HK40016585A - Fc-optimized anti-cd25 for tumor specific cell depletion - Google Patents

Description

Fc-optimized anti-CD 25 for tumor-specific cell depletion

Technical Field

The present invention is in the field of cancer immunotherapy and relates to methods of treating cancer, including methods of treating solid tumors, wherein the methods involve the use of antibodies against CD 25.

Background

Cancer immunotherapy involves the use of the subject's own immune system to treat or prevent cancer. Immunotherapy takes advantage of the fact that cancer cells typically 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 the challenge of the immune system to attack tumor cells by these target antigens. However, malignant tumors, particularly solid tumors or hematological cancers, can evade immune surveillance through various mechanisms inherent to tumor cells and mediated by components of the tumor microenvironment. In the latter, tumor infiltration by regulatory T cells (Treg cells or tregs), more specifically effector T cells (Teff) versus the unfavorable balance of tregs (i.e., low proportion of Teff to tregs) has been proposed as a key factor (Smyth M et al, 2014, immunol cell biol.92, 473-4).

Since their discovery, tregs have been found to be critical in mediating immune homeostasis and promoting the establishment and maintenance of peripheral tolerance. However, in the context of cancer, their effects are more complex. Since cancer cells express self-associated antigens and tumor-associated antigens, the presence of tregs in an attempt to suppress effector cell responses may promote tumor progression. Thus, infiltration of tregs in established tumors often represents one of the major obstacles to effective anti-tumor responses and cancer treatment. The inhibitory mechanisms adopted by tregs are thought to contribute significantly to the limitations or even failure of current therapies, especially immunotherapy relying on the induction or enhancement of anti-tumor responses (oneshi H et al,2012 anti.

Depletion of tregs as a therapeutic approach to the treatment of cancer is a method supported by studies showing the contribution of tregs to tumor formation and progression in mouse models. Furthermore, tumor infiltration by tregs is also associated with a poor prognosis in several human cancers (Shang B et al, 2015, Sci rep.5: 15179). Treg cells have been shown to contribute to tumor formation and progression in mouse models, and their deletion results in delayed tumor progression (Elpek et al, 2007J Immunol.178(11): 6840-8; Golgier et al, 2002; Eur J Immunol.32(11):3267-75, Jones et al, 2002Cancer Immun.22; 2: 1; Onizuka et al, 1999Cancer 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 to Treg cells) correlates with poor outcome in a variety of human cancers (Shang et al, 2015). In contrast, the Teff/Treg cell ratio is associated with a favorable response of immunotherapy in humans and mice (Hodiet al.,2008, proc. natl. acad. sci. usa,105, 3005-. However, Treg depletion in tumors is complex and there are differences in the results of this field of research.

CD is one of the potential molecular targets for achieving Treg depletion CD is also known as interleukin-2 high affinity receptor chain (IL-2Ra), which is constitutively expressed at high levels on Treg cells and is absent or expressed at low levels on T effector cells IL-2/CD interaction has been the target of several murine model studies, most of which involve the use of rat anti-rat CD mouse antibody PC (Setiady Y et al, 2010.EurJ Immunol.40:780-6), the CD binding and functional activity of which has been shown to be associated with and functional activity of a panel of monoclonal antibodies generated by different authors (Lowenthai. 1985.J.Immunol.,135, 3988-Buprofuse 3994; Moreau, J. L. Immunol., 1987. Eurr. J. Immunol.935, 20194, WO 9-11. J. Immunol.102, 1987. J. Immunol., 1987. J. Immunol., 1987. 1986. 1987. 1986. J. Mich. as anti-11. Mich.

For example, 7D4 is a rat IgM anti-mouse CD25 antibody, 7D4 has been widely used to detect CD25 positive cells in the presence of PC61 or after treatment with PC61 or in the presence of antibodies with similar binding properties (Onizuka S et al, 1999, cancer res.59, 3128-3133). Very little literature discloses any functional properties of the 7D4-IgM antibody alone or in comparison with PC61 (Kohm A et al, 2006, J Immunol.176: 3301-5; Hallett W et al, 2008.BiolBlood Marrow Transplant 14: 1088-. Indeed, the prior art does not teach the possibility to adapt or in some way modify the isoform or other structural features of 7D4 in order to obtain improved antibodies for cancer therapy.

However, the ability of 7D4-IgM antibodies (either by themselves or as engineered antibodies) or any anti-human CD25 antibody (e.g. 7G7B6 or M-a251) designed or characterized as having similar CD25 binding characteristics to that of 7D4 to mouse CD25 has been evaluated in detail in relation to optimal depletion of intratumoral Treg cells, either alone or in combination with other antibodies or other anti-cancer compounds, as discussed above, infiltration of Treg cells in tumors, in particular a low proportion of Teff cells, may lead to poor clinical results than Treg cells, CD25 has been identified as a Treg marker and thus may be an interesting target for therapeutic antibodies intended to deplete Treg, importantly, CD25 is a α subunit of the IL-2 receptor, IL-2 is a key cytokine for Teff response, anti-CD 25 antibodies that have been clinically tested so far, also block IL-2 signaling while depleting Treg cells, IL-2 signaling is now found to be a key cytokine for Teff response, the anti-CD 25 antibodies that have been clinically tested so far, while still blocking the anti-CD 632 signaling of treff cells, thus, the anti-IL-antibody-signaling is still required to provide a potent anti-CD-B cell depletion therapy method that is effective for the same.

Disclosure of Invention

The present invention provides the use of anti-CD 25 antibodies and anti-CD 25 antibodies, the anti-CD 25 antibodies being characterized by the structural elements: the structural elements allow binding of CD25 without substantially blocking the binding of interleukin 2(IL-2) to CD25 or the signaling of IL-2 through CD25, and allow efficient depletion of tregs, especially within tumors. The structural and functional characteristics of 7D4-IgM (as described with respect to mouse CD25) have been modified in order to provide antibodies with surprisingly improved characteristics in terms of depletion of tregs and anti-tumor efficacy when used alone or in combination with other anti-cancer drugs. The structural and functional characteristics of additional anti-CD 25 antibodies that do not block the binding of interleukin 2 to CD25 (and do not block IL-2 signaling through CD25) and that effectively deplete tregs have also been characterized. These findings can be used to define and generate additional anti-human CD25 antibodies that provide comparable anti-tumor effects in human subjects. Unless the context indicates otherwise, reference herein to "anti-CD 25 antibodies" and the like includes antigen-binding fragments thereof, as well as variants (including affinity matured variants).

In a primary aspect, the invention provides a method of treating a human subject having cancer, the method comprising the step of administering an anti-CD 25 antibody to a subject, wherein the subject has a tumor (preferably a solid tumor), wherein the antibody does not inhibit binding of interleukin-2 (IL-2) to CD 25.

Reference herein to "non-blocking," "non-IL-2 blocking," "non-blocking," and similar terms (with respect to not blocking IL-2 binding to CD25 in the presence of anti-CD 25 antibody) includes embodiments wherein the anti-CD 25 antibody does not block IL-2 signaling through CD 25. That is, the anti-CD 25 antibodies of the invention inhibit IL-2 signaling by CD25 by less than 50% compared to IL-2 signaling in the absence of the antibody. Preferably, the anti-CD 25 antibody inhibits IL-2 signaling by less than about 40%, 35%, 30%, preferably less than about 25% compared to IL-2 signaling in the absence of the antibody.

In one embodiment, the anti-CD 25 antibody competes with antibody 7G7B6 for binding to human CD 25; and/or competes with antibody MA251 for binding to human CD 25.

In one embodiment, the anti-CD 25 antibody binds to the same epitope recognized by antibody 7G7B6 and/or binds to the same epitope recognized by antibody MA 251.

In one embodiment, the anti-CD 25 antibody specifically binds to an epitope of human CD25, wherein the epitope comprises one or more amino acid residues comprised in one or more of the amino acid segments selected from amino acid 150 to 163 (YQCVQGYRALHRGP) of SEQ ID NO:1, amino acid 166 to 186 (SVCKMTHGKTRWTQPQLICTG) of SEQ ID NO:1, amino acid 42 to 56 (KEGTMLNCECKRGFR) of SEQ ID NO:1, amino acid 70 to 88 (NSSHSSWDNQCQCTSSATR) of SEQ ID NO: 1. 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 comprised in one or more amino acid segments selected from amino acid 150 to 163 (YQCVQGYRALHRGP) of SEQ ID NO:1, amino acid 166 to 186 (SVCKMTHGKTRWTQPQLICTG) of SEQ ID NO:1, amino acid 42 to 56 (KEGTMLNCECKRGFR) of SEQ ID NO:1, and/or amino acid 70 to 88 (NSSHSSWDNQCQCTSSATR) of SEQ ID NO: 1.

In one embodiment, the anti-CD 25 antibody specifically binds to an epitope of human CD25, wherein the epitope comprises at least one sequence selected from the group consisting of: amino acids 150 to 158 of SEQ ID NO:1 (YQCVQGYRA), amino acids 176 to 180 of SEQ ID NO:1 (RQTQP), 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-CD 25 antibody specifically binds to an epitope of human CD25, wherein the epitope comprises at least one sequence selected from amino acids, wherein the epitope comprises at least one sequence selected from the group consisting of: 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-CD 25 antibody specifically binds to an epitope of human CD25, wherein the epitope comprises at least one sequence selected from the group consisting of: 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-CD 25 antibody binds to an epitope comprising the sequence of amino acids 42 to 56 (KEGTMLNCECKRGFR) of SEQ ID NO: 1. In one embodiment, the anti-CD 25 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 (YQCVQGYRALH) of SEQ ID No. 1. In another embodiment, the anti-CD 25 antibody binds to an epitope comprising the sequence of amino acids 42 to 56 of SEQ ID NO:1(KEGTMLNCECKRGFR) and amino acids 74 to 88 of SEQ ID NO:1 (SSWDNQCQCTSSATR). In another embodiment, the anti-CD 25 antibody binds to an epitope comprising the sequence of amino acids 150 to 163 (YQCVQGYRALHRGP) of SEQ ID NO:1, amino acids 166 to 180 (SVCKMTHGKTRWTQP) of SEQ ID NO:1, amino acids 42 to 56 (KEGTMLNCECKRGFR) of SEQ ID NO:1, and amino acids 74 to 88 (SSWDNQCQCTSSATR) of SEQ ID NO: 1.

In one embodiment, the anti-CD 25 antibody binds to an epitope comprising the sequence of amino acids 176 to 180 (RWDQP) of SEQ ID NO: 1. In one embodiment, the anti-CD 25 antibody binds to an epitope comprising the sequence of amino acids 166 to 180 (SVCKMTHGKTRWTQP) of SEQ ID NO: 1. In one embodiment, the anti-CD 25 antibody binds to an epitope comprising the sequence of amino acids 176 to 186 (RWTQPQLICTG) of SEQ ID NO: 1.

In one embodiment, the anti-CD 25 antibody specifically binds to an epitope comprising the sequence of amino acids 150 to 158 of SEQ ID NO:1 (YQCVQGYRA) and amino acids 176 to 180 of SEQ ID NO:1 (RWGFP). In one embodiment, the anti-CD 25 antibody specifically binds to an epitope comprising the sequence of amino acids 150 to 158 of SEQ ID NO:1 (YQCVQGYRA) and amino acids 176 to 186 of SEQ ID NO:1 (RWTQPQLICTG). In one embodiment, the anti-CD 25 antibody specifically 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.

In one embodiment, the anti-CD 25 antibody binds to an epitope comprising the sequence of amino acids 74 to 84 (SSWDNQCQCTSSATR) of SEQ ID NO: 1. In one embodiment, the anti-CD 25 antibody binds to an epitope comprising the sequence of amino acids 70 to 84 (NSSHSSWDNQCQCTS) of SEQ ID NO: 1.

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

Such antibodies preferably have a dissociation constant (K) for CD25_d) Less than 10^-7M and/or dissociation constant of less than about 10 for at least one activated Fc gamma receptor^-6And M. Preferably, the antibody has a dissociation constant (K) for CD25_d) At 10^-8Or 10^-9Or 10^-10Or 10^-11Or 10^-12Or 10^-13Within or below the range. Most preferably, the antibody is a human IgG1 antibody that binds with high affinity to at least one activating Fc γ receptor and depletes tumor infiltrating regulatory T cells. Most preferablyPreferably, anti-CD 25 is characterized by other features associated with Fc γ receptors, in particular:

(a) binds to Fc γ receptor with an activation inhibition rate (a/I) superior to 1; and/or

(b) Binds Fc γ RIIa with higher affinity than Fc γ RIIb.

