those described in Figure 5. Growth curves for individual mice over time were established for each treatment group. The number of tumor-free survivors after 35 days is indicated in each figure. MC38 colon cancer cells for implantation were harvested 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 abdomen of each mouse. Tumors were monitored when the tumor volume approached the target range of 100 to 150 ^mm³ . 22 days after tumor implantation, on day 1 of the study, animals with individual tumor volumes ranging from 75 to 126 ^mm³ were divided into 9 groups (n = 10), with a group mean tumor volume of approximately 106 ^mm³ . Treatment was initiated on day 1 in mice with established MC38 tumors. On days 1, 2, 5, 9, and 12, the effects of each treatment were compared with those of the control group treated with PBS intraperitoneally. Anti-PD1 was administered twice weekly at 100 μg/animal for two weeks (biwk x 2), starting on day 2. 7D4m2a and PC61m2a were administered intraperitoneally once daily at 200 μg/animal on day 1. Tumor measurements were taken twice weekly. The study endpoint was a tumor volume of 4000 ^mm³ or 35 days, whichever came first (data points ending at earlier dates were due to mouse mortality; the number of surviving animals at the end of the experiment is indicated in each graph).

Figure 8 shows the MC38 tumor growth curves of individual mice that were untreated (PBS, solvent only) and treated with anti-mouse CD25 IgG2a antibodies that both consumed Tregs, but one was non-IL-2 blocking (7D4m2a) and the other was IL-2 blocking (PC61m2), or further treated with or without anti-mouse PD-L1 clone 10F.9G2 (aPDL1; clone 10F.9G2). The model, protocol, and data analysis were the same as in Figure 7.

Figure 9: Evaluation of the therapeutic activity of 7D4 mIgG2a alone (D) and in combination with an IL-2 neutralizing antibody (E) as a non-IL-2 blocking and Treg-depleting anti-mouse CD25 antibody, or a mouse IgG1 isotype IL-2 blocking and non-depleting anti-mouse CD25 antibody (PC61 mouse IgG1), in mice carrying CT26 syngeneic colon tumors from female BALB/c mice. The activities of mouse IgG2s control (A), IL-2 neutralizing antibody alone (B), and IL-2 blocking anti-CD25 antibody alone (C) were compared.

Figure 10: Shows the common sequence of human CD25 (Uniprot code P01589), referred to herein as SEQ ID NO:1. The extracellular domains of mature CD25 corresponding to amino acids 22 to 240 are underlined. Epitope positions from non-IL-blocking anti-CD25 antibodies were preliminarily identified as follows: epitope 1 (intact and short epitopes), epitope 2 (intact and short epitopes), epitope 3, and epitope 4 (intact and short epitopes). The positions of balithizumab and dalizumab epitopes (denoted as DAC) were also identified.

Figure 11: Characterization of (A)7D4, (B)PC61 and (C)2E4 binding to CD25 on CHO cells expressing CD25 at increased antibody concentrations and compared with mouse IgG2a isotype control.

Figure 12: SPR-based analysis of his-tagged anti-rmCD25 antibody performed on Biacore 2000, (A) 7D4, (B) 2E4.

Figure 13: Competitive analysis of his-tagged anti-rmCD25 antibodies performed on Octet96. Shows binding of 7D4 to the secondary antibody after capture at the sensor and antigen-association steps. Competitive binding to mCD25 was observed between 7D4 and 2E4 (A), but not between 7D4 and PC61 (B).

Figure 14: Characterization of 7D4, PC61, and 2E4 inhibition by blocking IL-2 signaling in a STAT5 phosphorylation assay using T cells isolated from C57BL/6 splenocytes, 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. The percentage of Treg cells limited to STAT5 phosphorylation was analyzed.

Figure 15: In vivo consumption of Tregs in balb/c mice carrying 4T1 tumors after administration of mouse anti-mouse CD25(7D4) antibody. (A) to (C): % of non-CD4, CD4+, and CD25+FoxP3+ cells in whole blood on day 3 post-administration. (D) to (F): % of non-CD4, CD4+, and CD25+FoxP3+ cells in tumors on day 3 post-administration. (G) to (I): % of non-CD4, CD4+, and CD25+FoxP3+ cells in whole blood on day 9 post-administration. (J) to (L): % of non-CD4, CD4+, and CD25+FoxP3+ cells in tumors on day 9 post-administration.

Figure 16: Characterization of CD25-binding mice (B) or chimeric (A, C, and D) anti-human CD25 clone 7G7B6 expressed on Karpas 299 cells (A), human in vitro differentiated Treg cells (B), SU-DHL-1 cells (C), or SR-786 cells (D) at increased antibody concentrations and compared with human IgG1 isotype control.

Figure 17: Characterization of 7G7B6 cells by blocking IL-2 signaling in a STAT5 phosphorylation assay using human-derived PBMCs, compared to mouse IgG2a isotype control, human IgG1 isotype control, dalizumab, or the absence of a primary antibody. Cells were incubated with 10 μg/ml antibody, followed by increasing the IL-2 concentration (as shown in the figure). The percentage of CD3-positive cells limited to phosphorylated STAT5 was analyzed.

Figure 18: Functional characterization of chimeric 7G7B6 cells using pan-universal T cells compared to a human IgG1 isotype control, dalizumab, 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, activated with CD3/CD28 beads for 72 h, and then analyzed by flow cytometry. Results show the percentage of granzyme B-positive proliferating CD4 T cells.

Figure 19: Characterization of the killing effect on CD25-positive cell lines and the function of chimeric 7G7B6 cells in ADCC assays compared to human IgG1 isotype controls. SU-DHL-1 (A) or SR-786 cells (B) with high or low CD25 expression were co-cultured with purified NK cells in the presence of different antibody concentrations (as shown in the figure). Target cell lysis was measured by releasing calcein into the supernatant 4 hours after calcein addition to NK cells. Data were normalized to a saponin-treated control.

Figure 20: Characterization of chimeric 7G7B6 function by phagocytosis of in vitro differentiated Treg cells compared to human IgG1 isotype in ADCP assay. Tregs were co-cultured with MCSF-differentiated macrophages in the presence of different antibody concentrations (as shown in the figure). Two-color flow cytometry analysis was performed on CD14+ stained macrophages and eFluor450-labeled Tregs. Residual target cells were defined as eFluor450-dye ⁺ / ^CD14- cells. Double-labeled cells (eFluor450-dye+/CD14+) were considered to represent macrophage phagocytosis of the target. Phagocytosis of target cells was calculated using the following equation: % phagocytosis = 100 × [(double positive %) / (double positive % + residual target percentage)].

Figure 21: Characterization of MA-251 binding to CD25 expressed on Karpas299 cells at increased antibody concentrations and compared with mouse IgG1 isotype control.

Figure 22: Characterization of MA-251 compared to mouse IgG1 isotype control, human IgG1 isotype control, dalizumab, or the absence of a primary antibody. Blockage of IL-2 signaling in the STAT5 phosphorylation assay was assessed using human PBMCs. Cells were incubated with 10 μg/ml antibody, followed by increasing the IL-2 concentration (as shown in the figure). The percentage of CD3-positive cells limited to phosphorylated STAT5 was analyzed.

Figure 23: Characterization of MA-251 and IL-2 binding to CD25. Interference with IL-2 ligand binding to CD25 was performed using a standard sandwich assay on a Forte BioOctet Red384 system (Pall Forte Bio Corp., USA). MA251 antibody was loaded onto the AHQ sensor, and unoccupied Fc binding sites on the sensor were blocked with an unrelated human IgG1 antibody. The sensor was exposed to 100 nM human CD25, followed by exposure to 100 nM human IL-2. Data were processed using Forte Bio DataAnalysis Software 7.0. Additional binding of human IL-2 after antigen association indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blocking (competitor).

Figure 24: Competitive analysis of anti-CD25 antibodies in Octet. The first Ab was bound to immobilized rhCD25, and then to either the first Ab (as a control) or the second Ab. The mAbs acting as non-blockers of IL-2 signaling competed with each other or with 7G7B6 and MA251, but not with the study dalizumab or the study baliximab (Figure 24(A) to (N)). (A) to (C) Competitive analysis of 7G7B6; (D) to (F) Competitive analysis of MA251; (G) to (I) and (N) Competitive analysis of antibody 3; (J) to (M) Competitive analysis of antibody 1. The mAb acting as an IL-2 signaling blocker (TSK031) did indeed compete with the study dalizumab and the study baliximab, but not with 7G7B6 (Figure 24(O) to (Q)).

Figure 25: In vivo models of tumor growth inhibition after drug administration: vectors (A) and (C); or antibody 1 (B), (D) and (E).

Figure 26: Affinity determination by SPR-based analysis of his-tagged anti-rhCD25 antibodies performed on a Biacore 2000. A) 7g7B6ch, B) MA251ch, C) Antibody 1, D) Antibody 3, and E) Dalizumab (control) or by biolayer interferometry on an Octet Red 96 instrument (F).

Figure 27: Characterization of antibody 1 that binds to CD25 expressed on in vitro differentiated Treg cells (A), SU-DHL-1 cells (B), or SR-786 cells (C) at increased antibody concentrations and compared with human IgG1 isotype control.

Figure 28: Characterization of antibody 1, which binds to CD25 expressed on CD3/CD28 bead-activated human (A) and (B) pan T cells and is then gated on CD4 ⁺ and CD8 ⁺ , at increased antibody concentrations and compared with human IgG1 isotype control.

Figure 29: Biolayer interference assays performed on an Octet Red384 using a standard sandwich assay show non-competitive binding of antibody 1 to IL-2 (A) and competitive binding of IL-2 to antibody 1 (B). Antibody 1 (anti-human CD25) was added to the AHQ sensor. The sensor was then exposed to 100 nM human CD25, followed by exposure to human IL-2. Additional binding of human IL-2 after antigen association indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blockade (competitor).

Figure 30: Biolayer interference assay performed on an Octet Red384 using a standard sandwich-type grouping assay, showing the non-competitive binding of antibody 1 and dalizumab to CD25. The reference monoclonal anti-human CD25 antibody, dalizumab, was added to the AHQ sensor. The sensor was then exposed to 100 nM human CD25 antigen, followed by exposure to the anti-human CD25 antibody (antibody 1). Additional binding of the secondary antibody after antigen association indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blockade (competitor).

Figure 31: Characterization of antibody 1 for blocking IL-2 signaling in the STAT5 phosphorylation assay using human-derived PBMCs, compared to human IgG1 isotype control, dalithiazide, or in the absence of a primary antibody. Cells were incubated with 10 μg/ml antibody, followed by increasing the concentration of IL-2 (as shown in the figure). The percentage of CD3-positive cells limited to phosphorylated STAT5 was analyzed.

Figure 32: Functional characterization of antibody 1 using pancreatic T cells, compared with human IgG1 isotype control, dalizumab, 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, then activated with CD3/CD28 beads for 72 h, followed by flow cytometry analysis. Results showed the percentage of granzyme B-positive proliferating CD4(A) or CD8(B) T cells.

Figure 33: Characterization of antibody 1 function regarding killing of CD25-positive cell lines in ADCC assays compared to human IgG1 isotype controls. SU-DHL-1 (A) or SR-786 (B) cells with high or low CD25 expression were co-cultured with purified NK cells in the presence of different antibody concentrations (as shown in the figure). Target cell lysis was measured by releasing calcein into the supernatant 4 hours after calcein addition to NK cells. Data were normalized to a saponin-treated control.

Figure 34: Characterization of antibody 1 function regarding phagocytosis of 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 different antibody concentrations (as shown in the figure). Two-color flow cytometry analysis was performed on CD14+ stained macrophages and eFluor450-labeled Treg cells. Residual target cells were defined as eFluor450-dye ⁺ / ^CD14- cells. Double-labeled cells (eFluor450-dye+/CD14+) were considered representative of macrophage phagocytosis of the target. Phagocytosis of target cells was calculated using the following equation: % phagocytosis = 100 × [(double positive %) / (double positive % + residual target percentage)].

Figure 35: Characterization of antibody 3 that binds to CD25 expressed on in vitro differentiated Treg cells (A), SU-DHL-1 cells (B), or SR-786 cells (C) at increased antibody concentrations and compared with human IgG1 isotype control.

Figure 36: Characterization of antibody 3, which binds to CD25 expressed on CD3/CD28 bead-activated human (A) and (B) or cynomolgus monkey (C) and (D) pancreatic T cells and is then gated on CD4 ⁺ and CD8 ⁺ cells, at increased antibody concentrations and compared with human IgG1 isotype control.

Figure 37: Biolayer interference assay performed on an Octet Red384 using a standard sandwich-type grouping assay, demonstrating the non-competitive binding of antibody 3 and IL-2. Antibody 3 (anti-human CD25) was added to the AHQ sensor. The sensor was then exposed to 100 nM human CD25, followed by exposure to human IL-2. The additional binding of human IL-2 after antigen association indicates an unoccupied epitope (non-competitor).

Figure 38: Biolayer interference assay performed on an Octet Red384 using a standard sandwich-type grouping assay, showing the non-competitive binding of antibody 3 and dalizumab to CD25. The reference monoclonal anti-human CD25 antibody, dalizumab, was added to the AHQ sensor. The sensor was then exposed to 100 nM human CD25 antigen, followed by exposure to the anti-human CD25 antibody (antibody 3). Additional binding of the secondary antibody after antigen association indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blockade (competitor).

Figure 39: Characterization of antibody 3 for blocking IL-2 signaling in the STAT5 phosphorylation assay using human-derived PBMCs, compared to human IgG1 isotype control, dalithiazide, or in the absence of a primary antibody. Cells were incubated with 10 μg/ml antibody, followed by increasing the concentration of IL-2 (as shown in the figure). The percentage of CD3-positive cells limited to phosphorylated STAT5 was analyzed.

Figure 40: Functional characterization of antibody 3 using pancreatic T cells, compared with human IgG1 isotype control, dalizumab, 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, then activated with CD3/CD28 beads for 72 h, followed by flow cytometry analysis. Results showed the percentage of granzyme B-positive proliferating CD4(A) or CD8(B) T cells.