The anti-CD 25 antibody may exhibit further preferred characteristics in view of the use of the anti-CD 25 antibody in a method of treatment. The anti-CD 25 antibody is preferably a monoclonal antibody, in particular a human, chimeric or humanized antibody. The antibody may be an affinity matured variant thereof, optionally a humanized or affinity matured variant of 7G7B6 or MA 251. Furthermore, given that the anti-CD 25 antibody interacts with immune cells and/or other components of the immune system to exert its activity, the anti-CD 25 antibody may further elicit an enhanced CDC, ADCC and/or ADCP response, preferably an increased ADCC and/or ADCP response, more preferably an increased ADCC response, compared to the existing anti-human CD25 clinical antibodies darlizumab and basiliximab. In some embodiments, the anti-CD 25 antibody may elicit a reduced CDC response, more preferably, the anti-CD 25 antibody does not elicit a CDC response, as compared to existing anti-human CD25 clinical antibodies, dallizumab and basiliximab.

The anti-CD 25 antibodies of the invention (as generally defined above and further detailed in the detailed description) can be used in methods of treating a human subject by administering the anti-CD 25 antibodies to the subject. In one embodiment, the subject has cancer. Preferably, the subject has a defined solid tumor (preferably, in a method further comprising the step of identifying a subject having a solid tumor). Such methods may further comprise administering an additional therapeutic agent to the subject. In one embodiment, the additional agent may be an immune checkpoint inhibitor for the subject, for example in the form of an antibody that binds and inhibits an immune checkpoint protein. Preferred immune checkpoint inhibitors are PD-1 antagonists, which may be anti-PD-1 antibodies or anti-PD-L1 antibodies. More generally, an anti-CD 25 antibody is useful in a method of depleting regulatory T cells in a solid tumor in a subject, the method comprising the step of administering the anti-CD 25 antibody to the subject.

In another aspect, the anti-CD 25 antibodies of the invention are useful for the preparation of a medicament for the treatment of cancer in a human subject, preferably wherein the subject has a tumor, preferably a solid tumor. The antibodies can be administered in combination with an additional therapeutic agent, preferably an additional cancer therapeutic agent, for example in combination with an immune checkpoint inhibitor (preferably a PD-1/PD-L1 pathway antagonist), a cancer vaccine, and/or in combination with standard of care therapy (e.g., chemotherapy or radiation therapy).

In another aspect, the invention provides a combination of an anti-CD 25 antibody as defined above and another anti-cancer compound (preferably an immune checkpoint inhibitor or other compound as set out in the detailed description), preferably wherein the subject has a solid tumor, for use in the treatment of cancer in a human subject, and the anti-cancer compound (e.g. an immune checkpoint inhibitor (e.g. a PD-1 antagonist) or a cytokine (e.g. interleukin 2)) may be administered simultaneously, separately or sequentially. Within this scope, the invention also provides a kit for treating cancer comprising an anti-CD 25 antibody as defined above and an anti-cancer compound (e.g., an immune checkpoint inhibitor (e.g., a PD-1 antagonist)).

In another aspect, the invention also provides a pharmaceutical composition comprising an anti-CD 25 antibody as defined above in a pharmaceutically acceptable medium. Such compositions may also comprise an anti-cancer compound (e.g., an immune checkpoint inhibitor (e.g., a PD-1 antagonist)).

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

(a) a first antigen-binding moiety that binds to CD 25; and

(b) a second antigen-binding moiety that binds to another antigen;

wherein the anti-CD 25 antibody does not inhibit the binding of interleukin-2 (IL-2) to CD25, and preferably, the bispecific antibody binds to at least one activating Fc γ receptor with high affinity and depletes tumor infiltrating regulatory T cells. Preferably, such second antigen-binding moiety binds an antigen selected from an immune checkpoint protein or a tumor-associated antigen, or may be or be based on an anti-human activated Fc receptor antibody (anti-FcgRI, anti-fcgriiia, anti-fcgriiii) or an antagonistic anti-human fcyriib antibody. Thus, the second antigen-binding moiety may bind FcRIIb. It may alternatively bind FcgRI, fcgriiia and/or fcgriiii with antagonistic activity.

Preferably, such bispecific antibodies comprise a second antigen-binding moiety that binds 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. Such an immune checkpoint protein is preferably expressed on tumor cells. Preferably, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1 and CTLA-4. The second antigen-binding moiety that binds to an immune checkpoint protein may be comprised in commercially available antibodies that are immune checkpoint inhibitors, such as:

(a) in the case of PD-1, the anti-PD-1 antibody may be nivolumab or pembrolizumab.

(b) In the case of PD-L1, anti-PD-L1 is atlizumab;

(c) in the case of CTLA-4, the anti-CTLA-4 is ipilimumab.

Such bispecific antibodies may be provided in any commercially available form, including molecular forms of Duobody, BiTE DART, CrossMab, Knobs-in-holes, Triomab, or other suitable bispecific antibodies and fragments thereof.

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

The combination of the anti-CD 25 antibody of the invention with another anti-cancer compound and a bispecific antibody as defined above can be used in a method of treating cancer, comprising the step of administering said combination or said bispecific antibody to a subject, in particular when the subject has a solid tumor, and for treating cancer in the subject.

Further objects of the invention are provided in the detailed description and examples, including further definitions of anti-human CD25 antibodies of the invention and their use in methods of treating cancer, in pharmaceutical compositions, in combination with other anti-cancer compounds, in bispecific antibodies.

Detailed Description

The present invention provides a method of treating or preventing cancer in a subject (preferably when the subject has a solid tumor), the method comprising administering to the subject an antibody that binds CD25, wherein the anti-CD 25 antibody is characterized by the structural elements: the structural elements allow binding of CD25 without interfering with interleukin 2 binding or signaling through CD25 and efficient depletion of tregs, particularly within tumors. Antibodies that bind CD25 as defined in the present invention are useful for the treatment or prevention of cancer, preferably solid tumors. Alternatively, the present invention provides the use of an antibody that binds CD25 and allows binding to CD25 without interfering with the binding of interleukin 2 to CD25 and efficiently depletes tregs for the manufacture of a medicament for the treatment or prevention of cancer, preferably solid tumors. The invention also provides the use of an antibody that binds CD25 and allows 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 present inventors have discovered that CD25 can be targeted using an anti-CD 25 antibody that does not inhibit (or does not substantially inhibit) the binding of interleukin 2 to CD25 or IL-2 signaling through CD25 to deplete regulatory T cells in a therapeutic setting (e.g., in established solid tumors). The present inventors have found that non-IL-2 blocking anti-CD 25 antibodies with an isotype that enhances their binding to activating Fc γ receptors result in efficient depletion of tumor infiltrating regulatory T cells while still allowing for an optimal Teff response, i.e., a therapeutic approach that can be associated (in combination or in a bispecific antibody), for example, with other cancer targeting compounds, such as those targeting immune checkpoint proteins, tumor associated antigens, or inhibitory Fc γ receptors. These findings also allow the combination of an anti-CD 25 antibody with interleukin-2 at appropriate doses for the treatment of cancer.

CD25 is the α chain of the IL-2 receptor and is present in activated T cells, regulatory T cells, activated B cells, some NK T cells, some thymocytes, myeloid 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 consensus sequence for 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 shown in SEQ ID NO: 2):

as used herein, "an antibody that binds to CD 25" refers to an antibody that is capable of binding to the CD25 subunit of the IL-2 receptor.

anti-CD 25 antibodies are antibodies capable of specifically binding to the CD25 subunit (antigen) of the IL-2 receptor. "Specific binding/Specific binding" is understood to mean that the dissociation constant of an antibody for an antigen of interest is less than about 10^-6M、10^-7M、10^-8M、10^-9M、10^-10M、10^-11M、10^-12M or 10^-13And M. In preferred embodiments, the dissociation constant is less than 10^-8M, e.g. at 10^-9M、10^-10M、10^-11M、10^-12M or 10^-13M is in the range of.

The term "antibody" as used herein refers to intact immunoglobulin molecules and fragments thereof that comprise an antigen binding site, and includes polyclonal, monoclonal, genetically engineered and other modified forms of antibodies, including but not limited to chimeric antibodies, humansHumanized, heteroconjugates, and/or multispecific antibodies (e.g., bispecific, diabodies, triabodies, and tetrabodies) and antigen-binding fragments of antibodies, including, for example, Fab ', F (ab')₂Fab, Fv, rlgG, polypeptide-Fc fusion, single-chain variants (scFv fragment, VHH, Trans-Shark single domain antibodies (shark single domain antibodies), single chain or tandem diabodiesVHH、A micro-antibody,Bicyclic peptides and other alternative immunoglobulin scaffolds). In some embodiments, the antibody may lack the covalent modifications (e.g., glycan linkages) that the antibody would have if it were naturally occurring. In some embodiments, the antibody may contain a covalent modification (e.g., a glycan linkage, a detectable moiety, a therapeutic moiety, a catalytic moiety, or other chemical group that provides improved stability or administration of the antibody, such as polyethylene glycol). In some embodiments, the antibody can be a masked antibody (e.g.,) In the form of (1). A masking antibody may comprise a blocking or "masking" peptide that specifically binds to the antigen-binding surface of the antibody and interferes with antigen binding of the antibody. The masking peptide is linked to the antibody by a cleavable linker (e.g., by a protease). Selective cleavage of the linker in the desired environment (i.e., in the tumor environment) allows the masking/blocking of peptide dissociation such that antigen binding occurs in the tumor, thereby limiting potential toxicity issues."antibody" may also refer to camelid antibodies (heavy chain only antibodies) and antibody-like molecules (e.g., anticalins (Skerra (2008) FEBS J275,2677-83)). In some embodiments, the antibodies are polyclonal or oligoclonal, produced as a set of antibodies, each associated with a single antibody sequence and binding to a more or less different epitope within the antigen (e.g., a different epitope within the extracellular domain of human CD25 associated with a different reference anti-human CD25 antibody). Polyclonal or oligoclonal antibodies can be provided in a single formulation for medical use as described in the literature (Kearns JD et al, 2015.MolCancer ther.14: 1625-36).

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 in a human subject. In aspects of the invention, the antibody may be an affinity matured humanized variant of 7G7B6 or MA 251. Affinity matured antibodies have at least 10% affinity for CD25 and/or the CDR sequences are at least 80% identical, preferably 90% identical to the CDRs of the parent sequence (across all sequences). Affinity matured antibodies are antibodies with one or more altered amino acids in one or more CDRs which result in an antibody with improved affinity for CD25 compared to the parent strain without the altered amino acids.

Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins with the same structural features. The immunoglobulins may be from any class, for example IgA, IgD, IgG, IgE or IgM. The immunoglobulin may be, for example, IgG₁、IgG₂、IgG₃Or IgG₄Any subclass of (a). In a preferred aspect of the invention, the anti-CD 25 antibody is from the IgG class, preferably the IgG1 subclass. In one aspect, the anti-CD 25 antibody is from the human IgG1 subclass. Alternatively, in one aspect, the anti-CD 25 antibody is from the human IgG2 subclass.

The Fc region of IgG antibodies interacts with several cellular Fc γ receptors (Fc γ rs) to stimulate and modulate downstream effector mechanisms. There are five activating receptors, namely Fc γ RI (CD64), Fc γ RIIa (CD32a), Fc γ RIIc (CD32c), Fc γ RIIIa (CD16a) and Fc γ RIIIb (CD16b), and an inhibitory receptor Fc γ RIIb (CD32 b). Communication of IgG antibodies to the immune system is controlled and mediated by Fc γ R, which relays information sensed and collected by the antibodies to the immune system, thereby providing a link between the innate and adaptive immune systems, particularly in the context of biological therapy (Hayes J et al, 2016.JInflamm Res 9: 209-219).

The IgG subclasses differ in their ability to bind Fc γ R, and this differential binding determines their ability to elicit a range of functional responses. For example, in humans, Fc γ RIIIa is the primary receptor involved in antibody-dependent cell-mediated cytotoxicity (ADCC) activation, and IgG1 followed by IgG3 showed the highest affinity for this receptor, reflecting their ability to efficiently induce ADCC. Although IgG2 has been shown to have weak binding to this receptor, anti-CD 25 antibody with the human IgG2 isotype has been found to deplete tregs efficiently.

In a preferred embodiment of the invention, the antibody binds Fc γ R with high affinity, preferably binds to the activating receptor with high affinity. Preferably, the antibody binds with high affinity to Fc γ RI and/or Fc γ RIIA and/or Fc γ RIIIA. In specific embodiments, the antibody binds to at least one activating Fc γ receptor at less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10The dissociation constants of M bind.

In one aspect, the antibody is an IgG1 antibody, preferably a human IgG1 antibody, which is capable of binding to at least one Fc-activated receptor. For example, the antibody can bind to one or more receptors selected from the group consisting of Fc γ RI, Fc γ RIIa, Fc γ RIIc, Fc γ RIIIa, and Fc γ RIIIb. In one aspect, the antibody is capable of binding to Fc γ RIIIA. In one aspect, the antibody is capable of binding to Fc γ RIIIA and Fc γ RIIA, and optionally Fc γ RI. In one aspect, the antibody is capable of binding to these receptors with high affinity, e.g., less than about 10^-7M、10^-8M、10^-9M or 10^-10The dissociation constants of M bind.