Figure 41: Functional characterization of antibody 3 in killing CD25-positive cell lines in ADCC assay compared with human IgG1 isotype control. CD25-high or low-expressing cells, SU-DHL-1 (A) or SR-786 cells (B), were co-cultured with purified NK cells in the presence of different antibody concentrations (as shown in the figure). Target cell lysis was measured 4 hours after the addition of NK cells by releasing calcein into the supernatant. Data were normalized to a saponin-treated control.

Figure 42: Characterization of antibody 3 function regarding phagocytosis of 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 different antibody concentrations (as shown in the figure). Two-color flow cytometry analysis was performed on CD14+ stained macrophages and eFluor450-labeled Treg cells. Residual target cells were defined as eFluor450-dye ⁺ / ^CD14- cells. Double-labeled cells (eFluor450-dye+/CD14+) were considered representative of macrophage phagocytosis of the target. Phagocytosis of target cells was calculated using the following equation: % phagocytosis = 100 × [(double positive %) / (double positive % + residual target percentage)].

Figure 43: Characterization of antibody 4 that binds to CD25 expressed on in vitro differentiated Treg cells (A), SU-DHL-1 cells (B), or SR-786 cells (C) at increased antibody concentrations and compared with human IgG1 isotype control.

Figure 44: Characterization of antibody 4 binding to unmodified CHO-S cells (negative control) (A) or macaque-CD25-CHO-S cells (B) at a 100 nM antibody concentration and compared with human IgG1 isotype control.

Figure 45: Biolayer interference assay performed on an Octet Red384 using a standard sandwich-type grouping assay, demonstrating the non-competitive binding of antibody 4 and IL-2. Antibody 4 (anti-human CD25) was added to the AHQ sensor. The sensor was then exposed to 100 nM human CD25, followed by exposure to human IL-2. The additional binding of human IL-2 after antigen association indicates an unoccupied epitope (non-competitor).

Figure 46: Biolayer interference assay performed on an Octet Red384 using a standard sandwich-type grouping assay, showing the non-competitive binding of antibody 4 and dalizumab to CD25. The reference monoclonal anti-human CD25 antibody, dalizumab, was added to the AHQ sensor. The sensor was then exposed to 100 nM human CD25 antigen, followed by exposure to the anti-human CD25 antibody (antibody 4). Additional binding of the secondary antibody after antigen association indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blockade (competitor).

Figure 47: Characterization of antibody 4 for blocking IL-2 signaling in the STAT5 phosphorylation assay using human-derived PBMCs, compared to human IgG1 isotype control, dalizumab, or in the absence of a primary antibody. Cells were incubated with 10 μg/ml antibody, followed by increasing the concentration of IL-2 (as shown in the figure). The percentage of CD3-positive cells limited to phosphorylated STAT5 was analyzed.

Figure 48: Functional characterization of antibody 4 using pancreatic T cells, compared with human IgG1 isotype control, dalizumab, 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, then activated with CD3/CD28 beads for 72 h, followed by flow cytometry analysis. Results showed the percentage of granzyme B-positive proliferating CD4(A) or CD8(B) T cells.

Figure 49: Characterization of antibody 4's function regarding killing of CD25-positive cell lines in ADCC assays compared to human IgG1 isotype controls. SU-DHL-1 (A) or SR-786 (B) cells with high or low CD25 expression were co-cultured with purified NK cells in the presence of different antibody concentrations (as shown in the figure). Target cell lysis was measured by releasing calcein into the supernatant 4 hours after calcein addition to NK cells. Data were normalized to a saponin-treated control.

Figure 50: Characterization of antibody 4 for ADCP induction compared to human IgG1 isotype control in reporter bioassay. SU-DHL-1 cells expressing CD25 were co-cultured with Jurkat T cells genetically engineered to express FcγRIIA and the NFAT-RE-luc2 responsive element driving luciferase expression (as shown in the figure) in the presence of various antibody concentrations.

Figure 51: Characterization of antibody 2 that binds to CD25 expressed on in vitro differentiated Treg cells (A), SU-DHL-1 cells (B), or SR-786 cells (C) at increased antibody concentrations and compared with human IgG1 isotype control.

Figure 52: Characterization of antibody 2, which binds to CD25 expressed on CD3/CD28 bead-activated human (A) and (B) or cynomolgus monkey (C) and (D) pancreatic T cells and is then gated on CD4 ⁺ and CD8 ⁺ cells, at increased antibody concentrations and compared with human IgG1 isotype control.

Figure 53: Biolayer interference assays performed on an Octet Red384 using a standard sandwich assay show non-competitive binding of antibody 2 to IL-2 (A) and competitive binding of IL-2 to antibody 2 (B). Antibody 2 (anti-human CD25) was added to the AHQ sensor. The sensor was then exposed to 100 nM human CD25, followed by exposure to human IL-2. The additional binding of human IL-2 after antigen association indicates an unoccupied epitope (non-competitor).

Figure 54: Biolayer interference assay performed on an Octet Red384 using a standard sandwich-type grouping assay shows the non-competitive binding of antibody 2 and dalizumab to CD25. The reference monoclonal anti-human CD25 antibody, dalizumab, was added to the AHQ sensor. The sensor was then exposed to 100 nM human CD25 antigen, followed by exposure to the anti-human CD25 antibody (antibody 2). Additional binding of the secondary antibody after antigen association indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blockade (competitor).

Figure 55: Characterization of antibody 2 for blocking IL-2 signaling in the STAT5 phosphorylation assay using human-derived PBMCs, compared to human IgG1 isotype control, dalithiazide, or in the absence of a primary antibody. Cells were incubated with 10 μg/ml antibody, followed by increasing the concentration of IL-2 (as shown in the figure). The percentage of CD3-positive cells limited to phosphorylated STAT5 was analyzed.

Figure 56: Characterization of antibody 2 function regarding killing of CD25-positive cell lines in ADCC assays compared to human IgG1 isotype controls. CD25-high or low-expressing cells SU-DHL-1 (A) or SR-786 (B) were co-cultured with purified NK cells in the presence of different antibody concentrations (as shown in the figure). Target cell lysis was measured by releasing calcein into the supernatant 4 hours after calcein addition to NK cells. Data were normalized to a saponin-treated control.

Figure 57: Characterization of antibody 2 function regarding phagocytosis of 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 different antibody concentrations (as shown in the figure). Two-color flow cytometry analysis was performed on CD14+ stained macrophages and eFluor450-labeled Treg cells. Residual target cells were defined as eFluor450-dye ⁺ / ^CD14- cells. Double-labeled cells (eFluor450-dye+/CD14+) were considered representative of macrophage phagocytosis of the target. Phagocytosis of target cells was calculated using the following equation: % phagocytosis = 100 × [(double positive %) / (double positive % + residual target percentage)].

Figure 58: Characterization of antibody 5 binding to CD25 expressed on Karpas 299 cells at increased antibody concentrations and compared with human IgG1 isotype control.

Figure 59: Characterization of antibody 5, which blocks IL-2 signaling in the STAT5 phosphorylation assay using human PBMCs. Mouse anti-human antibody MA-251 was used as a non-blocking control, while the clinical dalizumab high-yield method (DAC HYP) was used as a blocking control, along with mouse IgG1 isotype control, human IgG1 isotype control, or no primary antibody, respectively. Cells were incubated with 10 μg/ml antibody, followed by incubation with 10 U/ml IL-2. The percentage of CD3-positive cells limited to phosphorylated STAT5 was analyzed.

Figure 60: Competitive assay in Octet. Antibody 1 was bound to immobilized rhCD25, and then to either a first Ab (as a control) or a second Ab, and to either an IL-2 competitor (e.g., dalizumab and baliximab studies) or a non-IL-2 competitor (e.g., 7G7B6). Antibody 1 did not compete with the IL-2 signaling blockers baliximab (A) and dalizumab (B), but it did compete with 7G7B6 (a non-IL-2 blocker) (C).

Figure 61: Characterization of antibody 5 for ADCC induction with anti-human CD25 Fc silencing control antibody in reporter bioassay. SR-786 cells expressing CD25 were co-cultured with Jurkat T cells genetically engineered to express FcγRIIA and the NFAT-RE-luc2 responsive element driving luciferase expression (as shown in the figure) in the presence of various antibody concentrations.

Figure 62: Characterization of antibody 5 function regarding phagocytosis of 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 different antibody concentrations (as shown in the figure). Two-color flow cytometry analysis was performed on CD14+ stained macrophages and eFluor450-labeled Treg cells. Residual target cells were defined as eFluor450-dye ⁺ / ^CD14- cells. Double-labeled cells (eFluor450-dye+/CD14+) were considered representative of macrophage phagocytosis of the target. Phagocytosis of target cells was calculated using the following equation: % phagocytosis = 100 × [(double positive %) / (double positive % + residual target percentage)].

Figure 63: Characterization of antibodies 6, 7, 8 and 9 that bind to CD25 expressed on Karpas 299 cells at increased antibody concentrations and compared with human IgG1 isotype control.

Figure 64: Competitive assay in Octet. The first Ab (antibody 7) was bound to immobilized rhCD25, and then to either the first Ab (as a control) or the second Ab, dalizumab (A) or baliximab (B).

Figure 65: Characterization of antibody 7 for blocking IL-2 signaling in the STAT5 phosphorylation assay using human-derived PBMCs, compared to human IgG1 isotype control, dalizumab-Hyp, or the absence of a primary antibody. Cells were incubated with 10 μg/ml antibody, followed by incubation with 10 U/ml IL-2. The percentage of CD3-positive cells limited to phosphorylated STAT5 was analyzed.

Figure 66: Functional characterization of antibody 7 in response to ADCC in reporter bioassay with anti-human CD25 Fc silencing control antibody. SR-786 cells expressing CD25 were co-cultured with Jurkat T cells genetically engineered to express FcγRIIA and the NFAT-RE-luc2 responsive element driving luciferase expression (as shown in the figure) in the presence of various antibody concentrations.

Figure 67: Characterization of antibody 7 function regarding phagocytosis of in vitro differentiated Treg cells in ADCP assays compared to the anti-human CD25Fc silencing control antibody. Tregs were co-cultured with MCSF-differentiated macrophages in the presence of different antibody concentrations (as shown in the figure). Two-color flow cytometry analysis was performed on CD14+ stained macrophages and eFluor450-labeled Tregs. Residual target cells were defined as eFluor450-dye ⁺ / ^CD14- cells. Double-labeled cells (eFluor450-dye+/CD14+) were considered to represent macrophage phagocytosis of the target. Phagocytosis of target cells was calculated using the following equation: % phagocytosis = 100 × [(double positive %) / (double positive % + residual target percentage)].

Figure 68: Characterization of antibodies 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 that bind to CD25 expressed on Karpas 299 cells at increased antibody concentrations and compared with human IgG1 isotype control.

Figure 69: Competitive assay in Octet. The first Ab (antibody 19) was bound to immobilized rhCD25, and then to either the first Ab (as a control) or the second Ab, dalizumab (A) or baliximab (B).

Figure 70: Characterization of antibodies 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 in the STAT5 phosphorylation assay using human-derived PBMCs, compared to human IgG1 isotype control, dalizumab-Hyp, or the absence of a primary antibody. Cells were incubated with 10 μg/ml of antibody, followed by incubation with 10 U/ml of IL-2. The percentage of CD3-positive cells limited to phosphorylated STAT5 was analyzed.

Figure 71: Functional characterization of antibody 19 in response to ADCC in reporter bioassay with anti-human CD25 Fc silencing control antibody. SR-786 cells expressing CD25 were co-cultured with Jurkat T cells genetically engineered to express FcγRIIA and the NFAT-RE-luc2 responsive element driving luciferase expression (as shown in the figure) in the presence of various antibody concentrations.

Figure 72: Characterization of antibody 19 function regarding phagocytosis of in vitro differentiated Treg cells in ADCP assays compared to the anti-human CD25Fc silencing control antibody. Tregs were co-cultured with MCSF-differentiated macrophages in the presence of different antibody concentrations (as shown in the figure). Two-color flow cytometry analysis was performed on CD14+ stained macrophages and eFluor450-labeled Tregs. Residual target cells were defined as eFluor450-dye ⁺ / ^CD14- cells. Double-labeled cells (eFluor450-dye+/CD14+) were considered to represent macrophage phagocytosis of the target. Phagocytosis of target cells was calculated using the following equation: % phagocytosis = 100 × [(double positive %) / (double positive % + residual target percentage)].

Figure 73: Functional characterization of antibodies 19, 12, and 20 regarding phagocytosis of in vitro differentiated Treg cells in ADCP assays compared to the anti-human CD25Fc silencing control antibody. Tregs were co-cultured with MCSF-differentiated macrophages in the presence of different antibody concentrations (as shown in the figure). Two-color flow cytometry analysis was performed on CD14+ stained macrophages and eFluor450-labeled Tregs. Residual target cells were defined as eFluor450-dye ⁺ / ^CD14- cells. Double-labeled cells (eFluor450-dye+/CD14+) were considered representative of macrophage phagocytosis of the target. % phagocytosis of target cells was calculated using the following equation: % phagocytosis = 100 × [(double positive %) / (double positive % + residual target percentage)].

Figure 74: Therapeutic activity of non-IL-2 blocking anti-CD25 antibody 7D4 mouse IgG2a in combination with BVAB16 immunotherapy in a BVAB16 immunotherapy resistance model. Individual mice were treated with Gvax alone or in combination with 7D4.

Figure 75: Therapeutic activity of non-IL-2 blocking anti-CD25 antibodies (7D4 and 2E4) compared to the IL-2 blocking antibody (PC61) in a CT26 tumor model using female BALB/c mice. The non-IL-2 blocking anti-CD25 antibodies 7D4 and 2E4 exhibited effective therapeutic activity against solid tumors. Both 7D4 and 2E4 were more effective than the IL-2 blocking antibody PC61.

Figure 76: Evaluation of the therapeutic activity of the non-IL-2 blocking anti-CD25 antibody 7D4 mIgG2a in the MCA205 model under single and repeated injections in combination with anti-mouse PD-L1. * indicates mice that survived at the end of the experiment.

Specific Implementation

Example 1: In vitro characterization and preparation of recombinant anti-mouse CD25 and Treg-consuming antibodies, whether non-IL-2 blocking or IL-2 blocking.