In one aspect, the antibody binds the inhibitory receptor Fc γ RIIb with low affinity. In one aspect, the antibody is administered at greater than about 10^-7M, greater than about 10^-6M is greater than about 10^-5Dissociation constant of M binds Fc γ RIIb。

In a preferred embodiment of the invention, the anti-CD 25 antibody is from the IgG1 subclass and preferably has ADCC and/or ADCP activity, as discussed herein, in particular 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 Fc γ R subtypes with high inhibition of activation (a/I) (at least above 1). In contrast, other isoforms (e.g., the rgig 1 isoform) bind only with similar affinities to a single activating Fc γ R (Fc γ RIII) and to inhibitory Fc γ RIIb, resulting in a low a/I ratio (< 1). This lower a/I ratio can be associated with lower intratumoral Treg depletion and lower isotype antitumor therapeutic activity. Although the Fc γ R binding profile of the human IgG2 isotype antibody is known, significant Treg depletion can also be achieved with the human IgG2 isotype of the anti-CD 25 antibody. Thus, in one embodiment, the anti-CD 25 antibody is from the IgG2 subclass.

In preferred embodiments, an anti-CD 25 antibody as described herein binds to human CD25, preferably with high affinity. Still preferably, the anti-CD 25 antibody binds to the extracellular region of human CD25, as shown above. In one aspect, the invention provides an anti-CD 25 antibody as described herein. In particular, the examples provide experimental data for the production of antibodies secreted by the 7D4 hybridoma. As shown in the background of the invention, the antibody is specific for mouse CD25, as shown by comparing the monoclonal antibody panel (including PC61), the antibody binds to one of the three epitopes in mouse CD25 that are distinct from the binding site for IL-2, and does not block the binding of IL-2 to CD 25. For example, 7D4 has been shown to bind to mouse CD25 at an epitope comprising amino acids 184 to 194(REHHRFLASEE) in [ Uniprot sequence P01590] when the corresponding isoforms are associated. Assays involving 7D4 and mouse CD25 in the 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) and those disclosed in the examples include recombinant antibodies comprising the CD25 binding domain of 7D4 or non-IL-2 blocking anti-human CD25 antibodies named MA-251 and 7G7B6, which may be suitable for characterizing those human antibodies that recognize human CD25, which have the same functional characteristics of 7D4 at the level of interaction with CD25 (particularly by not blocking IL-2 binding) and with fcgamma receptor (particularly by preferentially binding one or more of human activating fcgamma 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 CD 25; and/or binds to the same epitope or epitope recognized by antibody 7G7B 6. 7G7B6 is a monoclonal antibody having the mouse IgG2a isotype that recognizes human CD 25. 7G7B6 comprises a heavy chain variable region having the sequence:

EVQLVESGGDLVQPRGSLKLSCAASGFTFSSYGMSWVRQTPDKRLELVATINGYGDTTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYFCARDRDYGNSYYYALDYWGQGTSVTVSS

(SEQ ID NO:3)，

and a light chain variable region having the sequence:

QIVLSQSPAILSASPGERVTMTCRASSSVSFMHWLQQKPGSSPKPWIYATSNLASGVSARFSGSGSGTSYSLTITRVEAEDAATYYCQQWSSNPPAFGGGTKLEIK

(SEQ ID NO:4)。

in one embodiment, the antibody comprises a heavy chain comprising: amino acid sequence GFTLDSYGVS (SEQ ID NO:7) as variable heavy chain CDR1, amino acid sequence GVTSSGGSAYYADSV (SEQ ID NO:8) as variable heavy chain CDR2, amino acid sequence DRYVYTGGYLYHYGMDL (SEQ ID NO:9) as variable heavy chain CDR 3; the light chain comprises: amino acid sequence RASQSISDYLA (SEQ ID NO:11) as variable light chain CDR1, amino acid sequence YAASTLPF (SEQ ID NO:12) as variable light chain CDR2, and amino acid sequence QGTYDSSDWYWA (SEQ ID NO:13) as variable light chain CDR 3. The antibody can compete with 7G7B6 for binding to human CD 25. Preferably, the antibody comprises a heavy chain comprising a heavy chain variable region comprising the sequence:

EVQLVESGGGLIQPGGSLRLSCAASGFTLDSYGVSWVRQAPGKGLEWVGVTSSGGSAYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRYVYTGGYLYHYGMDLWGQGTLVTVSS

(SEQ ID NO:10)，

the light chain comprises a light chain variable region comprising the sequence:

DIQMTQSPSSLSASVGDRVTITCRASQSISDYLAWYQQKPGKVPKLLIYAASTLPFGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQGTYDSSDWYWAFGGGTKVEI

(SEQ ID NO:14)。

in another embodiment, the antibody comprises a heavy chain comprising: amino acid sequence SGFSVDIYDMS (SEQ ID NO:15) as variable heavy chain CDR1, amino acid sequence YISSSLGATYYADSV (SEQ ID NO:16) as variable heavy chain CDR2, and amino acid sequence ERIYSVYTLDYYAMDL (SEQ ID NO:17) as variable heavy chain CDR3, and the light chain comprises: amino acid sequence QASQGITNNLN (SEQ ID NO:19) as the variable light chain CDR 1; the amino acid sequence YAASTLQS (SEQ ID NO:20) serves as the variable light chain CDR2, and the amino acid sequence QQGYTTSNVDNA (SEQ ID NO:21) serves as the variable light chain CDR 3. The antibody can compete with 7G7B6 for binding to human CD 25. Preferably, the antibody comprises a heavy chain comprising a heavy chain variable region comprising the sequence:

EVQLLESGGGLVQPGGSLRLSCAASGFSVDIYDMSWVRQAPGKGLEWVAYISSSLGATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERIYSVYTLDYYAMDLWGQGTLVTVSS

(SEQ ID NO:18)，

the light chain comprises a light chain variable region comprising the sequence:

DIQMTQSPSSLSASVGDRVTITCQASQGITNNLNWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYTTSNVDNAFGGGTKVEIK

(SEQ ID NO:22)。

in one embodiment, an antibody that can compete with 7G7B6 for binding to human CD25 comprises a heavy chain comprising the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRDYGNSYYYALDYWGQGTLVTVSS (SEQ ID NO:23), or

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYFCARDRDYGNSYYYALDYWGQGTLVTVSS(SEQ ID NO:24)；

The light chain comprises the following amino acid sequence:

EIVLTQSPGTLSLSPGERATLSCRASSSVSFMHWLQQKPGQAPRPLIYATSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCQQWSSNPPAFGQGTKLEIK (SEQ ID NO:25), or

QIVLTQSPGTLSLSPGERATLSCRASSSVSFMHWLQQKPGQSPRPLIYATSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCQQWSSNPPAFGQGTKLEIK(SEQ ID NO:26)。

In one aspect of the invention, the antibody competes with antibody MA251 for binding to human CD 25; and/or bind to the same epitope or epitope recognized by antibody MA 251. MA251 is a monoclonal antibody with the mouse isotype that recognizes human CD 25. The MA251 includes: a heavy chain variable region having the sequence:

QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGIQWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARAYGYDGSWLAYWGQGTLVTVSS

(SEQ ID NO:5)

and a light chain variable region having the sequence:

QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIFATSNLASGVPARFSGSGSGTSYSLTINRVEAEDADTYYCQQWSSNPPTFGGGTKLEIK

(SEQ ID NO:6)。

in one embodiment, an antibody that can compete with MA251 for binding to human CD25 comprises a heavy chain comprising a heavy chain variable region comprising the amino acid sequence:

QVQLVESGGGVVQPGGSLRLSCAVSGFSLTSYGIQWVRQAPGKGLEWVSVIWAGGSTNYNSALMSRFTISKDNSKSTLYLQMNSLRAEDTAVYYCARAYGYDGSWLAYWGQGTLVTVSS(SEQ ID NO:27)；

QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGIQWVRQPPGKGLEWIGVIWAGGSTNYNSALMSRVTISKDNSKSQFSLKLSSVTAADTAVYYCARAYGYDGSWLAYWGQGTLVTVSS (SEQ ID NO: 28); or

QVQLVESGGGVVQPGGSLRLSCAVSGFSLTSYGIQWVRQAPGKGLEWVSVIWAGGSTNYNSALMSRFTISKDNSKSTLYLQMNSLRAEDTAVYYCARAYGYDGSWLAYWGQGTLVTVSS(SEQ ID NO:29)；

The light chain comprises a light chain variable region comprising the amino acid sequence:

QIVLTQSPATLSLSPGERATLSCRASSSVSYMHWYQQKPGQAPRPLIFATSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPTFGGGTKLEIK(SEQ ID NO:30)；

QIVLTQSPATLSLSPGERATLSCRASSSVSYMHWYQQKPGQAPRPLIFATSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPTFGGGTKLEIK(SEQ ID NO:31)；

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIFATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIK (SEQ ID NO: 32); or

QIQLTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKSPKPLIFATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIK(SEQ ID NO:33)。

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

In one aspect, the antibody competes with antibody 7G7B6 and antibody MA251 for binding to human CD 25. In one aspect, the antibody binds to the same epitope recognized by 7G7B6 and recognized by MA 251.

Competition between the 7G7B6 antibody or the MA251 antibody and additional antibodies can be measured, for example, as discussed in the examples and known in the art. In some embodiments, competition between two antibodies (e.g., 7G7B6 or MA251 and an additional antibody) is determined by adding the additional antibody to the assay and measuring the interaction between the 7G7B6 or MA251 antibody and human CD 25. One such assay is an Octet-based assay in which simultaneous binding of 7G7B6 or MA251 antibody, an additional antibody, to recombinant human CD25 is determined. The antibodies were non-competitive if binding of both antibodies to recombinant human CD25 was detected. Alternatively, one such assay is an enzyme-linked immunosorbent assay (ELISA), in which the binding of 7G7B6 or MA251 antibody to recombinant human CD25 is detected. The latter antibody is a competitor to the 7G7B6 or MA251 antibody if the observed signal is reduced (e.g. by at least 75%) upon addition of the additional antibody. Simultaneous binding of the 7G7B6 or MA251 antibody and the additional antibody to human CD25 expressing cells can also be detected using flow cytometry.

In one aspect, the invention provides an anti-CD 25 antibody that specifically binds to an epitope of human CD25, wherein the epitope comprises one or more amino acid residues from one or more of the amino acid segments selected from amino acid 150 to 163 (YQCVQGYRALHRGP) of SEQ ID NO:1, amino acid 166 to 186 (SVCKMTHGKTRWTQPQLICTG) of SEQ ID NO:1, amino acid 42 to 56 (KEGTMLNCECKRGFR) of SEQ ID NO:1, amino acid 70 to 88 (NSSHSSWDNQCQCTSSATR) of SEQ ID NO: 1. 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: amino acids 150 to 158 of SEQ ID NO:1 (YQCVQGYRA), amino acids 176 to 180 of SEQ ID NO:1 (RQTQP), 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 are different from the IL-2 binding site in human CD25, and as described in the examples, antibodies that bind to such epitopes do not block the binding of IL-2 to CD 25.

In a preferred embodiment, the method of treating a human subject having cancer comprises the step of administering an anti-CD 25 antibody of the invention to the subject, wherein the subject preferably has a solid tumor, and wherein the anti-CD 25 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 the group consisting of fcyri (CD64), fcyriic (CD32c), and fcyriiia (CD16a) and depletes tumor infiltrating regulatory T cells. Preferably, the dissociation constant (K) of the anti-CD 25 antibody to CD25_d) Less than 10^-7M, preferably less than 10^-8And M. More preferably, the anti-CD 25 antibody binds to human CD25, providing an effect on IL-2 binding and Treg depletion, similar to the effect of 7D4 on mouse CD25 or the effect of 7G7B6 and MA251 on human CD 25. In the further stepIn embodiments, the anti-CD 25 antibody binds to fcgamma receptor with an activation inhibition rate (a/I) greater than 1 and/or binds to fcgamma RI (CD64), fcgamma RIIC (CD32c), fcgamma RIIIA (CD16a) and/or fcgamma RIIa (CD32a) with higher affinity than to fcgamma RIIb (CD32 b).

The CD25 binding domain of the 7D4 antibody has been cloned and expressed as a recombinant protein fused to an appropriate constant region. The sequence of the CD25 binding domain of the 7D4 antibody, as well as its specificity and/or other functional activity for different epitopes within the extracellular domain of CD25, can be used to compare candidate anti-CD 25 antibodies generated and screened by any suitable technique (by culturing a panel of hybridomas or generating a library of recombinant antibodies from rodents immunized with CD25, and then screening these antibody libraries with CD25 fragments for functional characterization as described herein). The anti-CD 25 antibodies thus identified may also be produced as recombinant antibodies, particularly as whole antibodies or as fragments or variants described herein.

Natural antibodies and immunoglobulins are typically heterotetrameric glycoproteins of about 150000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has a variable domain (V) at the amino terminus_H) Followed by a plurality of constant domains. Each light chain has a variable domain (V) at the amino terminus_L) And a constant domain at the carboxy terminus.

The 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. The variable region of the H chain or L chain contains an amino acid sequence capable of specifically binding to an antigen target. Among these are the smaller sequences called "hypervariable" because they have great variability between antibodies of different specificities. Such hypervariable regions are also referred to as "complementarity determining regions" or "CDR" regions.

These CDR regions explain the basic specificity of a particular antigenic determinant structure of an antibody. CDRs represent discrete amino acid segments within the variable region, however, regardless of species, the positional positioning of these key amino acid sequences within the variable heavy and light chain regions has been found to have similar positioning within the amino acid sequences of the variable chains. The variable heavy and light chains of all antibodies each have 3 CDR regions, each CDR region being discontinuous for the light (L) and heavy (H) chains (designated L1, L2, L3, H1, H2, H3). The accepted CDR regions have been previously described (Kabat et al, 1977.J Biol Chem 252, 6609-.

The antibodies of the invention may act 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 mechanism that allows targeting, blocking proliferation and/or depletion of Treg cells.

"complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by an antibody of the invention in the presence of complement.

"antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (fcrs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on target cells, resulting in lysis of the target cells.

"antibody-dependent cell-mediated phagocytosis" (ADCP) refers to a cell-mediated response in which Fc receptor (FcR) -expressing phagocytic cells (e.g., macrophages) recognize bound antibody on target cells resulting in phagocytosis of the target cells.

CDC, ADCC and ADCP can be measured using assay methods known and available in the art (Clynes et al (1998) Proc Natl Acad Sci USA95,652-6), as discussed in the examples. The constant regions of antibodies are important in the ability of the antibody to fix complement and mediate cell-dependent cellular cytotoxicity and phagocytosis. Thus, as discussed herein, the isotype of an antibody can be selected based on whether the antibody is required to mediate cytotoxicity/phagocytosis.

As discussed herein, in one embodiment of the invention, an anti-CD 25 antibody that does not inhibit interleukin 2 binding and results in Treg cell depletion is used. For example, anti-CD 25 antibodies that do not inhibit binding of interleukin 2 to CD25 and elicit a strong CDC response and/or strong ADCC and/or strong ADCP response may be used. Methods of increasing CDC, ADCC and/or ADCP are known in the art. For example, CDC response may be increased with mutations in the antibody that increase the binding affinity of C1q (ldusogenet al (2001) J lmmunol 166,2571-5).

Reference herein to "not inhibiting the binding of interleukin-2 to CD 25" may also be expressed as the anti-CD 25 antibody being a non-IL-2 blocking antibody or a "non-blocking" antibody (with respect to not blocking the binding of IL-2 to CD25 in the presence of an anti-CD 25 antibody), i.e. the antibody does not block the binding of interleukin-2 to CD25, in particular does not inhibit interleukin-2 signaling in cells expressing CD 25. Reference to "non-blocking," "non-IL 2-blocking," "non-blocking," or "non-blocking," etc. (with respect to the non-blocking nature of IL-2 binding to CD25 in the presence of an anti-CD 25 antibody) includes embodiments wherein the anti-CD 25 antibody of the invention does not block IL-2 signaling through CD 25. That is, the anti-CD 25 antibody inhibits IL-2 signaling by less than 50% compared to IL-2 signaling in the absence of the antibody. In particular embodiments of the invention as described herein, the anti-CD 25 antibody inhibits IL-2 signaling by less than about 40%, 35%, 30%, preferably by less than about 25% compared to IL-2 signaling in the absence of the antibody. The anti-CD 25 non-IL-2 blocking antibody allows binding to CD25 without interfering with IL-2 binding to CD25, or without substantially interfering with IL-2 binding to CD 25. The non-IL-2 blocking antibodies mentioned herein may also be expressed as anti-CD 25 antibodies that "do not inhibit the binding of interleukin-2 to CD 25" or as anti-CD 25 antibodies that "do not inhibit the signaling of IL-2".

Some anti-CD 25 antibodies may allow IL-2 to bind to CD25, but still block signaling through the CD25 receptor. Such anti-CD 25 antibodies are not within the scope of the invention. In contrast, non-IL-2 blocking anti-CD 25 antibodies allow IL-2 to bind to CD25 to promote at least 50% of the level of signaling through the CD25 receptor as compared to signaling in the absence of anti-CD 25 antibody.

IL-2 signaling through CD25 can be measured by methods discussed in the examples and known in the art. The comparison of IL-2 signaling in the presence and absence of an anti-CD 25 antibody agent may be performed under the same or substantially the same conditions.

In some embodiments, mayIL-2 signaling was 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 can include culturing PMBC cells in the presence of anti-CD 25 antibody at a concentration of 10ug/ml for 30 minutes, followed by addition of different concentrations of IL-2 (e.g., 10U/ml or different concentrations of 0.25U/ml, 0.74U/ml, 2.22U/ml, 6.66U/ml, or 20U/ml) for 10 minutes. The cells can then be permeabilized and the levels of STAT5 protein can then be measured with a fluorescently labeled antibody against the phosphorylated STAT5 peptide, which is analyzed by flow cytometry. The percentage of blocking IL-2 signaling can be calculated as follows: % blockade of 100 × [ (% Stat 5)⁺Cell antibody-free group [% Stat5⁺Cell 10ug/ml antibody panel)/(% Stat5⁺Cell no antibody group)]。

ADCC can be increased by methods that eliminate the fucose moiety from the antibody glycan, for example by generating antibodies in the YB2/0 cell line, or by introducing specific mutations on the Fc portion of human IgG1 (e.g., S298A/E333A/K334A, S239D/I332E/A330L, G236A/S239D/A330L/I332E) (Lazar et al, (2006) Proc Natl Acadsi USA 103, 2005-2010; Smith et al, (2012) Proc Natl 25Acad Sci USA 109,6181-6). ADCP can also be increased by introducing specific mutations in the Fc portion of human IgG1 (Richards et al (2008) MolCancer Ther 7,2517-27).

In a preferred embodiment of the invention, the antibodies are optimized to elicit an ADCC response, that is to say that the ADCC response is enhanced, increased or improved relative to other anti-CD 25 antibodies, including those that do not inhibit binding of interleukin 2 to CD25, such as the unmodified anti-CD 25 monoclonal antibody.

In a preferred embodiment of the invention, the antibodies are optimized to elicit an ADCP response, that is to say an enhanced, increased or improved ADCP response relative to other anti-CD 25 antibodies, including those that do not inhibit binding of interleukin 2 to CD25, such as the unmodified anti-CD 25 monoclonal antibody.

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

The antibodies according to the invention may also be partially or wholly synthetic, wherein at least part of the polypeptide chains of the antibody are synthetic and possibly optimized for binding to their cognate antigen. Such antibodies may be chimeric or humanized antibodies, and may be fully tetrameric in structure, or may be dimeric and comprise only a single heavy chain and a single light chain.

The antibody of the present invention may also be a monoclonal antibody. 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 by which it was produced.

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

anti-CD 25 antibodies having the characteristics described herein represent another object of the invention. anti-CD 25 antibodies are useful in medicine. In another embodiment, the invention provides a method of treating a disease in a subject comprising administering an anti-CD 25 antibody that does not inhibit binding of interleukin-2 (IL-2) to CD25 or IL-2 signaling through CD 25. Preferably, the disease is cancer, in particular for the treatment of solid tumors.

In another embodiment, the invention provides a nucleic acid molecule encoding an anti-CD 25 antibody as defined herein. In some embodiments, such provided nucleic acid molecules may contain codon-optimized nucleic acid sequences and/or may be contained in an expression cassette within an appropriate nucleic acid vector for expression in a host cell (e.g., bacterial, yeast, insect, fish, murine, simian, or human cell). In some embodiments, the invention provides a host cell comprising a heterologous nucleic acid molecule (e.g., a DNA vector) that expresses a desired antibody.

In some embodiments, the present invention provides methods of making an isolated anti-CD 25 antibody as defined above. In some embodiments, such methods can include culturing a host cell comprising the nucleic acid (e.g., a heterologous nucleic acid that can be contained in and/or delivered to the host cell by a vector). Preferably, the host cell (and/or heterologous nucleic acid sequence) is prepared and constructed such that the antibody or antigen-binding fragment or variant thereof is secreted from the host cell and isolated from the cell culture supernatant.

The antibodies of the invention may be monospecific, bispecific or multispecific. A "multispecific antibody" may have specificity for different epitopes of one target antigen or polypeptide, or may contain antigen binding domains specific for 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 herein, "bispecific antibody" refers to an antibody that has the ability to bind to two different epitopes on a single antigen or polypeptide or on two different antigens or polypeptides.

The bispecific antibodies of the invention as discussed herein can be produced by the following method: biological methods, such as somatic cell hybridization; or genetic methods, such as expression of non-native DNA sequences encoding the desired antibody structure in a cell line or organism; chemical means (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 a combination thereof.

Techniques and products allowing the generation of monospecificity or bispecific are known in the art, as extensively reviewed in the literature, as well as alternative formats, antibody-drug conjugates, antibody design methods, in vitro screening methods, constant regions, post-translational and chemical modifications, improved features for triggering Cancer cell death, such as Fc engineering (Tiller K and TessierP,2015Annu Rev Biomed eng.17: 191-216; Speiss C et al 2015, molecular immunology 6795-106; Weiner G,2015.Nat revcancer, 15: 361-370; Fan G et al 2015.J Hematol Oncol 8: 130). Such bispecific antibodies may 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, "epitope" or "antigenic determinant" refers to the site on an antigen to which an antibody binds. As is well known in the art, epitopes can be formed from contiguous amino acids (linear epitopes) or from noncontiguous amino acids juxtaposed by tertiary folding of a protein (conformational epitopes). Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, more typically at least 5 or 8 to 10 amino acids in a unique spatial conformation. Methods for determining spatial conformation of epitopes are well known in the art and include, for example, x-ray crystallography and 2-D nuclear magnetic resonance. See, e.g., epitopic Mapping Protocols in Methods in molecular Biology, Vol.66, Glenn E.Morris, Ed (1996). For example, an antibody of the invention can recognize a conformational epitope to which antibody 7G7B6 or MA251 binds. 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 (RQTFP), 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-CD 25 antibody can be included in an agent that further includes a conjugated payload (e.g., a therapeutic or diagnostic agent), which is particularly useful for cancer therapy or diagnosis. anti-CD 25 antibody conjugates with radionuclides or toxins may be used. Examples of radionuclides commonly used are, for example⁹⁰Y、¹³¹I and⁶⁷cu, etc., examples of commonly used toxins are doxorubicin and calicheamicin. In further embodiments, the anti-CD 25 antibody may be modified to have an altered half-life. Methods of achieving altered half-lives are known in the art. In some embodiments, the anti-CD 25 antibody is not conjugated to another therapeutic or diagnostic agent. In particular, in some embodiments, the anti-CD 25 antibody is not conjugated to a radionuclide, i.e., in some embodiments, the anti-CD 25 antibody is not radiolabeled.

In a preferred embodiment of the invention, the subject of any aspect of the invention as 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 the subject is a human. Thus, in all aspects of the invention described herein, the subject is preferably a human.

As used herein, the term "cancer," "cancerous," or "malignant" refers to or describes 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, blastoma, and sarcoma. More specific examples of such cancers include squamous cell cancer, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (HCC), hodgkin's lymphoma, non-hodgkin's lymphoma, Acute Myeloid Leukemia (AML), multiple myelomas, gastrointestinal (tract) 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, gastric cancer, bladder cancer, liver cancer, breast cancer, colon cancer and head and neck cancer.

In one aspect, the cancer involves a solid tumor. Examples of solid tumors are sarcomas (including cancers arising from transformed cells of mesenchymal origin in tissue (e.g., cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells or fibrous connective tissue)), carcinomas (including tumors arising from epithelial cells), mesotheliomas, neuroblastomas, retinoblastomas, and the like. 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, kidney cancer, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, oral cancer, sarcoma, eye cancer, thyroid cancer, urinary tract cancer, vaginal cancer, neck cancer, lymphoma, and the like.

In one aspect, the cancer involves tumors expressing 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, the 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 the cancer is one that has been identified as having a biomarker referred to as high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR). In addition, antibodies can be used when the identification of specific tumor-associated markers, antigens or biomarkers is used to define the precancerous, non-invasive state of the above-mentioned cancers (e.g., carcinoma in situ, smoldering myeloma, monoclonal gammopathy of unknown significance, cervical intraepithelial neoplasia, malthomas/GALTomes and various lymphoproliferative disorders) in a patient. Preferably, in some embodiments, the subject treated has a solid tumor.