Materials and Methods

The origin of antibodies and their recombination.

The heavy and light chain variable regions of the rat anti-mouse CD25 antibody PC61 were resolved from the PC-61.5.3 hybridoma (ATCC catalog number TIB-222) by rapid amplification of cDNA ends (RACE), and then cloned into the constant region and κ chain of mouse IgG2a (or the corresponding mouse IgG1 sequence isolated from the commercial plasmid (Invivogen).

Each antibody chain was then subcloned into a murine leukemia virus (MLV)-derived retroviral vector. For preliminary experiments, antibodies were generated using K562 cells transduced with vectors encoding both the heavy and light chains. Antibodies were purified from the supernatant using a Protein G HiTrapMabSelect column (GE Healthcare), dialyzed in phosphate-buffered saline (PBS), concentrated, and filtered sterilized.

The recloned anti-mouse CD25 heavy chain variable strand DNA from the PC-61.5.3 antibody (mouse IgG2a) encodes the following protein sequence:

METDTLLLWVLLLWVPGSTGEVQLQQSGAELVRPGTSVKLSCKVSGDTITAYYIHFVKQRPGQGLEWIGRIDPEDDSTEYAEKFKNKATITANTSSNTAHLKYSRLTSEDTATY FCTTDNMGATEFVYWGQGTLVTVSS

The recloned anti-mouse CD25 light chain variable strand DNA from the PC-61.5.3 antibody (mouse IgG2a) encodes the following protein sequence:

METDTLLLWVLLLWVPGSTGQVVLTQPKSVSASLESTVKLSCKLNSGNIGSYYMHWYQQREGRSPTNLIYRDDKRPDGAPDRFSGSIDISSNSAFLTINNVQTEDEAMYFCHSYDGRMYIFGGGTKLTV

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. Variable heavy and light chains were amplified using degenerate forward primers binding to the signal peptide or frame 1 region and reverse primers binding to the antibody constant region. The amplified genes were cloned and sequenced according to standard methods. cDNA was generated by reverse transcription, and homopolymer tails were added to the 3' end of the cDNA. The antibody variable domain gene was then amplified using gene-specific primers, followed by standard cloning and sequencing methods. The DNA was sequenced using routine Sanger sequencing, and the data were analyzed using DNASTAR Lasergene software. The signal peptide and variable domain sequences were identified by comparison with known sequences in the IMGT database.

Genes encoding the variable heavy chain domain and variable light chain domain were codon-optimized for expression in human cell lines and synthesized at the NheI and AvaI restriction sites at the 5' and 3' of the genes. Restriction digestion cloning was performed to insert the 7D4 variable heavy chain domain gene into a separate expression vector containing the mouse IgG1 and IgG2a constant domains. Restriction digestion cloning was performed to insert the 7F4 variable light chain gene into an expression vector containing the mouse κ constant domain. HEK293 cells in serum-free medium suspension were co-transfected with the heavy and light chain expression vectors and cultured for 6 days at 37°C with 5% CO2 and shaking at 140 rpm. The cultures were harvested by centrifugation at 4000 rpm and further purified by filtration through a 0.22 μM filter. The supernatant was loaded onto a Protein A column pre-equilibrated with pH 7.2 PBS, eluted with sodium citrate at pH 3.5, and equilibrated with 10% (v/v) 0.5 M Tris at pH 9.0. The neutralized antibody solution was exchanged for buffer in pH 7.2 PBS using a desalting column and concentrated using a centrifuge 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.

The recloned anti-mouse CD25 heavy chain DNA sequence from the 7D4 antibody (mouse IgG1) encodes the following protein sequence:

EVQLQQSGAALVKPGASVKMSCKASGYSFPDSWVTWVKQSHGKSLEWIGDIFPNSGATNFNEKFKGKATLTVDKSTSTAYMELSRLTSEDSAIYYCTRLDYGYWGQGVM VTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCK PCICTVPEVSSVFIFPPKPKDVLMISLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPILHQDWLNGKEFKCRVNSAAFPAPIEKTIS KTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

The recloned anti-mouse CD25 heavy chain variable strand DNA sequence from the 7D4 antibody (mouse IgG2a) encodes the following protein sequence:

EVQLQQSGAALVKPGASVKMSCKASGYSFPDSWVTWVKQSHGKSLEWIGDIFPNSGATNFNEKFKGKATLTVDKSTSTAYMELSRLTSEDSAIYYCTRLDYGYWGQGVMVT VSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCP PCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTI SKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

The recloned anti-mouse CD25κ light chain DNA sequences of 7D4(mIg1) and 7D4(mIg2a) antibodies (mouse IgG2a) encode the following protein sequences:

DVVLTQTPPTLSATIGQSVSISCRSSQSLLHSNGNTYLNWLLQRPGQPPQLLIYLASRLESGVPNRFSGSGSGTDFTLKISGVEAEDLGVYYCVQSSHFPNTFGVGTKL EIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

2E4 was generated from a 2E4 hybridoma (a gift from Dr. Ethan M. Shevach of the National Institutes of Health). Hybridoma sequencing was performed using proprietary next-generation sequencing (NGS) technology. RNA samples were used to generate cDNA libraries. The libraries were sequenced on the Illumina platform. De novo assembly was used to reconstruct the sample transcriptome from the raw data. Variable domain sequences were identified by comparison with known sequences.

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

EVQLVESGGGLVQPGRSLKLSCAASGFTFSDYGMAWVRQAPTKGLEWVASITNGGLNTYYRDSVKGRFTISRDNAKCTLYLQMDSLRSEDTATYYCATGGFSFWGQGTLVTVSS

The variable light chain domain protein sequence for anti-mouse CD25 2E4 (mIg1) has the following protein sequence:

DIVMTQSPTSMSISVGDRVTMNCKASQNVDSNVDWYQQKTGQSPKLLIYKASNRYTGVPDRFTGSGSGTDFTFTIRNMQAEDLAVYYCMQSNSYPLTFGSGTKLEIK

Assess the affinity of the recombinant antibody for mouse CD25.

ForteBio affinity measurements are typically performed on an Octet RED384 as previously described (see, for example, Estep Pet al., 2013. Mabs. 5(2), 270-8). In short, ForteBio affinity measurements are performed by online loading of IgG onto the AHQ sensor. The sensor is equilibrated offline in assay buffer for 30 minutes, then monitored online for 60 seconds to establish a baseline. The IgG-loaded sensor is exposed to 100 nM antigen for 3 minutes, then transferred to assay buffer for 3 minutes for dissociation rate measurement. All kinetics are analyzed using a 1:1 binding model.

result

Two mouse hybridomas were selected as reference antibodies to evaluate the CD25 binding and Treg depletion properties of anti-mouse CD25 (7D4 (mouse IgM isotype) and PC61 (mouse IgG1 isotype), respectively, with or without IL-2 blocking. The IL-2 binding-related properties described in the literature have been preliminarily confirmed using the original non-recombinant antibody and recombinant mouse IL-2 (Figure 1A). Recombinant variants of this antibody have been generated for testing, where the isotype is more active and relevant to functional studies (e.g., Treg depletion or effects on other immune cells). Furthermore, in the case of 7D4, isotype modification is necessary because the antibody aggregation properties of IgM antibodies can affect the assay results. Recombinant 7D4 (mIgG1), as the original non-recombinant IgM isotype antibody, still allows mouse IL-2 to bind to mouse CD25 (Figure 1B). 7D4 (mIgG1) also binds to mouse CD25 on the cell surface, similar to recombinant PC61 (IgG2a), while the reference anti-human CD25 antibody does not bind (Figure 1C).

DNA sequences encoding the 7D4 heavy chain variable domain (and the PC61 heavy chain variable domain) have also been cloned into a vector that allows expression of a mouse CD25-binding domain with a mouse IgG2a isotype (functionally corresponding to human IgG1). In this way, two recombinant anti-mouse CD25 antibodies with optimized ADCC activity can be compared. These recombinant anti-mouse CD25 antibodies effectively deplete intratumoral Tregs but exhibit unique properties regarding the binding of mouse IL-2 to mouse CD25. The CD25 affinity of the resulting recombinant anti-mouse CD25 antibodies has been tested. Different isotypes (mouse IgG2a or mouse IgG1) do not affect this property because the Kd values between the 7D4-based recombinants are similar (approximately 1 nM) and comparable to one of the PC61 (mIgG2a) recombinants, measured at 4.6 nM.

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 protease expressed by memory T cells and NK cells, as well as activated CD4 and CD8 T cells that 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 (>80% of which are proliferating and Gnz-expressing CD4 T cells) with anti-CD3 and anti-CD28 antibodies can be influenced by cytokines and antibodies. When this stimulation is combined with a neutralizing anti-IL-2 antibody, granzyme B production, but not proliferation, is inhibited: the frequency of proliferating and GnzB-producing cells decreases from >80% to <1%, while the frequency of proliferating cells remains >90%. This indicates that granzyme B production depends on IL-2 signaling, while cell proliferation does not. When PC61 (mIgG1) was added to stimulated T cells, a similar decrease in granzyme B-producing T cells was observed. However, 7D4 (mIgG1) largely preserved the ability of CD4 T cells to respond to anti-CD3 and anti-CD28 stimulation by producing GnzB (>65% of which were still GnzB-producing cells and proliferating). These results confirm that PC61-based antibodies block IL-2 signaling, while 7D4 has only a small effect on this signaling, and therefore can be used as an alternative antibody to evaluate the therapeutic potential of anti-human CD25 antibodies that do not affect IL-2 signaling, particularly in terms of Treg depletion and tumor specificity.

Example 2: Treg depletion and antitumor properties of recombinant anti-mouse CD25 and Treg-depleting antibodies, whether non-IL-2 blocking or IL-2 blocking.

Materials and Methods

mice

In vivo studies were conducted by Charles River Discovery Services in North Carolina (CR Discovery Services). At the start of the study, female BALB/c mice (BALB/cAnNcr1, Charles River) and female C57BL/6 mice (C57BL/6Ncr1, Charles River) were between 7 and 9 weeks of age. CR Discovery Services specifically adhered to the recommendations in the "Guidelines for the Care and Use of Laboratory Animals" regarding restriction, husbandry, surgery, feed and fluid regulation, and veterinary care. CR Discovery Services' animal care and use program is accredited by the International Association for Assessment and Accreditation of Laboratory Animal Management to ensure compliance with recognized standards of laboratory animal care and use.

Cell lines and tissue cultures

MCA205 tumor cells (3-methylcholanthrene-induced weakly immunogenic fibrosarcoma cells; from G. Kroemer, Gustave Roussy Cancer Institute) were cultured in Duchenne 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 from Gibco). MC38 mouse colon cancer cells (CR discovery services) were grown to mid-log phase in Duchenne Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 2 mM glutamine, 100 U/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 for antibody production were cultured in phenol red-free Iscove modified Dulbecco medium (IMDM) supplemented with 10% IgG-free FCS (Life Technologies).

In vivo tumor experiments

Cultured tumor cells were digested with trypsin (MCA205) or without trypsin digestion (MC38 and CT26), washed, resuspended in PBS, and subcutaneously injected (sc) into the flank (5 × ^10⁵ cells for the MCA205 and MC38 models in C57BL/6 mice; 3 × ^10⁵ cells for the CT26 model in BALB/c mice). Antibodies were injected intraperitoneally (ip) at the time points described in the illustration. For functional experiments, tumors and draining lymph nodes were harvested 12 days after tumor implantation and processed by flow cytometry for analysis as described (Simpson et al. (2013) J Exp Med 210, 1695-710). For therapeutic experiments, tumors were measured twice weekly, and the product of three orthogonal diameters was calculated as volume.

Flow cytometry

Collection 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 (groin, axillary, and humeral) and tumors from mice were excised and placed in serum-free RPMI. Lymph nodes were dispersed through a 70 μm filter, and tumors were mechanically destroyed using gentleMACS (Miltenyl Biotech). The tumors were then digested for 30 minutes at 37°C in serum-free RPMI with a mixture of 0.33 mg/ml DNase (Sigma-Aldrich) and 0.27 mg/ml Liberase TL (Roche). The tumors were filtered through a 70 μm filter, and the resulting tumor single-cell suspension was enriched with leukocytes via Ficoll-paque (GE Healthcare) gradient passage. The tumors and lymph nodes were washed in pure RPMI, resuspended in FACS buffer (500 mL PBS, 2% FCS, 2 mM EDTA), and placed in round-bottom 96-well plates. Prepare the surface antibody mix (mastermix) using the manufacturer's recommended dilution method: 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 viable dye (eFlour780, eBioscience) is also included in the surface mix. After permeabilization for 20 minutes using the intracellular fixation and permeabilization buffer kit (eBioscience), apply the intracellular staining assembly, which consists of the following antibodies used for the manufacturer's recommended dilution method: anti-granzyme B (clone GB11, Invitrogen), anti-FoxP3 (clone FJK-16s, eBiosciences), and Ki67 (CloneSolA15, eBiosciences, 48569882) antibodies.

result

The MCA205 sarcoma mouse model enables the generation of mice capable of evaluating the immune response and overall efficacy against solid tumors against a panel of immunomodulatory compounds within a short timeframe. Specifically, a recombinant anti-mouse CD25 antibody based on mouse IgG2a was tested to assess changes in T cell subsets present as tumor-infiltrating lymphocytes or in peripheral lymph nodes, as well as tumor growth and survival in mice exposed to MCA205. Another antibody (anti-mouse PD1) was included as a negative control for the immunological effects of Tregs.

Immunological analysis showed that the 7D4 antibody, when cloned into the mouse IgG2a backbone, exhibited a similar ability to deplete Tregs as PC61 (mouse IgG2a), and subsequently increased the Teff/Treg ratio in both tumors and the periphery, while anti-PD1, whether used alone or in combination, was ineffective (Figure 3). Therefore, any further effects measured using 7D4 (mIgG2a) as an alternative antibody to non-IL-2 blocking anti-human CD25 antibodies do not appear to be related to the altered Treg depletion properties.