In one aspect of the invention, the cancer is selected from melanoma, non-small cell lung cancer, renal cancer, ovarian cancer, bladder cancer, sarcoma, and colon cancer. In a preferred aspect of the invention, the cancer is selected from melanoma, ovarian cancer, non-small cell lung cancer and renal cancer. In one embodiment, the cancer is not melanoma, ovarian cancer, or breast cancer. In a preferred aspect, the cancer is a sarcoma, colon cancer, melanoma or colorectal cancer, or more generally, any human cancer in which the 4T1, MCA205, B16, CT26 or MC38 cell lines may represent preclinical models of compounds useful for their therapeutic management.

As used herein, the term "tumor" is applicable to subjects diagnosed with or suspected of having a tumor, cancer refers to a malignant or potentially malignant neoplasm or mass of tissue of any size, and includes both primary tumors and secondary neoplasms. The terms "cancer," "malignant tumor," "neoplasm," "tumor," and "cancer" are also used interchangeably herein to refer to tumors and tumor cells that exhibit relatively abnormal, uncontrolled, and/or autonomous growth, such that they exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. Typically, cells of interest for detection or treatment include precancerous (e.g., benign), malignant, pre-metastatic, and non-metastatic cells.

As used herein, a "solid tumor" is an abnormal growth or mass of tissue that generally does not contain cysts or fluid regions, particularly tumors and/or metastases (wherever located) other than leukemia or non-solid lymphoma. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell type in which they are formed and/or the tissue or organ in which they reside. Examples of solid tumors are sarcomas (including cancers arising from transformed cells of mesenchymal origin in tissue (e.g. cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells or fibrous connective tissue)), carcinomas (including tumors from epithelial cells), melanomas, lymphomas, mesotheliomas, neuroblastomas and retinoblastomas.

Particularly preferred cancers according to the present invention include cancers 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, it is preferred that the cancer is a solid tumor, i.e. the subject has a solid tumor (and not a non-solid tumor).

The definition of "treating" cancer as used herein defines achieving at least one positive therapeutic effect, such as a reduction in the number of cancer cells, a reduction in the size of the tumor, a reduction in the rate of cancer cell infiltration into peripheral organs, or a reduction in the rate of tumor metastasis or tumor growth.

The positive therapeutic effect of cancer can be measured in a number of ways (e.g., Weber (2009) J Nucl Med 50, 1S-10S). For example, with respect to tumor growth inhibition, T/C ≦ 42% is the lowest level of anti-tumor activity according to the National Cancer Institute (NCI) standard. T/C < 10% is considered a high level of anti-tumor activity, where T/C (%) ═ median tumor volume in the treated group/median tumor volume in the control group x 100. In some embodiments, the therapeutically effective amount of the achieved treatment is any one of Progression Free Survival (PFS), Disease Free Survival (DFS), or Overall Survival (OS). PFS, also referred to as "time to tumor progression," refers to the length of time during and after treatment that cancer does not grow, including the amount of time that a patient experiences a complete response or a partial response, and the amount of time that a patient experiences stable disease. DFS refers to the length of time a patient remains disease-free during and after treatment. OS refers to an extension of life expectancy compared to the original or untreated individual or patient.

As used herein, "prevention" (or prophylactic method) refers to delaying or preventing the onset of cancer symptoms. Prevention may be absolute (and thus no disease occurs) or effective only in certain individuals or for a limited period of time.

In a preferred aspect of the invention, the subject has a developed tumor, i.e., a subject that has had a tumor (e.g., a tumor classified as a solid tumor). Thus, the invention as described herein may be used when the subject already has a tumor (e.g., a solid tumor). Thus, the present invention provides treatment options that can be used to treat existing tumors. In one aspect of the invention, the subject has an existing solid tumor. The invention may be used as prophylaxis for a subject already having a solid tumor, or preferably as treatment for a subject already having a solid tumor. In one aspect, the invention is not used as a prophylactic or preventative method.

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

In one aspect of the invention, a method of treating or preventing cancer as described herein further comprises the step of identifying a subject having cancer, preferably a subject having a tumor (e.g. a solid tumor). In one embodiment, the method can include identifying a subject having a hematological cancer.

The dosage regimen of a therapy described herein effective to treat a cancer patient may vary depending on factors such as the disease state, age, and weight of the patient, as well as the ability of the treatment to elicit an anti-cancer response in the subject. The selection of an appropriate dosage is within the ability of one skilled in the art. For example 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 an amount is a unit dose (or an entire portion thereof) suitable for administration according to a dosing regimen (i.e., a therapeutic dosing regimen) that has been determined to correlate with a desired or beneficial result when administered to a relevant population.

The antibody according to any aspect of the invention as described herein may be in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluent or excipient. These compositions include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, or liposomes. In some embodiments, the preferred form may depend on the intended mode of administration and/or therapeutic application. The pharmaceutical composition containing the antibody may be administered by any suitable method known in the art, including, but not limited to, oral, mucosal, inhalation, topical, buccal, nasal, rectal, or parenteral (e.g., intravenous, infusion, intratumoral, intranodal, subcutaneous, intraperitoneal, intramuscular, intradermal, transdermal or other kinds of administration involving physical breakthrough into the tissue of the subject and administration of the pharmaceutical composition by breakthrough in the tissue). Such formulations may be, for example, in the form of injectable or infusible 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., perfusion) for at least a selected period of time, simultaneously with or between administration of the other compound.

In some embodiments, the antibody can be prepared with a carrier that protects it from rapid release and/or degradation, such as a controlled release formulation, e.g., an implant, a transdermal patch, and a microencapsulated delivery system. Biodegradable, biocompatible polymers may be used.

For example, one skilled in the art will appreciate that the route of delivery (e.g., oral versus intravenous versus subcutaneous versus intratumoral, etc.) can affect the dosage and/or that the desired dosage can affect the route of delivery. For example, focused delivery may be desirable and/or useful (e.g., intratumoral delivery in this example) where a particular high concentration of an agent in a specific site or location (e.g., intratumoral) is of interest. Other factors to be considered in optimizing the route and/or dosing regimen of a given treatment regimen may include, for example, the particular cancer to be treated (e.g., type, stage, location, etc.), the clinical condition of the subject (e.g., age, general health, etc.), the presence or absence of combination therapy, and other factors known to the physician.

Pharmaceutical compositions should generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of the antibody in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained release or biodegradable formulations as described herein. Sterile injectable preparations can be prepared using non-toxic parenterally acceptable diluents or solvents. Each pharmaceutical composition used according to the present invention may include pharmaceutically acceptable dispersing agents, wetting agents, suspending agents, isotonic agents, coating agents, antibacterial agents and antifungal agents, and the carrier, excipient, salt or stabilizer is non-toxic to the subject at the dosage and concentration used. Preferably, such compositions may further comprise a pharmaceutically acceptable carrier or excipient for the treatment of cancer, which carrier or excipient is compatible with a given method and/or site of administration, e.g. for parenteral (e.g. subcutaneous, intradermal or intravenous), intratumoral or peritumoral administration.

Although the invention is applicable toThe method of treatment or embodiment of the composition may not be effective to achieve a positive therapeutic effect in each subject, but it should achieve a positive therapeutic effect in using a pharmaceutical composition and dosing regimen 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, chi test²Test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpsra test and Wilcoxon test.

In the above and subsequent references to tumors, tumor diseases, cancers or cancers, metastasis in the original organ or tissue and/or in any other location may also be implied alternatively or additionally regardless of the location of the tumor and/or metastasis.

As discussed herein, the present invention relates to depleting regulatory T cells (tregs). Thus, in one aspect of the invention, anti-CD 25 antibodies that do not inhibit the binding of interleukin 2 to CD25 also deplete or reduce tumor infiltrating regulatory T cells. In one aspect, the depleting is by ADCC. In another aspect, the consumption is through ADCP.

Thus, the present invention provides a method for depleting regulatory T cells in a tumor in a subject, comprising administering to the subject an anti-CD 25 antibody that does not inhibit binding of interleukin 2 to CD 25. In a preferred embodiment, the tregs deplete solid tumors. By "depleting" is meant that the proportion or percentage of tregs is reduced relative to when no anti-CD 25 antibody is administered that does not inhibit binding of interleukin 2 to CD 25. In particular embodiments of the invention as described herein, more than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99% of the tumor-infiltrating regulatory T cells are depleted.

As used herein, "regulatory T cells" ("Treg/Treg cells/tregs") refer to the CD4+ T lymphocyte lineage that specifically controls autoimmunity, allergy, and infection. Typically, they modulate the activity of T cell populations, but they may also affect certain innate immune system cell types. Tregs are typically identified by the expression of the biomarkers CD4, CD25 and Foxp 3. Naturally occurring Treg cells typically account for about 5 to 10% of peripheral CD4+ T lymphocytes. However, within the tumor microenvironment (i.e. tumor infiltrating Treg cells), they may 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 through a perforin or granzyme B-dependent pathway, cytotoxic T lymphocyte-associated antigen 4(CTLA4+) Treg cells induce indoleamine 2, 3-dioxygenase (IDO) expression by APCs, which in turn inhibits T cell activation by reducing tryptophan, Treg cells can release interleukin-10 (IL-10) and transforming growth factor (TGF β) in vivo, thereby directly inhibiting T cell activation and inhibiting APC function by inhibiting expression of MHC molecules, CD80, CD86 and IL-12 Treg cells can also inhibit immunity by expressing high levels of CTLA4, CTLA4 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 a solid tumor is increased. In some embodiments, the ratio of effector T cells to regulatory T cells in the solid tumor is increased to greater than 5,10, 15, 20, 40, or 80.

Immune effector cells refer to immune cells involved in the effector phase of an 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.

For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes that express Fc α R are involved in specific killing of target cells and present antigens to other components of the immune system or bind to antigen-presenting cells.

In some embodiments, different agents that are anti-cancer may be administered in combination with the antibody by the same or different routes of delivery and/or according to different regimens. Alternatively or additionally, in some embodiments, one or more doses of the first active agent are administered substantially simultaneously with one or more other active agents, and in some embodiments, substantially simultaneously with one or more other active agents by a common route and/or as part of a single composition. One skilled in the art will further appreciate that a synergistic effect is achieved according to some embodiments of the combination therapies provided herein; in some such embodiments, the dosage of one or more agents used in the combination can be substantially different (e.g., lower) and/or can be delivered by an alternative, rather than standard, preferred, or necessary route when the agents are used in different treatment regimens (e.g., as a monotherapy and/or as part of a different combination therapy).

In some embodiments, when two or more active agents are used according to the present invention, these agents may be administered simultaneously or sequentially. In some embodiments, the administration of one agent is particularly timed relative to the administration of another agent. For example, in some embodiments, a first agent is administered such that a particular effect is observed (or expected to be observed, e.g., based on a population study showing a correlation between a given dosing regimen and a particular effect of interest). In some embodiments, the desired relative dosing regimen for the agents administered in combination may be empirically assessed or determined, e.g., using ex vivo, in vivo and/or in vitro models; in some embodiments, such assessment or empirical determination is made in vivo, in a patient population (e.g., to establish a correlation), or in a particular patient of interest.

In another aspect of the invention, anti-CD 25 antibodies that do not inhibit binding of interleukin 2 to CD25 have improved therapeutic efficacy when combined with immune checkpoint inhibitors. Combination therapy using anti-CD 25 antibodies and immune checkpoint inhibitors that do not inhibit binding of interleukin 2 to CD25 may have a synergistic effect in treating established tumors. The data in this example for PD-1/PD-L1 relate to interfering with the PD-1/PD-L1 interaction. Thus, the interaction between the PD-1 receptor and the PD-L1 ligand may be blocked, resulting in "PD-1 blockade". In one aspect, the combination may result in enhanced tumor regression, impaired or reduced tumor growth, and/or may result in prolonged survival, e.g., compared to an anti-CD 25 antibody or PD-1/PD-L1 blocking (either directly with an anti-PD 1 antibody, or indirectly with an anti-PD-L1 antibody), using the invention described herein. The combination therapy with the anti-CD 25 antibody and the immune checkpoint inhibitor may also further comprise administering interleukin-2 at a dosage suitable for treating cancer, when binding of interleukin 2 to CD25 is not inhibited by the anti-CD 25 antibody.

As used herein, an "immune checkpoint" or "immune checkpoint protein" refers to a protein that belongs to an inhibitory pathway in the immune system, in particular a protein for modulating T cell responses. Under normal physiological conditions, immune checkpoints are critical to prevent autoimmunity, particularly during responses to pathogens. Cancer cells can alter the regulation of immune checkpoint protein expression to avoid 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, and OX40, GITR, ICOS, 4-1BB, and HVEM. An immune checkpoint protein may also refer to a protein that binds to other immune checkpoint proteins. These proteins include PD-L1, PD-L2, CD80, CD86, HVEM, LLT1 and GAL 9.

By "immune checkpoint protein inhibitor" is meant any protein that can interfere with immune checkpoint protein-mediated signaling 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 the PD-1/PD-L1 interaction by an anti-PD-1 or anti-PD-L1 antibody.

Accordingly, the present invention also provides a method of treating cancer comprising administering to a subject an anti-CD 25 antibody that does not inhibit binding of interleukin 2 to CD25 and an additional therapeutic agent, preferably a checkpoint inhibitor. The invention also provides anti-CD 25 antibodies that do not inhibit binding of interleukin 2 to CD25 and an additional therapeutic agent, preferably an immune checkpoint inhibitor, for use in the treatment of cancer.

Furthermore, the present invention provides the use of an anti-CD 25 antibody that does not inhibit binding of interleukin 2 to CD25 and an additional therapeutic agent, preferably an immune checkpoint inhibitor, for the manufacture of a medicament for the treatment of cancer. The administration of the anti-CD 25 antibody and the additional therapeutic agent (e.g., an immune checkpoint inhibitor) that does not inhibit binding of interleukin 2 to CD25 may be simultaneous, separate, or sequential.

The present invention provides a combination of an anti-CD 25 antibody that does not inhibit binding of interleukin 2 to CD25 and an additional therapeutic agent, preferably an immune checkpoint inhibitor, for use in the treatment of cancer in a subject, wherein the anti-CD 25 antibody that does not inhibit binding of interleukin 2 to CD25 and the additional therapeutic agent (e.g. an immune checkpoint inhibitor) are administered simultaneously, separately or sequentially. Such anti-CD 25 antibodies that do not inhibit binding of interleukin 2 to CD25 and exhibit the human IgG1 isotype may be combined in particular with antibodies that target the point of immunosuppression but lack sequences that allow ADCC, ADCP and/or CDC.

In another aspect, the invention provides an anti-CD 25 antibody that does not inhibit binding of interleukin 2 to CD25 for use in the treatment of cancer, wherein the antibody is for administration in combination with an additional therapeutic agent, preferably an immune checkpoint inhibitor. The invention also provides the use of an anti-CD 25 antibody that does not inhibit binding of interleukin 2 to CD25 in the manufacture of a medicament for the treatment of cancer, wherein the medicament is for administration in combination with an additional therapeutic agent, preferably an immune checkpoint inhibitor.

The present invention provides a pharmaceutical composition comprising an anti-CD 25 antibody that does not inhibit the binding of interleukin 2 to CD25 and optionally an additional therapeutic agent, preferably an immune checkpoint inhibitor, in a pharmaceutically acceptable medium. As described above, the immune checkpoint inhibitor may be an inhibitor of PD-1, 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 and B cells. Interaction with its ligand has been shown to attenuate T cell responses both in vitro and in vivo. PD-1 binds two ligands PD-L1 and PD-L2. PD-1 belongs to the immunoglobulin superfamily. PD-1 signaling requires binding to PD-1 ligand in proximity to peptide antigens presented by the Major Histocompatibility Complex (MHC) (Freeman (2008) Proc Natl Acad Sci USA105,10275-6). Thus, proteins, antibodies or small molecules that prevent co-ligation of PD-1 and TCR on T cell membranes are useful antagonists of PD-1.

In one embodiment, the PD-1 receptor antagonist is an anti-PD-1 antibody or antigen-binding fragment thereof that specifically binds to PD-1 and blocks the binding of PD-L1 to PD-1. The anti-PD-1 antibody may be a monoclonal antibody. The anti-PD-1 antibody may be a human antibody or a humanized antibody. An anti-PD-1 antibody is an antibody that is capable of specifically binding to a 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 and/or block PD-1 ligand to interfere with or inhibit ligand binding to PD-1 receptor or bind directly and block PD-1 receptor without inducing inhibitory signal transduction through PD-1 receptor. In particular, the PD-1 antagonist includes a small molecule inhibitor of the PD-1/PD-L1 signaling pathway. Alternatively, the PD-1 receptor antagonist may bind directly to the PD-1 receptor without triggering inhibitory signal transduction, and also bind to a ligand of the PD-1 receptor to reduce or inhibit the triggering of signal transduction by the ligand through the PD-1 receptor. By reducing the number and/or amount of ligands that bind to PD-1 receptors and trigger inhibitory signaling, fewer cells are attenuated by the negative signal delivered by PD-1 signaling, and a stronger immune response can be achieved.

In one embodiment, the PD-1 receptor antagonist is an anti-PD-L1 antibody or antigen-binding fragment thereof that specifically binds to PD-L1 and blocks the binding of PD-L1 to PD-1. The anti-PD-L1 antibody may be a monoclonal antibody. The anti-PD-L1 antibody can be a human or humanized antibody, such as atlizumab (MPDL 3280A).

The invention also provides methods of treating cancer comprising administering to a subject an anti-CD 25 antibody that does not inhibit binding of interleukin 2 to CD25 and an antibody that is an agonist of the T cell activation co-stimulatory pathway. Antibody agonists for the T cell activation co-stimulatory pathway include, but are not limited to, agonist antibodies to ICOS, GITR, OX40, CD40, LIGHT, and 4-1 BB.

Additional methods of treating cancer include administering an anti-CD 25 antibody that does not inhibit interleukin 2 binding to CD25 and a compound that reduces, blocks, inhibits and/or antagonizes fcyriib (CD32 b). Such Fc γ RIIb antagonists may be small molecules that interfere with Fc γ RIIb-induced intracellular signaling, modified antibodies that do not engage inhibitory Fc γ RIIb receptors, or anti-human Fc γ RIIb (anti-CD 32b antibodies). For example, antagonistic anti-human Fc γ RIIb antibodies have also been characterized for their anti-tumor properties (Roghanian a et al, 2015, Cancer cell.27, 473-488; Rozan C et al, 2013, mol Cancer ther.12: 1481-91; WO 2015173384; WO 2008002933).

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

(a) a first antigen-binding moiety that binds to CD 25; and

(b) a second antigen-binding portion that binds to an immune checkpoint protein, a tumor-associated antigen, an anti-human activating Fc receptor antibody (FcgRI, fcgriiia, fcgriiii), or an antagonistic anti-human fcyriib antibody;

wherein the anti-CD 25 antibody does not inhibit binding of interleukin-2 (IL-2) to CD25, and is preferably an IgG1 bispecific antibody that binds with high affinity to at least one activating Fc γ receptor and depletes tumor infiltrating regulatory T cells. In a preferred embodiment, the second antigen-binding moiety binds to PD-L1.

As used herein, "tumor-associated antigens" refer to antigens expressed on tumor cells that distinguish them from non-cancer cells adjacent to them and include, but are not limited to, CD20, CD38, PD-L1, EGFR, EGFRV3, CEA, TYRP1, and HER 2. Various review articles have been published describing relevant tumor-associated antigens and corresponding therapeutically useful anti-tumor antibody drugs (see, e.g., Sliwkowski & Mellman (2013) Science 341,192-8). Such antigens and corresponding antibodies include, but are not limited to, CD22 (bornauzumab), CD20 (rituximab, tositumomab), CD56 (rituximab (Lorvotuzumab)), CD66e/CEA (Labetuzumab)), CD152/CTLA-4 (Ipilimumab)), CD221/IGF1R (MK-0646), CD326/Epcam (Edrecolomab)), CD 340/2 (trastuzumab, pertuzumab), and EGFR (cetuximab, panitumumab).

In one aspect, a bispecific antibody according to the invention as described herein results in ADCC, or in one aspect, in 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 to specific epitopes on immune checkpoint proteins or tumor associated antigens as defined herein. In a preferred embodiment, the second antigen-binding moiety binds to PD-L1. In a preferred aspect, the present invention provides a bispecific antibody comprising:

(a) a first antigen-binding moiety that binds to CD25 and does not affect the binding of IL-2 to CD 25; and

(b) a second antigen-binding moiety that binds to an immune checkpoint protein expressed on a tumor cell.

In a specific embodiment, the immune checkpoint protein expressed on the tumor cell is PD-L1, VISTA, GAL9, B7H3 or B7H 4. Still preferably, the anti-CD 25 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 Fc γ receptor and depletes tumor infiltrating regulatory T cells. Alternatively, the anti-CD 25 antibody is a human IgG2 antibody that depletes tumor infiltrating regulatory T cells. In one embodiment, the anti-CD 25 antibody is a human IgG2 antibody that binds with high affinity to at least one activated Fc γ receptor, preferably Fc γ RIIa.

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

In one aspect, the bispecific antibody can bind to CD25 and to immunomodulatory receptors (e.g., CTLA4, ICOS, GITR, 4-1BB, or OX40) expressed at high levels on tumor-infiltrating tregs.

The invention also provides a kit comprising an anti-CD 25 antibody as described herein and an additional therapeutic agent, preferably an immune checkpoint inhibitor, preferably a PD-1 antagonist as discussed herein (either directly using the anti-PD 1 antibody, or indirectly using the anti-PD-L1 antibody). In one aspect, the immune checkpoint inhibitor is anti-PD-L1. In another embodiment, the kit comprises an anti-CD 25 antibody as described herein and an antibody that is an agonist of a T cell activation co-stimulatory pathway. The kit may comprise instructions for use.

Any aspect of the invention as described herein may be performed in combination with additional therapeutic agents, in particular other cancer therapies. In particular, the anti-CD 25 antibody and the optional immune checkpoint inhibitor according to the present invention may be administered in combination with a costimulatory antibody, chemotherapy and/or radiotherapy (by application of radiation in vitro or by administration of a radiation conjugated compound), cytokine-based therapy, targeted therapy, monoclonal antibody therapy, vaccine or adjuvant, or any combination thereof.

Contemplated chemotherapeutic agents include, but are not limited to, alkylating agents, anthracyclines, epothilones, nitrosoureas, ethylenimine/methylmelamine, alkyl sulfonates, alkylating agents, antimetabolites, pyrimidine analogs, epipodophyllotoxins, enzymes (e.g., L-asparaginase), biological response modifiers, such as IFN α, 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, adrenocortical inhibitors (e.g., mitotane (o, p' -DDD) and aminoacetyladrenaline), hormones and antagonists, including steroid antagonists, such as prednisone and equivalents, dexamethasone and aminoacetylimines, progestogens, such as hydroxyprogesterone, medroxyprogesterone, and medroxyprogesterone acetate, testosterone, such as testosterone, and antiandrogen, such as antiandrogen, testosterone, and antiandrogen, such as, testosterone, and/or a non-steroidal hormone releasing androgen.

Additional cancer therapies may also include administration of cancer vaccines. As used herein, "cancer vaccine" refers to a therapeutic cancer vaccine administered to a cancer patient and is intended to eradicate cancer cells by boosting 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 genetic vaccines (DNA, RNA and virus based vaccines). Thus, in principle, therapeutic cancer vaccines can be used to inhibit further growth of advanced cancers and/or recurrent tumors that are refractory to conventional therapies (e.g., surgery, radiation therapy, and chemotherapy). Tumor cell-based vaccines (autologous and allogeneic) include genetically modified secretion of soluble immune stimulators such as cytokines (IL-2, IFN-g, IL12, GMCSF, FLT3L), single chain Fv antibodies against immunoregulatory receptors (PD-1, CTLA-4, GITR, ICOS, OX40, 4-1BB), and/or ligands that express immune stimulators on their tumor cell membranes (e.g., ICOS ligands, 4-1BB ligands, GITR ligands, and/or OX40 ligands), and the like. In one embodiment, the cancer vaccine may be a GVAX anti-tumor vaccine.

Additional cancer therapies may be other antibodies or small molecule agents that reduce immune modulation within the peripheral and tumor microenvironment, such as molecules targeting the TGF β pathway, IDO (indoleamine deoxyase), arginase, and/or CSF 1R.

"combination" may refer to the administration of an additional therapy prior to, simultaneously with, or subsequent to the administration of any aspect of the invention.

The invention will now be further described by the following examples, which are intended to assist those of ordinary skill in the art in carrying out the invention, with reference to the accompanying drawings, and are not intended to limit the scope of the invention in any way.