MCA205 model mice treated with 7D4 also showed a higher percentage of GnzB-positive cells, such as proliferating CD4-positive and CD8-positive T cells, involving not only anti-PD1 treatment but also blocking PC61's IL-2 (mIg2a). In such treated mice, not only did 7D4 (mIg2a), but PC61 (mIg2a) did not affect Teff cells, but it also increased the frequency of Teff cells compared to PC61 (mIg2a), indicating even higher antitumor activity of anti-human CD25 antibodies that do not block IL-2/CD25 interaction (Figure 4).

The use of anti-human CD25, functionally equivalent to 7D4 (mIg2a), for immunotherapy in cancers, particularly solid tumors, can be tested in the MCA205 mouse model and other models such as CT26 and MC38 (colon cancer) or B16 (melanoma) models. When combined with anti-PD1 antibodies, both IgG2a and anti-mouse CD25 antibodies showed therapeutic activity against established CT26 tumors. Interestingly, when used as monotherapy, non-IL-2 blocking 7D4 (mIg2a) antibodies showed significantly higher therapeutic activity than PC61-based antibodies of the same isotype. At the end of the experiment, all mice treated with 7D4 (mIg2a) showed tumor growth controlled to less than 50 ^mm³ , while none of the mice treated with PC61 (mIg2a) showed tumors smaller than 50 ^mm³ , with 8 out of 10 mice even reaching a stop-loss point of 2000 ^mm³ . This is also illustrated by the difference in survival rates: all mice treated with 7D4 (mIg2a) survived on day 50, while only 2 out of 10 mice treated with PC61 (mIg2a) survived. In fact, at least at this concentration, if the potency of PC61 (mIg2a) is significantly improved by combination with anti-PD1, the potency of 7D4 (mIg2a) is not further improved.

Since these 7D4- and PC61-based antibodies exhibit similar Treg-depleting abilities (see Figure 3), this difference in potency can be explained at least in part by the effect of 7D4(mIg2a) on the lower interaction between IL-2 and its receptor. This suggests that not only is the lack of IL-2/IL-2 receptor blocking activity harmless to therapeutic activity, but it can also provide a therapeutic advantage. Therefore, this data supports the selection of non-IL-2/IL-2 receptor antibodies that block CD25-targeting therapies for cancer treatment. These advantageous properties of the 7D4(mIg2a) antibody were also confirmed when used in the same CT26 mouse model with anti-mouse PD-L1 (Figure 6) or when used in the same combination with the antibody in the MC38 mouse model (Figures 7 and 8).

These data indicate that Treg depletion and CD25 binding properties of antibodies based on 7D4 characteristics and with appropriate isotypes can be used in combination with other anticancer compounds, such as antibodies targeting immune checkpoint proteins (e.g., antibodies against PD-1 and anti-PD-L1) or antibodies against other cancer-related targets. This approach can be achieved by generating and administering novel mixtures of monospecific antibodies or as two products of novel bispecific antibodies. This method, involving the construction of bispecific antibodies combining two antigen-binding properties and a treatment-related isotype (e.g., human IgG1), can be validated using Duobody technology, which allows efficient binding of the single-chain heavy and light chains from two different monospecific antibodies, which are generated separately and contain a single matching point mutation in the CH3 domain, thus allowing Fab exchange within a single heteropolymer (Labrijn AF et al., Nat Protoc. 2014, 9:2450-63). The functional properties of such 7D4-based Duobody products (e.g., including anti-PD1 or anti-PD-L1) can be evaluated using models of cell interactions and consumption of 7D4-based antibodies and antibody combinations as described above.

These results also suggest that the binding properties of 7D4 to mouse CD25 without interfering with the interaction between IL-2 and its receptor, and with IL-2 signaling in CD25-expressing cells, can be utilized in the selection of isotypes (e.g., human IgG1) of anti-human CD25 with similar mechanisms of action. In fact, several other properties can be considered for screening anti-human CD25 antibody candidates that offer further improvements in the preparation, use, and/or administration for the treatment of cancer, particularly solid tumors.

These properties can also be defined based on known anti-human CD25 characteristics, such as Humax-TAC, balithiumab, or dalizumab, which all have nanomolar Kd against human CD25 but block the binding of human IL-2 to human CD25 (using clone M-A251 as a potential reference non-IL-2 blocking anti-human CD25 antibody to be included in the selection of anti-human CD25 in this invention).

These functions can be one or more of the following:

- Affinity to recombinant or isolated monoclonal human CD25, wherein _KD is less than 25 nM, preferably less than 10 nM, and even more preferably less than 1 nM (e.g., established using techniques such as Octet, Kinexa, ELISA or other techniques).

- Cross-reactivity with recombinant and isolated monomeric cynomolgus CD25, wherein _KD is below 75 nM, preferably below 30 nM, and even more preferably below 3 nM (e.g., established using Octet, Kinexa, ELISA, etc.).

- Affinity to recombinant human CD25 on the surface of CHO or MJ cells, wherein _KD is less than 100 nM, preferably less than 10 nM, and even more preferably less than 1 nM (established using techniques such as flow cytometry, cell-based ELISA, etc.).

- Affinity to the recombinant rhesus macaque CD25 monomer on the surface of CHO cells, wherein _KD is less than 300 nM, preferably less than 30 nM, and even more preferably less than 3 nM (established using techniques such as flow cytometry, cell-based ELISA, etc.).

- Human Treg cells bind to KD at less than 100 nM, preferably less than 10 nM, and even more preferably less than 1 nM (using techniques such as flow cytometry, cell-based ELISA, etc.)

- Cynomolgus monkey Treg cells bind to KD at less than 300 nM, preferably less than 30 nM, or even more preferably less than 3 nM (using techniques such as flow cytometry, cell-based ELISA, etc.)

- Lack of inhibition of the interaction between recombinant human IL-2 and recombinant human CD25 in biochemical assays (as described in Example 1, less than 25% of IL-2 binding to CD25 was blocked during screening);

- Lack of IL-2-induced signaling in cell-based assays, such as STAT5 phosphorylation assays in activated CD8-positive or CD4-positive T cells or CD25-expressing cell lines, or granzyme B upregulation upon activation in CD4-positive T cells (as described in Example 1, less than 25% of baseline signal is suppressed); and/or

- Relevant power assessment in cell-based assays, such as ADCC, ADCP and/or CDC assays in cell lines expressing human CD25 or primary Treg cells (EC50 less than 10 nM, preferably less than 1 nM, even more preferably less than 0.1 nM).

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: Female BALB/c mice obtained from Charles River were subcutaneously injected with 3 × ^10⁵ CT26 tumor cells in 0% Matrigel, n = 15 per group. Animals were randomly assigned to treatment groups based on their body weight on day 1. Treatment began on day 6, with each animal receiving 200 μg/injection of each antibody (mouse IgG2a isotype, IL-2 neutralizing antibody, PC61mIgG1 (IL-2 signaling blocking anti-mouse CD25 antibody against mouse IgG1 isotype) and 7D4 mIgG2a (IL-2 signaling blocking anti-mouse CD25 antibody against mouse IgG2a isotype). Animals received monotherapy with one group of each antibody, or a combination of 7D4 mIgG2a and IL-2 neutralizing antibody or 7D4 mIgG2a and PC61 mIgG1 antibody. Mice were euthanized when the tumor volume reached 2000 ^mm³ or after 50 days, whichever came first.

Therapeutic activity of non-IL-2 blocking antibodies compared to blocking antibodies

Three × ^10⁵ CT26 cells were subcutaneously implanted into the lateral ventricular region. Matching was performed on day 0, when the tumor reached 30–60 ^mm³ , and treatment began. Treatment consisted of intraperitoneal administration of 10 mg/kg every two weeks from day 1 onwards. Patients were treated with the IL-2 neutralizing antibody PC61-m2a, the non-IL-2 blocking antibody 7D4, the non-IL-2 blocking antibody 2E4, or received no treatment.

Therapeutic activity of combination of non-IL-2 blocking antibodies and aPDL1 therapy

As shown in the figure, mice were subcutaneously injected with 50,000 MCA205 tumor cells, with n=10 or n=5 per group. Animals were randomly assigned to treatment groups. Animals received single therapy with 7D4 mIgG2a or aPD-L1 (clone 10F.9G2), combination therapy with 7D4 mIgG2a and PD-L1 (clone 10F.9G2), or no treatment. Group acceptance: a7D4 mIgG2a alone - day 10 (200ug), aPD-L1 rIgG2b (10F.9G2) - days 6, 9, and 12 (200ug), aPD-L1 + a7D4 combination (aPDL-1 received on days 6, 9, and 12, a7D4 received on day 10), or aPD-L1 + a7D4 combination (aPDL-1 received on days 6, 9, and 12, and a7D4 received on day 10), - additional injection of a7D4 on day 15 + aPD-L1 on day 18 (5 mice only).

result

The anti-CD25-consuming non-IL-2 blocking antibody 7D4 mIgG2a induced tumor rejection in treated mice, while other antibodies showed no effect as monotherapy compared to isotype control mouse IgG2a. Combination with an IL-2 blocking antibody (PC61mIgG1 or IL2nAb) eliminated the therapeutic activity of the non-IL-2 blocking antibody 7D4 mIgG2a (Figure 13). This indicates that the non-IL-2 blocking characteristic of 7D4 mIgG2a is key to its therapeutic activity. It also suggests that the therapeutic activity of this antibody depends on an anti-tumor immune response mediated by T effector cells that are dependent on IL-2 signaling for optimal activity. These results suggest that optimal therapeutic activity of CD25-targeting antibodies does not require IL-2/CD25 blocking activity and support the use of anti-CD25 non-IL-2 blocking antibodies as described herein in cancer therapy.

These results further demonstrate that the lack of IL-2/CD25 blocking activity is harmless to antibody therapeutic activity and support the use of anti-CD25 non-IL-2 blocking antibodies as described in this article in cancer treatment.

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

Results showed that single or repeated injections of the non-IL2-blocking aCD25 antibody 7D4 after initiation of aPDL1 therapy enhanced the antitumor response. The aCD25 antibody prevented Teff cell activation following aPDL1 treatment (Figure 76).

Example 4: Epitope Characterization of Anti-CD25 Non-IL-2 Blocking Antibody

Epitopebinning

Antibody epitope grouping was performed on a Forte Bio Octet Red384 system (Pall Forte Bio Corporation, Menlo Park, CA) using a standard sandwich-format grouping assay. Anti-mouse CD25PC61 antibody was added to the AMC sensor, and unoccupied Fc binding sites on the sensor were blocked with unrelated mouse IgG1 antibody. The sensor was then exposed to 15 nM target antigen, followed by exposure to 7D4 antibody. Data were processed using ForteBio's data analysis software 7.0. Additional binding of the secondary antibody after antigen binding indicated unoccupied epitopes (non-competitors), while no binding indicated epitope blocking (competitors).

Epitope mapping of anti-CD25 non-IL-2 blocking antibodies

Linear, monocyclic, β-turn mimics, disulfide mimics, discontinuous disulfide mimics, and discontinuous epitope mimics representing different groups of the human CD25 sequence (Uniprot record P01589) were synthesized using a solid-phase Fmoc synthesis method (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). The binding of antibodies to each synthetic peptide was tested in an ELISA (Pepscan, The Netherlands). The peptide array was incubated with the primary antibody solution overnight at 4°C. After washing, the peptide array was incubated with a 1/1000 dilution of a suitable antibody peroxidase conjugate (2010-05; Southern Biotech) at 25°C for 1 hour. After washing, peroxidase substrate 2,2'-azono-di-3-ethylbenzothiazoline _sulfonate (ABTS) and 20 μl/ml of 3% _H₂O₂ were added. Colorimetric analysis was performed after 1 hour. Colorimetric analysis was quantified using a charge-coupled device (CCD) camera and image processing system. Values obtained from the CCD camera ranged from 0 to 3000 mAU, similar to a standard 96-well plate ELISA reader. To verify the quality of the synthetic peptides, a set of separate positive and negative control peptides were synthesized in parallel and screened with unrelated control antibodies.

result

Epitope grouping was performed to determine whether the antibody bound to an epitope overlapping with that of the commercially available mouse anti-human non-IL-2 blocking CD25 antibody 7G7B6. The antibody was further characterized to identify the epitopes of the non-IL-2 blocking antibody. The epitopes of the anti-mouse CD25 blocking antibody PC61 were determined for comparison. The epitope plotting results for the anti-human CD25 antibody (Table 1) are shown in Table 1, and the results for the anti-mouse CD25 antibody (Table 2) are shown below:

Table 1 - Anti-human CD25 antibodies:

*Secondary table position

The amino acid (aa) sequence number is based on the human CD25 sequence obtained from Uniproto accession number P01589.

Table 2 - Anti-mouse CD25 antibodies:

The amino acid (aa) sequence numbering was based on mouse CD25 derived from the sequence published under Uniprot accession number P01590.

Epitope mapping studies using Pepscan technology showed that the anti-human antibody binds to human CD25 on an epitope that does not overlap with the IL-2 binding site on CD25. The epitope bound by the anti-human antibody is different from that of balithimab and dalizumab. The epitope bound by balithimab and dalizumab contains residues in the region of amino acid 137 to 143 (SEQ ID NO: 1), which overlaps with the CD25-IL-2 interaction side (Binder Me et al., Cancer Res 2007 vol 67(8):3518-23). The anti-mouse CD25 non-blocking antibodies 2E4 and 7D4 recognize epitopes different from those of PC61.

Example 5: Characterization of mouse anti-CD25 antibody

Antibody binds to CHO cells expressing mouse CD25

Binding to CD25-expressing CHO cells was examined by staining test samples (anti-CD25 primary antibody, 7D1, PC61, and 2E4) with 30 mg/ml antibody, followed by semi-logarithmic serial dilutions (7 spots) and incubation on ice for 30 minutes. Then, the samples were stained on ice for 30 minutes with 1 mg/ml secondary antibody (Alexa Fluor647-AffiniPure Fab fragment goat anti-human IgG (H+L) - (Jackson Immuno Research)). All samples were stained in duplicate. Viable cells were gated using FSC parameters via flow cytometry during sample collection. The mean fluorescence intensity (MFI) of stained cells was plotted on an XY plot, MFI was plotted against the logarithm of concentration, and the data were fitted to a non-linear regression curve from which EC50 was calculated. The results, shown in Figure 11, confirmed the binding of the anti-mouse CD25 antibody to mouse CD25-expressing CHO cells.