Drawings

Figure 1 shows the characterization of the 7D4 and PC61 anti-mouse CD25 antibodies used in the examples. The effect on IL-2 binding was performed using a tandem cross-blocking assay (tandem format cross-blocking assay). Biotinylated mouse CD25 was loaded onto the SA sensor. The sensor was then exposed to 100nM mouse IL-2 followed by anti-mouse CD25 antibody at 150 seconds. Additional binding of the antibody after IL-2 association indicates that anti-mouse CD25 does not block IL-2 binding, while no further binding indicates ligand blocking. In contrast to 7D4(A), PC-61mIgG2a showed interference of the interaction of mouse IL-2 with mouse CD 25. The mouse IL-2/mouse CD25 interaction in the presence of recombinant anti-mouse CD 257D 4(mIgG1) was evaluated using a standard sandwich cross-blocking assay. 7D4(mIgG1) was loaded onto the AHQ sensor and the unoccupied Fc binding sites on the sensor were blocked with an irrelevant human IgG1 antibody. The sensor was then exposed to 100nM recombinant mouse CD25(R & D Systems; catalog number 2438-RM-050), followed by recombinant mouse IL-2 (Peprotech; catalog number 212-12). Additional binding by mouse IL-2 following association of 7D4(mIgG1) mouse CD25 indicates an unoccupied epitope, where neither 7D4(mIgG1) nor mouse IL-2 competed for an epitope in mouse CD25 due to simultaneous binding with mouse CD 25. (B) Binding to mouse CD25 was determined using CHO cells expressing mouse CD25 against anti-human CD25 binding antibodies Daclizumab (DAC), PC61(mIgG2a) antibody (primary anti-CD 25 obtained from clone PC-61 with murine IgG2a and kappa constant regions associated with ADCC) and a7D4(mIgG1) antibody (anti-D25 obtained from clone 7D4 with murine IgG1 and kappa constant regions). Anti-mouse CD25IgG bound to cell-expressed mCD 25. CHO-mCD25 was aliquoted into 96 well assay plates (50000 cells/well) and incubated with a solution containing 0.1mL of antibody (100 nM antibody + 0.1% bovine serum albumin in PBS) for 15 minutes at 25 ℃. Cells were washed three times with ice-cold PBS + 0.1% bovine serum albumin, then labeled with goat anti-human IgG (gamma chain specific) R-PE (Southern Biotech, catalog No. 2040-09) and analyzed using flow cytometry (propidium iodide was used to distinguish dead cells). DAC binding was not detected, whereas both PC61(mIgG2a) and 7D4(mIgG1) showed significant binding to these cells (C).

FIG. 2 shows the effect of anti-mouse CD25 antibody on the induction of granzyme B expression by CD4T cells following stimulation with anti-CD 3 and anti-CD 28, total CD4 positive T cells isolated from mouse lymph nodes and spleen using CD4 MicroBeads, and CellTrace^TMPurple dye (ThermoFisher) was labeled for measuring cell proliferation. The 96-well plates were seeded with labeled T cells (105) and feeder cells (105; CD90.2 negative fraction using the mouse pan T Dynabeads kit). anti-CD 3 (clone 145-2C11, BioXcell catalog number BE 0001-1; 1. mu.g/ml) and anti-CD 28 (clone 37.51 catalog number BioXcell BE 0015-1; 0.5. mu.g/ml) were added to the wells (except for the control sample of unstimulated labeled T cells) to activate CD4T cells and induce proliferation and granzyme B production. The following antibodies (at 25 μ g/mL) were then further added to wells containing labeled CD4T cells and anti-CD 3 and anti-CD 28 antibodies (wells containing only labeled T cells and anti-CD 3 and anti-CD 28 antibodies served as negative controls): PC61(mIgG2a), 7D4(mIgG1) or neutralizing anti-mouse IL-2 antibody (clone Jes6-1A12, BioXcell6032988564) were used as positive controls to show the effect of blocking the interaction between IL-2 and its receptor on T cell activation. The sample of labeled T cells was then incubated for about 84 hours. Cells were then fixed and permeabilized, then stained with anti-mouse granzyme B antibody (clone GB11, Invitrogen). Cells were then analyzed by flow cytometry for granzyme B expression and CellTrace purple dye dilution (BV 450). The percentage of T cells proliferating and expressing the marker for granzyme b (GnzB) is shown in the upper left quadrant of each treatment specificity graph (Q9), while the percentage of T cells proliferating but not expressing the marker for GnzB is shown in the lower left quadrant of each treatment specificity graph (Q12).

FIG. 3 shows blocking or non-blocking of administration in the presence or absence of anti-mouse PD1(aPD 1; clone RMP1-14)In vivo effects of anti-mouse CD25 antibody (mouse IgG2a) of the same isotype on immune cells for the interaction between CD25 and IL-2. On day 0, MCA205 tumor cells (5X 10)⁵15) Six groups of mice were injected subcutaneously and treated individually as shown in the figure. In four groups, anti-mouse CD25 antibody (aPC61 mIgG2a or a7D4mIgG2 a; 200. mu.g) was injected intraperitoneally on the fifth day. In three groups, APD1(100 μ g) was injected intraperitoneally with aPD1(100 μ g) on the sixth and ninth days. Tumors and lymph nodes were harvested on day 12, then treated according to the desired type to stain cells, and analyzed by flow cytometry using the following antibodies as indicated in the figures: anti-CD 3 (clone 17A2, Biolegend), anti-CD 4 (clone RM4-5, BDbiosciences), anti-CD 8 (clone 53-6.7, Biolegend) and anti-FoxP 3 (clone FJK-16s, eBiosciences). Endonuclear staining of FoxP3 was performed using a FoxP3 transcription factor staining buffer set (eBioscience). The percentage of CD4 positive/Foxp 3 positive regulatory T cells and CD4 positive/Foxp 3 negative effector CD4T cells (CD4Teff) and the ratio of effector CD8 positive T cells/Treg cells and CD4 Teff/Treg in LN and TIL are shown. Data analysis was performed in Flowjo 10.0.8 edition (TreeStar Inc.). Statistical analysis was performed in Prism 6(graphpad software, Inc.); p-value (ns ═ p) was calculated using Kruskall-Wallis analysis of variance and Dunn's post hoc test>0.05；****＝p<0.0001)。

FIG. 4 shows the effect of an anti-mouse CD25 antibody (IgG2a) of the same isotype, with or without blocking the interaction between CD25 and IL-2, on the production of granzyme B by proliferating T cells in vivo, in the presence or absence of anti-mouse PD1(aPD 1; clone RMP 1-14). Cell samples were generated in six treatment groups as shown in figure 3 using the MCA 205-based model. The cells were then stained according to the desired type and analyzed by flow cytometry using the following antibodies as indicated in the figures: anti-CD 3(PeCy7, clone 145-2C11, Ebioscience, 25003182), anti-CD 4(V500, clone RM4-5, BDbiosciences, 560782), anti-CD 8(BV785, clone 53-6.7, Biolegend, 100750), anti-granzyme B (APC, clone GB 11; Invitrogen, grb05) and Ki67(V450, clone SolA 15; eBiosciences, 48569882). Intranuclear staining of Ki67 and granzyme B was performed using the FoxP3 transcription factor staining buffer kit (eBioscience, 00-5523-00). The percentage of GnzB positive was compared to the total number of GnzB positive proliferating (as indicated by Ki67 positivity) CD4 positive or CD8 positive T cells. Statistical analysis was performed as in fig. 3 (ns ═ p > 0.05;. p < 0.01;. p < 0.001;. p < 0.0001).

FIG. 5 shows the effect of anti-mouse CD25(IgG2a isotype) administered with or without anti-PD-1 (clone RMP1-14) in combination on eradication of established tumors in a CT26 mouse model. Both 7D4m2a and PC61m2a were anti-mouse CD25 and abrogated Treg antibodies, but one was non-IL-2 blocked (7D4m2a) and the other was IL-2 blocked (PC61m 2). Growth curves of individual mice over time were formed for each treatment group. The number of tumor-free survivors after 50 days is indicated in each figure. CT26 cells were harvested for implantation during log phase growth and resuspended in cold PBS. On study day 1 (D1), 3 × 10 injections were administered subcutaneously in the right flank of each mouse⁵Individual cells (0.1mL cell suspension). Anti-mouse CD25(10mg/kg) was injected intraperitoneally on day 6 (when palpable tumors were detected). Anti-mouse PD1 was injected intraperitoneally on days 7, 10, 14, and 14. Tumors were measured twice weekly in two dimensions with calipers to detect growth. Tumor size (mm)³) Calculated from the following formula: tumor volume (w2 × 1)/2, where w is the width of the tumor and l is the length of the tumor (mm). The end point of the study was a tumor volume of 4000mm³Or 50 days, whichever came first (data points stopped at different earlier dates were due to mouse death; number of animals surviving at the end of the experiment is indicated in each figure).

Figure 6 shows the CT26 tumor growth curves of individual mice that were untreated (PBS, vehicle only), treated with anti-mouse CD25IgG2a antibody that all consumed tregs but one was a non-IL-2 block (7D4m2a) and the other was an IL-2 block (PC61m2), or further treated with or without combination with anti-mouse PD-L1 clone 10f.9g2(aPDL 1; clone 10 f.9g2). The model, protocol and data analysis were the same as for fig. 5.

FIG. 7 shows that anti-mouse CD25(IgG2a isotype) administered with or without combination with anti-PD-1 (clone RMP1-14) has eradicated MC38 in a mouse modelThe role of the formed tumor. The antibodies tested were those depicted in figure 5. Growth curves of 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 log phase growth and resuspended in cold PBS. Injection was subcutaneously injected 5X 10 to the right flank of each mouse⁵Tumor cells (0.1mL cell suspension). When the tumor volume approaches 100 to 150mm³The tumor is monitored at the target range. Individual tumor volumes were between 75 and 126mm on study day 1, 22 days after tumor implantation³The animals in the range of (1) were divided into 9 groups (n-10), the group mean tumor volume being about 106mm³. On day 1, treatment was initiated in mice with an established MC38 tumor. The effect of each treatment was compared to vehicle treated control groups that received PBS intraperitoneally on day 1, day 2, day 5, day 9, and day 12. anti-PD 1 was administered twice weekly at 100 μ g/animal for two weeks (biwk x 2) starting on day 2. 7D4m2a and PC61m2a were administered intraperitoneally at 200. mu.g/animal on day 1. Tumor measurements were performed twice weekly. The end point of the study was a tumor volume of 4000mm³Or 35 days, whichever came first (data points stopped at different earlier dates were due to mouse death; number of animals surviving at the end of the experiment is indicated in each figure).

FIG. 8 shows the MC38 tumor growth curves of individual mice that were untreated (PBS, vehicle only), treated with anti-mouse CD25IgG2a antibody that all consumed Tregs but one was a non-IL-2 block (7D4m2a) and the other was an IL-2 block (PC61m2), or further treated with or without combination with anti-mouse PD-L1 clone 10F.9G2(aPDL 1; clone 10 F.9G2). The model, protocol and data analysis were the same as for fig. 7.

FIG. 9: the therapeutic activity of 7D4mIgG2a as a non-IL-2 blocking and Treg depleting anti-mouse CD25 antibody alone (D) and in combination with an IL-2 neutralizing antibody (E), or an IL-2 blocking and non-depleting anti-mouse CD25 antibody of the mouse IgG1 isotype (PC61 mouse IgG1) was evaluated in mice bearing CT26 syngeneic colon tumors in female BALB/c mice. The activities of a test mouse IgG2s control (A), IL-2 neutralizing antibody alone (B), and IL-2 blocking anti-CD 25 antibody alone (C) were compared.

FIG. 10: the consensus sequence of human CD25 (Uniprot code P01589), referred to herein as SEQ ID NO:1, is shown. The extracellular domain of mature CD25 corresponding to amino acids 22 to 240 is underlined. Epitope positions from non-IL blocking anti-CD 25 antibodies were initially identified as follows: epitope 1 (full and short epitopes), epitope 2 (full and short epitopes), epitope 3 and epitope 4 (full and short epitopes)). The positions of the basiliximab and dallizumab epitopes (denoted as DACs) were also identified.

FIG. 11: characterization of (a)7D4, (B) PC61, and (C)2E4 bound to CD25 on CHO cells expressing CD25 at increased antibody concentrations and in comparison to a mouse IgG2a isotype control.

FIG. 12: SPR-based analysis of his-tagged anti-rmCD 25 antibody on Biacore 2000, (a)7D4, (B) on 2E 4.

FIG. 13: competition assay for his-tag labeled anti-rmCD 25 antibody was performed on Octet 96. Binding by secondary antibody after capture of 7D4 on the sensor and antigen association step is shown. Competitive binding to mCD25 was observed between 7D4 and 2E4(a), but not between 7D4 and PC61 (B).

FIG. 14: characterization of 7D4, PC61 and 2E4 for blocking IL-2 signaling in a STAT5 phosphorylation assay, compared to a mouse IgG2a isotype control or in the absence of primary antibody, using T cells isolated from C57BL/6 splenocytes. Cells were incubated with 50. mu.g/ml antibody and then 50U/ml IL-2. The analysis was limited to the percentage of Treg cells that phosphorylated STAT 5.

FIG. 15: in vivo depletion of tregs in balb/c mice bearing 4T1 tumors after administration with mouse anti-mouse CD25(7D4) antibody. (A) To (C): non-CD 4, CD4+ and CD25+ FoxP3+ cells% in whole blood on day 3 post-dose. (D) To (F): non-CD 4, CD4+ and CD25+ FoxP3+ cells% in tumors on day 3 post-dose. (G) To (I): non-CD 4, CD4+ and CD25+ FoxP3+ cells% in whole blood on day 9 post-dose. (J) To (L): non-CD 4, CD4+ and CD25+ FoxP3+ cells% in tumors at day 9 post-dose.