Affinity measurement of anti-mCD25 antibody

At an ambient experimental temperature of 25°C, using a CM-5 sensor chip in a Biacore 2000, the _KD of anti-mouse CD25 antibodies 7D4, PC61, and 2E4 was measured by SPR to determine their affinity. Initially, the anti-mouse antibodies were immobilized across all flowing cells in assay buffer (pH 7.4, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20) and held at RU 16,000 to 18,000 for 10 minutes. The ligand (antibody test item) was then sequentially added to capture levels between 119 and 163 RU. The analyte (his-labeled recombinant mouse CD25) was then associated with assay buffer diluted 2-fold starting at 800 nM (minimum concentration 3.13 nM) for 6 minutes. Dissociation was performed in assay buffer over 10 minutes. The regeneration step between sample concentrations was performed for 10 minutes in 10 mM glycine at pH 1.7. The entire process was maintained at a flow rate of 25 μl/min. Kinetic data were fitted using global modeling software for bivalent analytes provided by Biacore with reference subtraction. SPR-based analysis is shown in Figure 12. The Kd values established for the anti-mouse CD25 antibody in this assay were as follows: 2.6 × ^10⁻⁹ M for 7D4; 114 × ^10⁻⁹ M for 2E4; and 3.6 × ^10⁻⁹ M for PC61 (results not shown).

Competition against anti-mouse antibodies in Octet

Antibody competition was performed on a Forte Bio Octet Red96 system (Pall Forte Bio Corp., USA) using a standard sandwich assay. 10 nM anti-mouse CD25 antibody was added to the MC sensor for 900 seconds, and unoccupied Fc binding sites on the sensor were blocked with unrelated mouse IgG2a antibody. The sensor was then exposed to 15 nM of the target antigen (his-tagged mouse CD25) for 600 seconds, followed by exposure to the secondary anti-CD25 antibody (also 10 nM). Data were processed using ForteBio data analysis software 9.0. Additional binding of the secondary antibody after antigen binding indicated unoccupied epitopes, while no binding indicated epitope blocking.

Competitive binding with mCD25 was observed between 7D4 and 2E4 (Fig. 13(A)), but not between 7D4 and PC61 (Fig. 13(B)).

In vitro IL-2 signaling as determined by STAT5 phosphorylation:

ubiquitous T cells were isolated from spleen cells using the Invitrogen FlowComp™ Mouse ubiquitous T (CD90.2) Kit (Cat: 11465D). Cells were plated at 200,000 cells per plate and incubated at 37°C for 2 hours. 50 μg/ml antibody was added, and the cells were incubated at 37°C for 30 minutes, followed by stimulation with IL-2 (50 U/ml) at 37°C for 10 minutes.

Cell fixation and permeabilization with eBioscience™ Foxp3/transcription factor staining buffer kit (Invitrogen) followed by treatment with BD Phosflow Perm Buffer III (BD Biosciences) stopped IL-2-induced STAT5 phosphorylation. Cells were then simultaneously stained with antibodies labeled with surface and intracellular fluorescent dyes (STAT5-Alexa Fluor 647 clone 47/stat5/pY694 BD Biosciences, CD3-PerCP-Cy5.5 clone 17A2 Biolegend, CD4-PE clone RM4-5 Biolegend, FoxP3-AF488 clone FJK-16s Ebiosciences). Samples were obtained on a Fortessa LSR X20 flow cytometer (BD Biosciences) and analyzed using BD FACSDIVA software. Double peaks were excluded using FCS-H versus FCS-A, and lymphocytes were defined using SSC-A versus FCS-A parameters. CD3 ⁺ T cells were defined using CD3 PerCP-Cy5.5-A versus FCS-A plots, and gates were plotted on a histogram showing the counts of STAT5 Alexa Fluor 647-A to determine the STAT5 ⁺ CD3 ⁺ T cell population. The percentage of IL-2 signaling blockade was calculated as follows: %blockade = 100 × [(%Stat5 ⁺ cells in the Ab-free group - 50ug/ml %Stat5 ⁺ cells in the Ab-free group) / (%Stat5 ⁺ cells in the Ab-free group)]. Further analysis of STAT5 phosphorylation by different cell subsets (CD4 ⁺ , CD8 ⁺ , CD4 ⁺ FoxP3-) was also evaluated by gating the corresponding subsets and analyzed as described above. Image and statistical analyses were performed using GraphPad Prism v7 (results not shown). Results are shown in Figure 14.

result :

To further evaluate the ability of anti-mouse antibodies 7D4 and 2E4 to bind CD25 without interfering with IL-2 signaling in CD25-expressing target cells, the following assessments were conducted. The non-IL-2 blockers 7D4 and 2E4 competitively bound to CD25, while PC61 (an IL-2 signaling blocker) did not compete with either 2E4 or 7D4 for CD25 binding (Figure 12).

STAT5 assays confirmed that 7D4 and 2E4 do not block IL-2 signaling, while IL-2 signaling is blocked by the “blocking” antibody PC61 (Figure 14).

Example 6: In vivo consumption of Treg

One × ^10⁵ 4T1 cells were implanted into the second pectoral fat pad tissue of Balb/c mice in 200 μl of RPMI 1640 medium. When the tumor reached 50 to 100 ^mm³ , the mice were randomly assigned and administered a single, gradual intraperitoneal dose of 2 μg, 20 μg, or 200 μg of mouse anti-mouse CD25 (7D4) antibody to each mouse. On days 3 and 9, tumor tissue and whole blood were isolated for immunophenotyping.

result :

Based on post-dose analyses performed on days 3 and 9 using immunophenotyping, antibody 7D4 exhibited Treg-depleting activity in whole blood and tumor tissue (Figure 15).

Example 7: Characterization of anti-CD25 antibody 7G76B

Binding of anti-CD25 antibody to human cells expressing CD25:

7G76B was evaluated by binding to human lymphoma cell lines Karpas 299, SU-DHL-1, and SR-786, as well as in vitro differentiated Treg cells. Binding to CD25-expressing human cell lines (SU-DHL-1 and SR-786) was assessed by first blocking cells with Trustain (Biolegend), followed by incubation at 4°C for 30 min with serially titrated anti-CD25 antibody at a half-logarithmic dilution from the highest concentration of 20 μg/ml, then washing and incubation with PE-conjugated anti-human IgG Fc antibody (Biolegend). Cells were washed again and resuspended in FACS buffer containing DAPI and collected on an Intellicyt iQue. Viable cells were gated using FSC parameters via flow cytometry during sample collection. The geomean intensity of stained cells was plotted on an XY plot, the geomean intensity was plotted against the logarithm of concentration, and the data were fitted to a nonlinear regression curve from which EC50 was calculated.

Binding to CD25-expressing Karpas 299 cells and in vitro differentiated Tregs was detected by the following: The test sample (anti-CD25 primary antibody) was stained with 30 mg/ml antibody, followed by a semi-logarithmic serial dilution (7 spots) and incubated on ice for 30 minutes. Then, the sample was stained on ice for 30 minutes with a 1 mg/ml secondary antibody (Alexa Fluor 647-AffiniPure Fab fragment goat anti-human IgG (H+L) - (Jackson ImmunoResearch)). All samples were stained in duplicate. Live cells were gated using FSC parameters via flow cytometry during sample collection. The mean fluorescence intensity (MFI) of stained cells was plotted on an XY plot, MFI was plotted against the logarithm of concentration, and the data were fitted to a non-linear regression curve from which EC50 was calculated. The results shown in Figures 16 and 21 confirmed the binding of the anti-CD25 antibody to CD25-expressing cells.

In vitro IL-2 signaling as determined by STAT5 phosphorylation:

IL-2 blockade was characterized using a STAT5 phosphorylation assay to examine IL-2 signaling. Previously frozen PBMCs (Stemcell Technologies) were cultured for 30 minutes in 96-U wells in the presence of 10 U/ml anti-CD25 antibody, followed by the addition of different concentrations of IL-2 (Peprotech) at 0.1 U/ml, 1 U/ml, or 10 U/ml to RPMI 1640 (Life Technologies) containing 10% FBS (Sigma), 2 mM L-glutamine (Life Technologies), and 10000 U/ml Pen-Strep (Sigma). IL-2-induced STAT5 phosphorylation was stopped when cells were fixed, permeabilized with eBioscience™ Foxp3/transcription factor staining buffer kit (Invitrogen), and treated with BD Phosflow Perm buffer III (BD Biosciences). IL-2-induced STAT5 phosphorylation was stopped when cells were fixed, permeabilized with eBioscience™ Foxp3/transcription factor staining buffer (Invitrogen), and treated with BD Phosflow Perm Buffer III (BD Biosciences). Cells were then simultaneously stained with antibodies labeled with surface and intracellular fluorescent dyes (STAT5-Alexa Fluor 647 clone 47/stat5/pY694 BD Biosciences, CD3-PerCP-Cy5.5 clone UCHT1Biolegend, CD4-BV510 clone SK3 BD Biosciences, CD8-Alexa Fluor 700 clone RPA-T8 Invitrogen, CD45RA-PE-Cy7 clone HI100 Invitrogen, FoxP3-Alexa Fluor 488 clone 236A/E7 Invitrogen). Samples were obtained using a Fortessa LSR X20 flow cytometer (BD Biosciences) and analyzed using BD FACSDIVA software. Binocular peaks were excluded using FCS-H versus FCS-A, and lymphocytes were defined using SSC-A versus FCS-A parameters. CD3 ⁺ T cells were defined using CD3 PerCP-Cy5.5-A versus FCS-A plots, and gating was performed on a histogram showing the counts of STAT5 Alexa Fluor 647-A to determine the STAT5 ⁺ CD3 ⁺ T cell population. The percentage of IL-2 signaling blockade was calculated as follows: %blockade = 100 × [(%Stat5 ⁺ cells in the Ab-free group - 10ug/ml %Stat5 ⁺ cells in the Ab-free group) / (%Stat5 ⁺ cells in the Ab-free group)]. Further analysis of STAT5 phosphorylation by different cell subsets (CD4 ⁺ , CD8 ⁺ , CD4 ⁺ FoxP3+, naive and memory T cells) was also evaluated by gating the corresponding subsets and analyzed as described above. Image and statistical analyses were performed using GraphPad Prism v7 (results not shown). Results are shown in Figures 17 and 22.

In vitro T cell activation assay:

The effect of IL-2 signaling on the Teff response was characterized in an assay examining the upregulation of intracellular granzyme B (GrB) and the proliferation of T cells. Following the manufacturer's recommendations, previously frozen primary human ubiquitous T cells (Stemcell Technologies) were labeled with eFluor450 cell proliferation dye (Invitrogen) and added at 1 × ^10⁵ cells/well to RPMI 1640 (Life Technologies) containing 10% FBS (Sigma), 2 mM L-glutamine (Life Technologies), and 10000 U/ml Pen-Strep (Sigma) in 96-U well plates. Cells were then treated with 10 μg/ml anti-CD25 antibody or control antibody, followed by human T activator CD3/CD28 (cells to beads ratio 20:1; Gibco), and incubated at 37°C in a 5% _CO₂ humidified incubator for 72 h. To assess T cell activation, cells were stained with eBioscience FixableViability Dye efluor780 (Invitrogen) followed by antibodies labeled with fluorescent dyes targeting 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). Cells were then fixed and permeabilized with the eBioscience™ Foxp3/transcription factor staining buffer kit (Invitrogen) and stained for intracellular GrB and nuclear FoxP3 (granulase 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. Binocular peaks were excluded using FCS-H versus FCS-A, and lymphocytes were defined using SSC-A versus FCS-A parameters. CD4+ and CD8+ T cell subsets gated from live CD3 ⁺ lymphocytes were assessed using GrB-PE-A and proliferating eFluor450-A plots. Results are expressed as the percentage of proliferating GrB ^{-positive cells in the total CD4+ T cell} population. Graphs and statistical analyses were performed using GraphPad Prism v7. Results are shown in Figure 18.

In vitro ADCC assay:

Antibody-dependent cell-mediated cytotoxicity assays (ADCC) were performed using SU-DHL-1 or SR-786 (CD25-positive) human cell lines as target cells and human NK cells as effector cells to characterize anti-human CD25 antibodies. NK cells were isolated from PBMCs of healthy donors 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 plated with calcein-AM (Thermofisher) in the presence of anti-CD25 or allotype antibodies, in quadruple replicates for each condition, and incubated at 37°C and 5% _CO2 for 30 min. After incubation, NK cells were added at a target-effect (T:E) ratio of 10,000 target cells to 100,000 effector cells and incubated at 37°C and 5% _CO2 for 4 h. Calcein fluorescence in the supernatant was read using a BMG Fluostar plate reader. The percentage of specific lysis was calculated relative to target cells alone (0% lysis) and target cells treated with 0.1% saponin (100% lysis). Raw data plots were generated using Graphpad Prism v7 to produce dose-response curves. The percentage of target cell lysis was plotted on an XY plot, the normalized percentage release of calcein AM was plotted against the logarithm of concentration, and the data were fitted to a nonlinear regression curve from which EC50 was calculated. The results are shown in Figure 19.

In vitro ADCP assay:

In vitro differentiated Tregs were used as target cells, and monocyte-derived macrophages were used as effector cells for antibody-dependent cell-mediated phagocytosis (ADCP) assays. PBMCs were isolated from leukocyte 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 50 ng/ml M-CSF in 10% FBS (Sigma), 2 mM L-glutamine (Life Technologies), and 10000 U/ml penicillin-streptomycin (Sigma), followed by fresh medium containing M-CSF after 3 days. Regulatory T cells (Tregs) were isolated using a human Treg cell differentiation kit (R&D Systems). These cells were cultured for 5 days in a 37°C, 5% _CO2 humidified incubator and labeled with eFluor450 dye (Invitrogen) as recommended by the manufacturer. On day 5, macrophages and Tregs labeled with eFluor450 dye were co-cultured for 4 hours at an effector-to-target ratio of 10:1 in the presence of anti-CD25 antibody or a control, as described below. Target cells (Tregs) were added at 1 × ^10⁴ cells/well, and effector cells (macrophages) were added at 1 × ^10⁵ cells/well, i.e., an effector-to-target ratio of 10:1. Anti-CD25 antibody was then added at the highest concentration of 1 μg/ml, followed by a logarithmic serial dilution (7 spots). Cells and antibody were incubated at 37°C and 5% _CO₂ for 4 hours. To assess ADCP, cells were placed on ice, stained with the cell surface marker CD14 (clone MFP9 of CD14-PerCP-Cy5.5, 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 with eFluor450-dye ⁺ / ^CD14- . Macrophages were defined as CD14 ⁺ . Double-labeled cells (eFluor450-dye+/CD14+) were considered to represent macrophage phagocytosis of the target. Phagocytosis of target cells was calculated using the following equation: % phagocytosis = 100 × [(double positive %) / (double positive % + residual target percentage)]. The results are shown in Figure 20.

statistics:

Curve fitting was performed using Prism software (GraphPad) to determine the EC50 value and maximum activity.

Human antibodies do not block IL2-CD25 interaction

Interference between IL2 ligand and CD25 binding was determined using a standard sandwich assay on a Forte Bio Octet Red384 system (Pall Forte BioCorp., USA). MA251 antibody was loaded onto the AHQ sensor, and unoccupied Fc binding sites on the sensor were blocked with an unrelated human IgG1 antibody. The sensor was exposed to 100 nM human CD25, followed by exposure to 100 nM human IL-2. Data were processed using Forte Bio Data Analysis Software 7.0. Additional binding of human IL2 after antigen association indicated an unoccupied epitope (non-competitor), while no binding indicated epitope blockade (competitor).

result :

To further evaluate the ability of the 7G7B6 antibody to not interfere with IL-2 signaling and to kill CD25-expressing target cells, the antibody was evaluated. In the STAT5 assay, 7G7B6 did not block IL-2 signaling regardless of the IL-2 concentration tested, while IL-2 signaling was completely blocked by the reference antibody dalizumab (Fig. 17). Dalizumab, 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, Neurootherapeutics, 10(1): 55–67), binds to an epitope different from that of 7G7B6 (Fig. 10 and Fig. 24B), which may explain why dalizumab blocked IL-2 signaling while 7G7B6 did not in the STAT5 phosphorylation assay (Fig. 17). Furthermore, dalizumab reduces the effector response of activated T cells, possibly because it blocks IL-2 signaling, while 7G7B6, which does not block IL-2 signaling, has no negative impact on T cell responses (Figure 18). Finally, when compared with IgG1 isotype antibodies, the 7G7B6 chimeric antibody kills CD25-expressing cells, i.e., tumor cells or regulatory T cells, through ADCC (Figure 19) and ADCP (Figure 20).

In summary, 7G7B6 has been characterized as a chimeric antibody and demonstrated to effectively kill CD25-positive cells (Tregs or cancer cell lines) without interfering with IL-2 signaling and therefore without inhibiting the T-effect response. Therefore, 7G7B6 is a Treg-depleting antibody that can be used as a monotherapy or in combination for cancer treatment.

To further evaluate the ability of the MA-251 antibody to not interfere with IL-2 signaling, the MA-251 antibody was evaluated in an IL2-CD25 Octet competitive assay. Simultaneous IL2 binding and MA251 binding to CD25 were observed (Figure 23), indicating that MA251 binds in a non-competitive manner. In the STAT5 assay, regardless of the tested IL-2 concentration, MA-251 did not block IL-2 signaling, while IL-2 signaling was completely blocked by the reference antibody daclizumab (Figure 22). Dalizumab, 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 (Figures 10 and 24(E)). This could explain why dalizumab blocks IL-2 signaling in STAT5 phosphorylation assays while MA251 does not (Figure 22).

Example 8: Competitive assay of anti-human CD25 Ab

Antibody competition was performed using a standard sandwich assay on a Forte Bio Octet Red96 system (Pall Forte Bio Corp., USA). 26.8 nM his-labeled recombinant human CD25 was added to the Ni-NTA sensor for 200 seconds. After the baseline step, the sensor was exposed to 66.6 nM of the first antibody for 600 or 1800 seconds, followed by exposure to the second anti-CD25 antibody (also 66.6 nM, for 600 or 1800 seconds). Data were processed using Forte Bio Data Analysis Software 9.0. Additional binding to the secondary antibody indicated an unoccupied epitope (no epitope competition), while no binding indicated epitope blocking (epitaxy competition).

result

The non-IL-2 signaling blocker mAbs (antibody 1 and antibody 3) competed with each other or with 7G7B6 and MA251, but not with the study dalizumab or the study baliximab (Examples (A) to (N), Figure 24). The IL-2 signaling blocker (i.e., TSK031) did compete with the study dalizumab and the study baliximab, but not with 7G7B6 (Examples (O) to (Q), Figure 24).

Example 9: Therapeutic Analysis of Non-Blocking Antibodies

On day 0, 1 × ^10⁷ SU-DHL-1 cells from 200 μl RPMI1640 were implanted into the right ventral region. On day 12, mice with palpable tumors were randomly assigned to be treated with the solvent or with antibody 1 twice weekly at 2 mg/kg. On day 15, mice with tumors ranging from 100 to 200 ^mm³ were randomly assigned to be treated with the solvent, antibody 1 at 2 mg/kg twice weekly, or a single dose of antibody 1 at 10 mg/kg.

result

Antibody 1, administered twice weekly at a dose of 2 mg/kg, prevented growth in 9/10 mice with palpable tumors (Fig. 25(A)-(B)). In mice with tumors ranging from 100 to 200 ^mm³ , tumor growth was also prevented by administration of 2 mg/kg twice weekly and by a single dose of 10 mg/kg (Fig. 25(C)-(E)).

Example 10: Affinity Measurement of Anti-Human CD25 Antibody

At an ambient experimental temperature of 25°C, the affinity of anti-human CD25 antibodies was determined using a Biacore 2000 BLCEL instrument with a CM-5 sensor chip, measuring the _KD of the antibody via SPR. Initially, the anti-human antibody was immobilized across all flowing cells in assay buffer (pH 7.4, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20) and held for 10 minutes at a RU of 12,000 to 14,000. The ligand (antibody test item) was then sequentially added to capture levels between 145 and 190 RU. The analyte (his-labeled recombinant human CD25) was then associated with assay buffer diluted 2-fold starting at 400 nM (minimum concentration 3.13 nM) and held for 6 minutes. Dissociation was performed in assay buffer over 10 minutes. Regeneration steps between sample concentrations were performed for 10 minutes at 10 mM glycine pH 1.7. The entire process was maintained at a flow rate of 25 μl/min. Figures 26(C) and 26(D) show the kinetic data fitted using global two-state reaction conformational change analysis software provided by Biacore with reference difference. Figures 26(A), 26(B), and 26(E) show the 1:1 Langmuir model with reference difference.

ForteBio affinity measurements are typically performed on an Octet RED384 as previously described (see, for example, Estep Petal., 2013. Mabs. 5(2), 270-8).

Alternatively, the affinity of the anti-human CD25 antibody was determined by measuring the K_{_D} of the anti-human CD25 antibody using biolayer interferometry on an Octet Red 96 system (Pall Forte Bio Corp., USA). The sensor was equilibrated offline in kinetic buffer for 10 min, followed by online monitoring for 60 s to establish a baseline. 13.32 nM of antibody was loaded into the AHC biosensor for 200 s, followed by the addition of different concentrations of his-labeled rhCD25 (serial dilutions of 1:3, 50 nM to 0.54 nM) for 600 s, and then allowed to dissociate in kinetic buffer for 400 s. The kinetic data were fitted using global 1:1 analysis software provided by Pall Forte Bio with reference difference. The results are shown in Figure 26(F).

result:

The results are shown in Figure 26. The Kd values established for the anti-CD25 antibodies in this assay are as follows: 3.2 × ^10⁻⁹ M for antibody 1; 3.8 × ^10⁻⁹ M for antibody 3; and 0.61 × ^10⁻⁹ M for dalizumab.

Example 11: Characterization of anti-CD25 antibodies - Antibody 1 to Antibody 21

Binding of anti-CD25 antibody to cells expressing CD25:

Candidates were evaluated by combining them with human lymphoma cell lines such as Karpas299, SU-DHL-1, and SR-786 cells, in vitro differentiated Treg cells, activated human or cynomolgus monkey PBMCs, HSC-F cynomolgus monkey T cell lines, and CHO cells.

Binding to CD25-expressing human cell lines (SU-DHL-1 and SR-786) was detected by the following method: Cells were first blocked with Trustain (Biolegend), then incubated with anti-CD25 antibody serially titrated from the highest concentration of 20 μg/ml in a half-logarithmic dilution at 4°C for 30 min, followed by washing and incubation with PE-conjugated anti-human IgG Fc antibody (Biolegend). Cells were washed again and resuspended in FACS buffer containing DAPI and collected on an Intellicyt iQue. Viable cells were gated using FSC parameters via flow cytometry during sample collection. The geomean intensity of stained cells was plotted on an XY plot, the geomean intensity was plotted against the logarithm of concentration, and the data were fitted to a nonlinear regression curve from which EC50 was calculated.

Binding to CD25-expressing Karpas 299 cells and in vitro differentiated Tregs was detected by the following: The test sample (anti-CD25 primary antibody) was stained with 30 mg/ml antibody, followed by a semi-logarithmic serial dilution (7 spots) and incubated on ice for 30 minutes. Then, the sample was stained on ice for 30 minutes with a 1 mg/ml secondary antibody (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 Immuno Research)). All samples were stained in duplicate. Live cells were gated using FSC parameters via flow cytometry during sample collection. The mean fluorescence intensity (MFI) of stained cells was plotted on an XY plot, MFI was plotted against the logarithm of concentration, and the data were fitted to a non-linear regression curve from which EC50 was calculated.

Binding to activated human or cynomolgus monkey PMBCs expressing CD25 was detected by the following: The test sample was stained with 20 mg/ml antibody (anti-CD25 primary antibody), followed by a semi-logarithmic serial dilution (7 spots) and incubated on ice for 30 minutes. Then, it was stained on ice for 30 minutes with a 1 mg/ml secondary antibody (rabbit anti-human Fcg F(ab')2- (Jackson ImmunoResearch)). All samples were stained in triplicate. To minimize cell death induced by cross-linking mediated by secondary antibody binding, cell lines were examined in stained populations of four test samples at a time. Viable lymphocytes were gated using FSC against SSC parameters via flow cytometry during sample collection. The mean fluorescence intensity (MFI) of the gated CD4 ⁺ and CD8 ⁺ T cell subsets was plotted on an XY plot, MFI was plotted against the logarithm of concentration, and the data were fitted to a non-linear regression curve from which EC50 was calculated.

Binding to the CD25-expressing HSC-F cynomolgus T cell line was detected by staining the test material (anti-CD25 primary antibody) with 20 mg/ml antibody, followed by a semi-logarithmic serial dilution (7 spots) and incubation on ice for 30 minutes. All samples were stained in triplicate. To minimize cell death induced by cross-linking mediated by secondary antibody binding, cell lines were examined in a stained cohort of four test items at a time. Live lymphocytes were gated to SSC parameters using FSC via flow cytometry during sample collection. The mean fluorescence intensity (MFI) of live cells was plotted on an XY plot, MFI was plotted against the logarithm of concentration, and the data were fitted to a nonlinear regression curve from which EC50 was calculated.

Binding to CD25-expressing CHO cells was also examined. Approximately 100,000 cells overexpressing the antigen were washed with wash buffer and incubated with 100 μl of 100 nM IgG for 15 min at room temperature. Cells were then washed twice with wash buffer and incubated with 100 μl of 1:100 human PE on ice for 15 min. Cells were then washed twice with wash buffer and analyzed on a FACSCanto II analyzer (BD Biosciences). Unmodified CHO cell lines were also used as a negative control.

In vitro IL-2 signaling as determined by STAT5 phosphorylation:

IL-2 blockade was characterized using a STAT5 phosphorylation assay to examine IL-2 signaling. Previously frozen PBMCs (Stemcell Technologies) were cultured for 30 minutes in 96-U wells in the presence of 10 U/ml anti-CD25 antibody, followed by the addition of different concentrations of IL-2 (Peprotech) at 10 U/ml or 0.25 U/ml, 0.74 U/ml, 2.22 U/ml, 6.66 U/ml, or 20 U/ml to RPMI 1640 (Life Technologies) containing 10% FBS (Sigma), 2 mM L-glutamine (Life Technologies), and 10000 U/ml Pen-Strep (Sigma). IL-2-induced STAT5 phosphorylation was stopped when cells were fixed, permeabilized with the eBioscience TMFoxp3/transcription factor staining buffer kit (Invitrogen), and treated with BD Phosflow Perm Buffer III (BDBiosciences). Cell fixation and permeabilization with eBioscience TMFoxp3/transcription factor staining buffer (Invitrogen) followed by treatment with BD Phosflow Perm Buffer III (BD Biosciences) stopped IL-2-induced STAT5 phosphorylation. Cells were then simultaneously stained with antibodies labeled with surface and intracellular fluorescent dyes (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 obtained using a Fortessa LSR X20 flow cytometer (BD Bioscience) and analyzed using BD FACSDIVA software. Binocular peaks were excluded using FCS-H versus FCS-A, and lymphocytes were defined using SSC-A versus FCS-A parameters. CD3 ⁺ T cells were defined using CD3 PerCP-Cy5.5-A versus FCS-A plots, and gating was performed on a histogram showing the counts of STAT5 Alexa Fluor 647-A to determine the STAT5 ⁺ CD3 ⁺ T cell population. The percentage of IL-2 signaling blockade was calculated as follows: %blockade = 100 × [(%Stat5 ⁺ cells in the Ab-free group - 10ug/ml %Stat5 ⁺ cells in the Ab-free group) / (%Stat5 ⁺ cells in the Ab-free group)]. Further analysis of STAT5 phosphorylation by different cell subsets (CD4 ⁺ , CD8 ⁺ , CD4 ⁺ FoxP3+, naive and memory T cells) was also evaluated by gating the corresponding subsets and analyzed as described above. Image and statistical analyses were performed using GraphPad Prism v7.

In vitro T cell activation assay:

The effect of IL-2 signaling on the Teff response was characterized in an assay examining the upregulation of intracellular granzyme B (GrB) and the proliferation of T cells. Following the manufacturer's recommendations, previously frozen primary human ubiquitous T cells (Stemcell Technologies) were labeled with eFluor450 cell proliferation dye (Invitrogen) and added at 1 × ^10⁵ cells/well to RPMI 1640 (Life Technologies) containing 10% FBS (Sigma), 2 mM L-glutamine (Life Technologies), and 10000 U/ml Pen-Strep (Sigma) in 96-U well plates. Cells were then treated with 10 μg/ml anti-CD25 antibody or control antibody, followed by human T activator CD3/CD28 (cells to beads ratio 20:1; Gibco), and incubated at 37°C in a 5% _CO₂ humidified incubator for 72 h. To assess T cell activation, cells were stained with eBioscience FixableViability Dye efluor780 (Invitrogen) followed by antibodies labeled with fluorescent dyes targeting 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). Cells were then fixed and permeabilized with the eBioscience™ Foxp3/transcription factor staining buffer kit (Invitrogen) and stained for intracellular GrB and nuclear FoxP3 (granulase 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. Binocular peaks were excluded using FCS-H versus FCS-A, and lymphocytes were defined using SSC-A versus FCS-A parameters. Samples were acquired using a Fortessa LSR X20 flow cytometer (BD Bioscience) and analyzed using BD FACSDIVA software. Binocular peaks were excluded using FCS-H versus FCS-A, and lymphocytes were defined using SSC-A versus FCS-A parameters. CD4 ⁺ and CD8 ⁺ T cell subsets gated from live CD3 ⁺ lymphocytes were assessed using GrB-PE-A and proliferating eFluor450-A plots. Results are expressed as the percentage of proliferating GrB-positive cells in the total CD4 ⁺ or CD8 ⁺ T cell population. Graphs and statistical analyses were performed using GraphPad Prism v7.

In vitro ADCC assay:

Antibody-dependent cell-mediated cytotoxicity assays (ADCC) were performed using SU-DHL-1 or SR-786 (CD25-positive) human cell lines as target cells and human NK cells as effector cells to characterize anti-human CD25 antibodies. NK cells were isolated from PBMCs of healthy donors 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 plated with calcein-AM (Thermofisher) in the presence of anti-CD25 or allotype antibodies, in quadruple replicates for each condition, and incubated at 37°C and 5% _CO2 for 30 min. After incubation, NK cells were added at a target-effect (T:E) ratio of 10,000 target cells to 100,000 effector cells and incubated at 37°C and 5% _CO2 for 4 h. Readings of calcein fluorescence in the supernatant were performed using a BMG Fluostar plate reader. The percentage of specific lysis was calculated relative to target cells alone (0% lysis) and target cells treated with 0.1% saponin (100% lysis). Raw data plots were generated using Graphpad Prism v7 to produce dose-response curves. The percentage of target cell lysis was plotted on an XY plot, the normalized percentage release of calcein AM was plotted against the logarithm of concentration, and the data were fitted to a nonlinear regression curve from which EC50 was calculated.

ADDCs were also determined in a luciferase reporter system assay. SR786 cells expressing CD25 (referred to herein as target (T) cells) were incubated at 37°C with different concentrations of anti-CD25 mAb (or control IgG) in low-IgG, FBS-supplemented medium (RPMI containing 4% FBS) for 20 minutes. ADDC effector cells (E) were then added to the cell-mAb mixture at a 1:1 E:T ratio. Effector cells were Jurkat cells stably transfected with a luciferase reporter system and overexpressing CD16/FcγRIIIA (Promega). After overnight incubation at 37°C, cells were lysed, and luciferase activity was measured by the luminescent release of a specific luciferase substrate via hydrolysis, following the manufacturer's instructions (Promega Bio-Glow protocol).

In vitro ADCP assay using in vitro differentiated macrophages and Treg cells:

In vitro differentiated Tregs were used as target cells, and monocyte-derived macrophages were used as effector cells for antibody-dependent cell-mediated phagocytosis (ADCP) assays. PBMCs were isolated from leukocyte 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 50 ng/ml M-CSF in 10% FBS (Sigma), 2 mM L-glutamine (Life Technologies), and 10000 U/ml penicillin-streptomycin (Sigma), followed by fresh medium containing M-CSF after 3 days. Regulatory T cells (Tregs) were isolated using a human Treg cell differentiation kit (R&D Systems). These cells were cultured for 5 days in a 37°C, 5% _CO2 humidified incubator and labeled with eFluor450 dye (Invitrogen) as recommended by the manufacturer. On day 5, macrophages and Tregs labeled with eFluor450 dye were co-cultured for 4 hours at an effector-to-target ratio of 10:1 in the presence of anti-CD25 antibody or a control, as described below. Target cells (Tregs) were added at 1 × ^10⁴ cells/well, and effector cells (macrophages) were added at 1 × ^10⁵ cells/well, i.e., an effector-to-target ratio of 10:1. Anti-CD25 antibody was then added at the highest concentration of 1 μg/ml, followed by a logarithmic serial dilution (7 spots). Cells and antibody were incubated at 37°C and 5% _CO₂ for 4 hours. To assess ADCP, cells were placed on ice, stained with the cell surface marker CD14 (clone MFP9 of CD14-PerCP-Cy5.5, 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 with eFluor450-dye ⁺ / ^CD14- . Macrophages were defined as CD14 ⁺ . Double-labeled cells (eFluor450-dye+/CD14+) were considered to represent macrophage phagocytosis of the target. Phagocytosis of target cells was calculated using the following equation: % phagocytosis = 100 × [(double positive %) / (double positive % + residual target percentage)].

In vitro ADCP assay using FcγRIIa-H Reporter

ADCP bioassay effector cells (FcγRIIa-H) were obtained from Promega (Cat#G9881/5; Lot#0000261099). SUDHL-1 target cells (25 μl/well) were seeded at 5000 cells/well using a 96-well white polystyrene plate (Costar; Cat#3917). The assay antibody was serially diluted 3-fold with 25 μl added to the cells. 50,000 μl effector cells were added to each well (25 μl volume) to achieve an effector-to-target cell ratio of 10:1. All target cells, antibody, and effector cells were seeded using cell culture medium. The plates were incubated at 37°C for 18 hours. The plates were then removed from the incubator and held at room temperature for 20 minutes. 60 μl of Bio-Glo luciferase assay substrate buffer was added to each well, followed by incubation for 30 minutes, and luminescence was measured using a GloMax Multi detection system (Promega).

statistics:

Curve fitting was performed using Prism software (GraphPad) to determine the EC50 value and maximum activity.

result

Antibody 1 was evaluated for its ability to not interfere with IL-2 signaling and its ability to kill CD25-expressing target cells. Results of binding to rhCD25 and competitive binding analysis with IL-2 are shown in Figures 27 and 28. Ligand binding assays using Octet showed that antibody 1 did not affect the binding of IL-2 to CD25 (Figure 29). This was confirmed in a STAT5 assay, where antibody 1 did not block IL-2 signaling regardless of the tested IL-2 concentration, while IL-2 signaling was completely blocked by the reference antibody dalizumab (Figure 31). Dalizumab binding, 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), differs from the epitope of antibody 1 (Figure 30), which may explain why dalizumab blocks IL-2 signaling while antibody 1 does not (Figure 31). Furthermore, dalithiazide reduced the effector response of activated T cells, possibly due to its blocking of IL-2 signaling, while antibody 1, which does not block IL-2 signaling, had no negative impact on T cell responses (Figure 32). Finally, when compared with the IgG1 isotype antibody, antibody 1 killed CD25-expressing cells, i.e., tumor cells or regulatory T cells, through ADCC (Figure 33) and ADCP (Figure 34).

In summary, antibody 1 has been characterized and demonstrated to effectively kill CD25-positive cells (Tregs or cancer cell lines) without interfering with IL-2 signaling and therefore without inhibiting the T-effect response. Therefore, antibody 1 is a Treg-depleting antibody that can be used as a monotherapy or in combination for cancer treatment.

Antibody 3 was evaluated for its ability to not interfere with IL-2 signaling and its ability to kill CD25-expressing target cells. Results of binding to rhCD25 and competitive binding analysis with IL-2 are shown in Figures 35 and 36. Ligand binding assays using Octet showed that antibody 3 did not affect the binding of IL-2 to CD25 (Figure 37). This was confirmed in a 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 dalizumab (Figure 39). Dalizumab binding, which has been shown to block the interaction between CD25 and IL-2 via a so-called “Tac” epitope, differs from that of antibody 3 (Figure 38), which may explain why dalizumab blocks IL-2 signaling while antibody 3 does not (Figure 39). Furthermore, dalithiazide reduced the effector response of activated T cells, possibly due to its blocking of IL-2 signaling, while antibody 3, which does not block IL-2 signaling, had only minimal effect on T cell responses (if any) when compared to the absence of antibody or with an allotype control (Figure 40). Finally, when compared to the IgG1 allotype antibody, antibody 3 killed CD25-expressing cells, i.e., tumor cells or regulatory T cells, via ADCC (Figure 41) and ADCP (Figure 42).

In summary, antibody 3 has been characterized and demonstrated to effectively kill CD25-positive cells (Tregs or cancer cell lines) without interfering with IL-2 signaling and therefore without inhibiting the T-effect response. Therefore, antibody 3 is a Treg-depleting antibody that can be used as a monotherapy or in combination for cancer treatment.

Antibody 4 was evaluated for its ability to not interfere with IL-2 signaling and its ability to kill CD25-expressing target cells. Results of binding to rhCD25 and competitive binding analysis with IL-2 are shown in Figures 43 and 44. Ligand binding assays using Octet showed that antibody 4 did not affect the binding of IL-2 to CD25 (Figure 45). This was confirmed in a STAT5 assay, where antibody 4 did not block IL-2 signaling regardless of the tested IL-2 concentration, while IL-2 signaling was completely blocked by the reference antibody dalizumab (Figure 47). Dalizumab binding, which has been shown to block the interaction between CD25 and IL-2 via a so-called “Tac” epitope, differs from that of antibody 4 (Figure 46), which may explain why dalizumab blocks IL-2 signaling while antibody 4 does not (Figure 47). Furthermore, dalithiazide reduced the effector response of activated T cells, possibly due to its blocking of IL-2 signaling, while antibody 4, which does not block IL-2 signaling, had no negative impact on T cell responses (Figure 48). Finally, when compared with IgG1 isotype antibodies, antibody 4 killed CD25-expressing cells, i.e., tumor cells or regulatory T cells, through ADCC (Figure 49) and ADCP (Figure 50).

In summary, antibody 4 has been characterized and demonstrated to effectively kill CD25-positive cells (Tregs or cancer cell lines) without interfering with IL-2 signaling and therefore without inhibiting the T-effect response. Therefore, antibody 4 is a Treg-depleting antibody that can be used as a monotherapy or in combination for cancer treatment.

Antibody 2 was evaluated for its ability to not interfere with IL-2 signaling and its ability to kill CD25-expressing target cells. Results of binding to rhCD25 and competitive binding analysis with IL-2 are shown in Figures 51 and 52. Ligand binding assays using Octet showed that antibody 2 did not affect the binding of IL-2 to CD25 (Figure 53). This was confirmed in a 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 dalizumab (Figure 55). Dalizumab binding, which has been shown to block the interaction between CD25 and IL-2 via the so-called “Tac” epitope, differs from that of antibody 2 (Figure 54), which may explain why dalizumab blocks IL-2 signaling while antibody 2 does not (Figure 55). Finally, when compared with the IgG1 isotype antibody, antibody 2 kills CD25-expressing cells, namely tumor cells or regulatory T cells, through ADCC (Fig. 56) and ADCP (Fig. 57).

In summary, antibody 2 has been characterized and demonstrated to effectively kill CD25-positive cells (Treg or cancer cell lines) without interfering with IL-2 signaling and therefore without inhibiting the T-effect response. Therefore, antibody 2 is a Treg-depleting antibody that can be used as a monotherapy or in combination for cancer treatment.

Antibody 5 is characterized by comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the following sequence:

EVQLVESGGGLIQPGGSLRLSCAAS GFTLDSYGVS WVRQAPGKGLEW V GVTTSSGGSAYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRYVYTGGYLYHYGMDL WGQGTLVTVSS(SEQ ID NO:10);

The light chain variable region contains the following sequence:

DIQMTQSPSSSLSASVGDRVTITC RASQSISDYLA WYQQKPGKVPKLLI YAASTLPF GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QGTYDSSDWYW A FGGGTKVEIK (SEQ ID NO: 14).

As mentioned above, the sequence and frame regions (FRs) of the complementarity determination regions (CDRs, namely CDR1, CDR2 and CDR3) are defined according to the Kabat numbering scheme.

The antibody 5 was evaluated for its ability to not interfere with IL-2 signaling and its ability to kill CD25-expressing target cells. The results of binding to rhCD25 are shown in Figure 58. STAT5 assays showed that antibody 5 did not block the tested IL-2 signaling, while IL-2 signaling was completely blocked by the antibody dalizumab (Figure 59). Competition assays showed that antibody 5 did not compete with the IL-2 signaling blockers dalizumab or baliximab (Figure 60(A) and (B)), but it did compete with 7G7B6 (a non-IL-2 blocker) (Figure 60(C)). Finally, when compared with an anti-human CD25 Fc silencing control antibody, antibody 5 killed CD25-expressing cells, i.e., tumor cells or regulatory T cells, via ADCC (Figure 61) and ADCP (Figure 61).

In summary, antibody 5 has been characterized and demonstrated to effectively kill CD25-positive cells (Tregs or cancer cell lines) without interfering with IL-2 signaling and therefore without inhibiting the T-effect response. Therefore, antibody 5 is a Treg-depleting antibody that can be used as a monotherapy or in combination for cancer treatment.

Antibodies 6, 7, 8, and 9 are characterized by containing the following sequences:

Antibody 6 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the following sequence:

EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYGMS WVRQAPGKGLEL VS TINGYGDTTYYPDSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA R DRDYGNSYYYALDY WGQGTLVTVSS(SEQ ID NO:23);

The light chain variable region contains the following sequence:

EIVLTQSPGTLSLSPGERATLSC RASSSVSFMH WLQQKPGQAPRPLI YA TSNLAS GIPDRFSGSGSGTDYTLTISRLEPEDFAVYYC QQWSSNPPA FGQGT KLEIK (SEQ ID NO: 25).

Antibody 7 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the following sequence:

EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYGMS WVRQAPGKGLEL VS TINGYGDTTYYPDSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA R DRDYGNSYYYALDY WGQGTLVTVSS(SEQ ID NO:23);

The light chain variable region contains the following sequence:

QIVLTQSPGTLSLSPGERATLSC RASSSVSFMH WLQQKPGQSPRPLI YA TSNLAS GIPDRFSGSGSGTDYTLTISRLEPEDFAVYYC QQWSSNPPA FGQGT KLEIK (SEQ ID NO: 26).

Antibody 8 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the following sequence:

EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYGMS WVRQAPGKGLEL VS TINGYGDTTYYPDSVK GRFTISRDNAKNTLYLQMNSLRAEDTAVYFCA R DRDYGNSYYYALDY WGQGTLVTVSS(SEQ ID NO:24);

The light chain variable region contains the following sequence:

EIVLTQSPGTLSLSPGERATLSC RASSSVSFMH WLQQKPGQAPRPLI YA TSNLAS GIPDRFSGSGSGTDYTLTISRLEPEDFAVYYC QQWSSNPPA FGQGT KLEIK (SEQ ID NO: 25).

Antibody 9 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the following sequence:

EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYGMS WVRQAPGKGLEL VS TINGYGDTTYYPDSVK GRFTISRDNAKNTLYLQMNSLRAEDTAVYFCA R DRDYGNSYYYALDY WGQGTLVTVSS(SEQ ID NO:24);

The light chain variable region contains the following sequence:

QIVLTQSPGTLSLSPGERATLSC RASSSVSFMH WLQQKPGQSPRPLI YA TSNLAS GIPDRFSGSGSGTDYTLTISRLEPEDFAVYYC QQWSSNPPA FGQGT KLEIK (SEQ ID NO: 26).

As mentioned above, the sequence and frame regions (FRs) of the complementarity determination regions (CDRs, namely CDR1, CDR2 and CDR3) are defined according to the Kabat numbering scheme.

Epitope mapping results showed that antibodies 6, 7, 8, and 9 bound human CD25 in the regions of amino acids 150 to 163 (YQCVQGYRALHRGP) and 166 to 180 (SVCKMTHGKTRWTQP) of SEQ ID NO:1, and bound to the extracellular protein sequence of human CD25 with Kd values of ^10⁻⁸ M to ^10⁻¹⁰ M.

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. STAT5 assays showed that the antibodies did not block the tested IL-2 signaling, while IL-2 signaling was completely blocked by the antibody dalizumab (Figure 65). Competition assays showed that antibody 7 did not compete with the IL-2 signaling blockers dalizumab or baliximab (Figure 64(A) and (B)). Finally, when compared with an anti-human CD25 Fc silencing control antibody, antibody 7 killed CD25-expressing cells, i.e., tumor cells or regulatory T cells, via ADCC (Figure 66) and ADCP (Figure 67).

In summary, antibodies 6, 7, 8, and 9 have been characterized and demonstrated to effectively kill CD25-positive cells (Tregs or cancer cell lines) without interfering with IL-2 signaling and therefore without inhibiting the T-effect response. Therefore, these antibodies are Treg-depleting and can be used as monotherapy or in combination for cancer treatment.

Antibodies 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 are characterized by containing a heavy chain variable region comprising the following sequence:

As mentioned above, the sequence and frame regions (FRs) of the complementarity determination regions (CDRs, namely CDR1, CDR2 and CDR3) are defined according to the Kabat numbering scheme.

Epitope mapping results showed that antibodies 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 bound to human CD25 in the regions of amino acids 150 to 163 (YQCVQGYRALHRGP) and 166 to 180 (SVCKMTHGKTRWTQP) of SEQ ID NO:1, and bound to the extracellular protein sequence of human CD25 with Kd values of ^10⁻⁸ M to ^10⁻¹⁰ M.

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

In summary, antibodies 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 have been characterized and demonstrated to effectively kill CD25-positive cells (Tregs or cancer cell lines) without interfering with IL-2 signaling and therefore without inhibiting the T-effect response. Therefore, these antibodies are Treg-depleting and can be used as monotherapy or in combination for cancer treatment.

Example 12: Therapeutic Analysis in Combination with Cancer Vaccines

The therapeutic activity of a combination of non-IL-2 blocking anti-CD25 antibody IgG2a and GVAX in a BVAB16 immunotherapy-resistant mouse model was determined. On day 0, 50 × ^10³ B16BL6 cells were implanted intradermally. On day 5, 200 μg of non-IL-2 blocking anti-CD25 antibody was administered intraperitoneally, or no administration was given. On days 6, 9, and 12, mice were treated with 1 × ^10⁶ irradiated (150 Gy) B16B16 cells supplemented with GM-CSF (GVAX), or no treatment was given. Tumor growth and mouse survival were monitored until day 33. The results are shown in Figure 74.

A synergistic effect of the combination of GVAX and the 7D4 non-blocking anti-CD25 antibody was observed in the B16B16 model. Therefore, 7D4 administration concurrently with cancer vaccines enhanced the vaccine-induced anti-tumor response. These results suggest that non-IL2-blocking depleting anti-CD25 antibodies can be combined with cancer vaccines for the treatment of human cancers. Furthermore, this data indicates that non-IL2-blocking depleting antibodies can enhance vaccine-induced immune responses, suggesting broader applications beyond cancer.

All publications mentioned in the foregoing specification are incorporated herein by reference. Various modifications and variations of the methods and systems 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 conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. Indeed, various modifications to the modes of carrying out the invention, as will be apparent to those skilled in molecular biology, cellular immunology, or related fields, are intended to fall within the scope of the appended claims.

Claims (36)

1. Use of a human IgG1 anti-CD25 antibody in the preparation of a medicament for treating a human subject suffering from a solid tumor, wherein the antibody inhibits less than 25% of IL-2 signaling compared to the absence of the antibody, wherein IL-2 signaling is measured by the level of phosphorylated STAT5 protein in cells as determined by a STAT5 phosphorylation assay, wherein the STAT5 phosphorylation assay comprises: - PMBC cells were cultured for 30 minutes in the presence of anti-CD25 antibody at a concentration of 10 ug/ml, and then different concentrations of IL2 were added for 10 minutes. - To make cells permeable; and - STAT5 protein levels were measured using fluorescently labeled antibodies against phosphorylated STAT5 peptides obtained by flow cytometry. 2. The use according to claim 1, characterized in that the anti-CD25 antibody competes with antibody 7G7B6 for binding to human CD25; and/or It competes with antibody MA251 for binding to human CD25. 3. The use according to claim 1 or 2, characterized in that the anti-CD25 antibody binds to the same epitope recognized by antibody 7G7B6 and/or the epitope recognized by antibody MA251. 4. The use according to claim 1 or 2, characterized in that the anti-CD25 antibody specifically binds to an epitope of human CD25, wherein the epitope comprises one or more amino acid residues selected from one or more amino acid segments selected from amino acid segments of SEQ ID NO:1 (YQCVQGYRALHRGP), SEQ ID NO:1 (SVCKMTHGKTRWTQPQLICTG), SEQ ID NO:1 (KEGTMLNCECKRGFR), and SEQ ID NO:1 (NSSHSSWDNQCQCTSSATR). 5. The use according to claim 4, characterized in that the anti-CD25 antibody specifically binds to an epitope of human CD25, wherein the epitope comprises at least one sequence selected from: amino acids 150 to 158 of SEQ ID NO:1 (YQCVQGYRA), amino acids 176 to 180 of SEQ ID NO:1 (RWTQP), amino acids 42 to 56 of SEQ ID NO:1 (KEGTMLNCECKRGFR), and amino acids 74 to 84 of SEQ ID NO:1 (SSWDNQCQCTS). 6. The use according to claim 4, characterized in that the anti-CD25 antibody specifically binds to an epitope of human CD25, wherein the epitope comprises at least one sequence selected from: amino acids 150 to 158 of SEQ ID NO:1 (YQCVQGYRA), amino acids 166 to 180 of SEQ ID NO:1 (SVCKMTHGKTRWTQP), amino acids 176 to 186 of SEQ ID NO:1 (RWTQPQLICTG), amino acids 42 to 56 of SEQ ID NO:1 (KEGTMLNCECKRGFR), and amino acids 74 to 84 of SEQ ID NO:1 (SSWDNQCQCTS). 7. The use according to claim 4, characterized in that the anti-CD25 antibody specifically binds to an epitope of human CD25, wherein the epitope comprises at least one sequence selected from: amino acids 42 to 56 of SEQ ID NO:1 (KEGTMLNCECKRGFR), amino acids 70 to 84 of SEQ ID NO:1 (NSSHSSWDNQCQCTS), and amino acids 150 to 158 of SEQ ID NO:1 (YQCVQGYRA). 8. The use according to claim 4, wherein the anti-CD25 antibody binds to an epitope comprising the sequence of amino acids 42 to 56 (KEGTMLNCECKRGFR) of SEQ ID NO:1. 9. The use according to claim 4, characterized in that the anti-CD25 antibody binds to an epitope comprising the sequence of amino acids 42 to 56 of SEQ ID NO:1 (KEGTMLNCECKRGFR) and amino acids 150 to 160 of SEQ ID NO:1 (YQCVQGYRALH). 10. The use according to claim 4, characterized in that the anti-CD25 antibody binds to an epitope comprising the sequence of amino acids 42 to 56 (KEGTMLNCECKRGFR) of SEQ ID NO:1 and amino acids 74 to 84 (SSWDNQCQCTSSATR) of SEQ ID NO:1. 11. The use according to claim 4, characterized in that the anti-CD25 antibody binds to an epitope comprising the sequence of amino acids 150 to 163 (YQCVQGYRALHRGP), amino acids 166 to 180 (SVCKMTHGKTRWTQP), amino acids 42 to 56 (KEGTMLNCECKRGFR), and amino acids 74 to 84 (SSWDNQCQCTSSATR) of SEQ ID NO:1. 12. The use according to claim 4, characterized in that the anti-CD25 antibody binds to an epitope of the sequence comprising amino acids 150 to 158 (YQCVQGYRA) and amino acids 176 to 180 (RWTQP) of SEQ ID NO:1. 13. The use according to claim 4, characterized in that the anti-CD25 antibody binds to an epitope comprising the sequence of amino acids 150 to 158 (YQCVQGYRA) of SEQ ID NO:1 and amino acids 176 to 186 (RWTQPQLICTG) of SEQ ID NO:1. 14. The use according to claim 4, characterized in that the anti-CD25 antibody binds to an epitope comprising the sequence of amino acids 150 to 163 (YQCVQGYRALHRGP) of SEQ ID NO:1 and amino acids 166 to 180 (SVCKMTHGKTRWTQP) of SEQ ID NO:1. 15. The use according to claim 4, characterized in that the anti-CD25 antibody binds to an epitope of the sequence comprising amino acids 74 to 84 (SSWDNQCQCTSSATR) of SEQ ID NO:1. 16. The use according to claim 4, characterized in that the anti-CD25 antibody binds to an epitope of the sequence comprising amino acids 70 to 84 (NSSHSSWDNQCQCTS) of SEQ ID NO:1. 17. The use according to claim 4, wherein the anti-CD25 antibody binds to an epitope comprising the sequence of amino acids 176 to 180 (RWTQP) of SEQ ID NO:1. 18. The use according to claim 4, wherein the anti-CD25 antibody binds to an epitope comprising the sequence of amino acids 166 to 180 (SVCKMTHGKTRWTQP) of SEQ ID NO:1. 19. The use according to claim 4, wherein the anti-CD25 antibody binds to an epitope comprising the sequence of amino acids 176 to 186 (RWTQPQLICTG) of SEQ ID NO:1. 20. The use according to claim 1, 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 consumes tumor-infiltrating regulatory T cells. 21. The use according to claim 1, characterized in that the anti-CD25 antibody: (a) Binding to Fcγ receptors with an activation inhibition rate (A/I) greater than 1; and/or (b) Bind FcγRIIa with a higher affinity than binding FcγRI, FcγRIIc and/or FcγRIIb. 22. The use according to claim 1, wherein the antibody is selected from: (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. 23. The use according to claim 1, wherein the dissociation constant (Kd) of the anti-CD25 antibody against CD25 is less than ^10⁻⁷ M. 24. The use according to claim 1, wherein the anti-CD25 antibody is a monoclonal antibody. 25. The use according to claim 1, wherein the anti-CD25 antibody is a human antibody, a chimeric antibody, or a humanized antibody. 26. The use according to claim 1, wherein the antibody is an affinity-matured variant of the antibody. 27. The use according to claim 1, wherein the antibody is a humanized variant of 7G7B6 or MA251 and/or an affinity-matured variant. 28. The use according to claim 1, wherein the anti-CD25 antibody induces enhanced CDC, ADCC and/or ADCP responses. 29. The use according to claim 28, wherein the anti-CD25 antibody induces an increased ADCC and/or ADCP response. 30. The use according to claim 29, characterized in that the anti-CD25 antibody induces an increased ADCC response. 31. The use according to claim 1, characterized in that the anti-CD25 antibody is administered to a subject with an established tumor. 32. Use of the combination of the anti-CD25 antibody as defined in claim 1 with another therapeutic agent in the preparation of a medicament for treating cancer in a human subject, characterized in that the subject suffers from a solid tumor, and the anti-CD25 antibody and the other therapeutic agent are administered simultaneously, separately, or sequentially. 33. The use according to claim 32, wherein the additional therapeutic agent is an immune checkpoint inhibitor. 34. The use according to claim 33, wherein the immune checkpoint inhibitor is a PD-1 antagonist. 35. The use according to claim 32, wherein the additional therapeutic agent is a cancer vaccine. 36. The use according to claim 35, wherein the cancer vaccine is a GVAX cancer vaccine.