FIG. 16: characterization of mouse (B) or chimeric (A, C and D) anti-human CD25 clone 7G7B6, binding to CD25 expressed on Karpas299 cells (a), human in vitro differentiated Treg cells (B), SU-DHL-1 cells (C) or SR-786 cells (D), at increased antibody concentrations and in comparison to human IgG1 isotype control.

FIG. 17: characterization of 7G7B6 for blocking IL-2 signaling in STAT5 phosphorylation assay compared to mouse IgG2a isotype control, human IgG1 isotype control, daclizumab, or in the absence of primary antibody using human-derived PBMC. Cells were incubated with 10. mu.g/ml antibody and the concentration of IL-2 was then increased (as shown). The analysis was limited to the percentage of CD3 positive cells that phosphorylated STAT 5.

FIG. 18: chimeric 7G7B6 was functionally characterized using pan T cells compared to human IgG1 isotype control, daclizumab, or a commercially available mouse anti-human IL-2 neutralizing antibody (clone: AB12-3G4) as a positive control. Cells were incubated with 10ug/ml antibody and then activated with CD3/CD28 beads for 72 hours before flow cytometry analysis. The results show the percentage of proliferating CD4T cells that were granzyme B positive.

FIG. 19: chimeric 7G7B6 function was characterized for killing of CD25 positive cell lines in ADCC assay compared to human IgG1 isotype control. SU-DHL-1(A) or SR-786 (B) cells expressing high or low CD25 were co-cultured with purified NK cells, respectively, in the presence of different concentrations of antibody (as shown). Target cell lysis was measured by the release of calcein into the supernatant 4 hours after calcein was added to NK cells. Data were normalized to saponin treated controls.

FIG. 20: chimeric 7G7B6 function was characterized in comparison to the human IgG1 isotype in the ADCP assay with respect to phagocytosis of in vitro differentiated Treg cells. Tregs were co-cultured with MCSF differentiated macrophages in the presence of different concentrations of antibody (as shown). Two-color flow cytometry analysis was performed with CD14+ stained macrophages and eFluor450 dye labeled tregs. Residual target cells are defined as being eFluor450-dye⁺/CD14^-A cell. Double labeled cells (eFluor 450-dye +/CD14+) are thought to represent phagocytosis of the target by macrophages. Using the following equationCalculating phagocytosis of target cells: % phagocytosis ═ 100 × [ (double positive%)/(double positive% + percent residual target)]。

FIG. 21: MA-251 bound to CD25 expressed on Karpas299 cells was characterized at increased antibody concentrations and compared to a mouse IgG1 isotype control.

FIG. 22: characterization of MA-251 compared to mouse IgG1 isotype control, human IgG1 isotype control, daclizumab, or in the absence of primary antibody. Blockade of IL-2 signaling in STAT5 phosphorylation assays was assessed using human PBMCs. Cells were incubated with 10. mu.g/ml antibody and the concentration of IL-2 was then increased (as shown). The analysis was limited to the percentage of CD3 positive cells that phosphorylated STAT 5.

FIG. 23: MA-251 and IL2 bound to CD25 were characterized. Interference with binding of IL2 ligand to CD25 was performed on a Forte bioictet Red384 system (Pall Forte Bio corp., USA) using a standard sandwich packet assay. The MA251 antibody was loaded onto the AHQ sensor and the unoccupied Fc binding sites on the sensor were blocked with the irrelevant human IgG1 antibody. The sensor was exposed to 100nM human CD25, followed by 100nM human IL-2. Data were processed using Forte Bio data analysis Software 7.0. Additional binding of human IL2 following antigen association indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blocking (competitor).

FIG. 24: competition assay for anti-CD 25 antibody in Octet. The primary Ab was allowed to bind to immobilized rhCD25 before binding to the primary Ab (as a control) or the secondary Ab. Mabs that were non-blockers of IL-2 signaling either competed with each other or with 7G7B6 and MA251, and did not compete with study dallizumab or study basiliximab (fig. 24(a) to (N)). (A) Competition assays to (C)7G7B 6; (D) competition assay to (F) MA 251; (G) competitive assay to (I) and (N) antibody 3; (J) competition assay to (M) antibody 1. mAb as a blocker of IL-2 signaling (TSK031) did compete with study dallizumab and study basiliximab, and did not compete with 7G7B6 (fig. 24(O) to (Q)).

FIG. 25: in vivo models showing inhibition of tumor growth after dosing: carriers (A) and (C); or antibodies 1(B), (D), and (E).

FIG. 26: the affinity was determined by SPR-based analysis of his-tagged anti-rhCD 25 antibody performed on Biacore 2000. A)7g7B6ch, B) MA251ch, C) antibody 1, D) antibody 3 and E) daclizumab (control) or by biolayer interferometry (F) on an Octet Red96 instrument.

FIG. 27 is a schematic view showing: characterization of antibody 1 binding 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 to human IgG1 isotype control.

FIG. 28: binding to CD25 expressed on human (A) and (B) Pan T cells (Pan T cells) activated with CD3/CD28 beads followed by CD4 at increased antibody concentrations and compared to human IgG1 isotype control⁺And CD8⁺Characterization of up-gated antibody 1.

FIG. 29: biolayer interferometry assays were performed on Octet Red384 by using standard sandwich format grouping assays, showing noncompetitive binding of antibody 1 and IL-2 (A) and competitive binding of IL-2 competing antibody to IL-2 (B). Anti-human CD25 antibody 1 was loaded onto the AHQ sensor. The sensor was then exposed to 100nM human CD25, followed by human IL-2. Additional binding of human IL2 following antigen association indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blocking (competitor).

FIG. 30: biolayer interferometry was performed on Octet Red384 by using a standard sandwich format grouping assay, showing non-competitive binding of antibody 1 and daclizumab to CD 25. The reference monoclonal anti-human CD25 antibody darlizumab was loaded onto the AHQ sensor. The sensor was then exposed to 100nM human CD25 antigen, followed by anti-human CD25 antibody (antibody 1). Additional binding of the secondary antibody after antigen association indicates unoccupied epitopes (non-competitors), while no binding indicates epitope blocking (competitors).

FIG. 31: antibody 1 was characterized for blocking IL-2 signaling in a STAT5 phosphorylation assay using human-derived PBMCs, as compared to human IgG1 isotype control, daclizumab, or in the absence of primary antibody. Cells were incubated with 10. mu.g/ml antibody and the concentration of IL-2 was then increased (as shown). The analysis was limited to the percentage of CD3 positive cells that phosphorylated STAT 5.

FIG. 32: antibody 1 was functionally characterized using pan-T cells, compared to human IgG1 isotype control, daclizumab, or a commercially available mouse anti-human IL-2 neutralizing antibody (clone: AB12-3G4) as a positive control. Cells were incubated with 10ug/ml antibody and then activated with CD3/CD28 beads for 72 hours before flow cytometry analysis. The results show the percentage of granzyme B positive proliferating CD4(a) or CD8(B) T cells.

FIG. 33: in the ADCC assay, antibody 1 was functionally characterized with respect to killing of CD25 positive cell line compared to human IgG1 isotype control. SU-DHL-1(A) or SR-786 (B) cells, which are high or low expressing CD25, were co-cultured with purified NK cells, respectively, in the presence of different concentrations of antibody (as shown). Target cell lysis was measured by the release of calcein into the supernatant 4 hours after calcein was added to NK cells. Data were normalized to saponin treated controls.

FIG. 34: antibody 1 function was characterized in the ADCP assay for phagocytosis of in vitro differentiated Treg cells compared to the human IgG1 isotype control. Tregs were co-cultured with MCSF differentiated macrophages in the presence of different concentrations of antibody (as shown). Two-color flow cytometry analysis was performed with CD14+ stained macrophages and eFluor450 dye labeled tregs. Residual target cells are defined as being eFluor450-dye⁺/CD14^-A cell. Double labeled cells (eFluor 450-dye +/CD14+) are thought to represent phagocytosis of the target by macrophages. Phagocytosis of target cells was calculated using the following equation: % phagocytosis ═ 100 × [ (double positive%)/(double positive% + percent residual target)]。

FIG. 35: characterization of antibody 3 binding 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 to human IgG1 isotype control.

FIG. 36: binding to CD25 expressed on human (A) and (B) or cynomolgus monkey (C) and (D) pan-T cells activated with CD3/CD28 beads followed by CD4 at increased antibody concentrations and compared to human IgG1 isotype control⁺And CD8⁺Characterization of up-gated antibody 3.

FIG. 37: biolayer interferometry was performed on Octet Red384 by using a standard sandwich format grouping assay, showing noncompetitive binding of antibody 3 and IL-2. Anti-human CD25 antibody 3 was loaded onto the AHQ sensor. The sensor was then exposed to 100nM human CD25, followed by human IL-2. Additional binding of human IL2 following antigen association indicates an unoccupied epitope (non-competitor).

FIG. 38: biolayer interferometry was performed on Octet Red384 by using a standard sandwich format grouping assay, showing non-competitive binding of antibody 3 and daclizumab to CD 25. The reference monoclonal anti-human CD25 antibody darlizumab was loaded onto the AHQ sensor. The sensor was then exposed to 100nM human CD25 antigen, followed by anti-human CD25 antibody (antibody 3). Additional binding of the secondary antibody after antigen association indicates unoccupied epitopes (non-competitors), while no binding indicates epitope blocking (competitors).

FIG. 39: antibody 3 was characterized for blocking IL-2 signaling in a STAT5 phosphorylation assay using human-derived PBMCs, as compared to human IgG1 isotype control, daclizumab, or in the absence of primary antibody. Cells were incubated with 10. mu.g/ml antibody and the concentration of IL-2 was then increased (as shown). The analysis was limited to the percentage of CD3 positive cells that phosphorylated STAT 5.

FIG. 40: antibody 3 was functionally characterized using pan-T cells, compared to human IgG1 isotype control, daclizumab, or a commercially available mouse anti-human IL-2 neutralizing antibody (clone: AB12-3G4) as a positive control. Cells were incubated with 10ug/ml antibody and then activated with CD3/CD28 beads for 72 hours before flow cytometry analysis. The results show the percentage of granzyme B positive proliferating CD4(a) or CD8(B) T cells.

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

FIG. 42: antibody 3 function was characterized in the ADCP assay for phagocytosis of in vitro differentiated Treg cells compared to the human IgG1 isotype control. Tregs were co-cultured with MCSF differentiated macrophages in the presence of different concentrations of antibody (as shown). Two-color flow cytometry analysis was performed with CD14+ stained macrophages and eFluor450 dye labeled tregs. Residual target cells are defined as being eFluor450-dye⁺/CD14^-A cell. Double labeled cells (eFluor 450-dye +/CD14+) are thought to represent phagocytosis of the target by macrophages. Phagocytosis of target cells was calculated using the following equation: % phagocytosis ═ 100 × [ (double positive%)/(double positive% + percent residual target)]。

FIG. 43: characterization of antibody 4 binding 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 to human IgG1 isotype control.

FIG. 44: characterization of antibody 4 binding to unmodified CHO-S cells (negative control) (A) or cynomolgus-CD 25-CHO-S cells (B) at 100nM antibody concentration and compared to human IgG1 isotype control.

FIG. 45: biolayer interferometry was performed on Octet Red384 by using a standard sandwich format grouping assay, showing noncompetitive binding of antibody 4 and IL-2. Anti-human CD25 antibody 4 was loaded onto the AHQ sensor. The sensor was then exposed to 100nM human CD25, followed by human IL-2. Additional binding of human IL2 following antigen association indicates an unoccupied epitope (non-competitor).

FIG. 46: biolayer interferometry was performed on Octet Red384 by using a standard sandwich format grouping assay, showing non-competitive binding of antibody 4 and daclizumab to CD 25. The reference monoclonal anti-human CD25 antibody darlizumab was loaded onto the AHQ sensor. The sensor was then exposed to 100nM human CD25 antigen, followed by anti-human CD25 antibody (antibody 4). Additional binding of the secondary antibody after antigen association indicates unoccupied epitopes (non-competitors), while no binding indicates epitope blocking (competitors).

FIG. 47: antibody 4 was characterized for blocking IL-2 signaling in a STAT5 phosphorylation assay using human-derived PBMCs, as compared to human IgG1 isotype control, daclizumab, or in the absence of primary antibody. Cells were incubated with 10. mu.g/ml antibody and the concentration of IL-2 was then increased (as shown). The analysis was limited to the percentage of CD3 positive cells that phosphorylated STAT 5.

FIG. 48: antibody 4 was functionally characterized using pan-T cells, compared to human IgG1 isotype control, daclizumab, or a commercially available mouse anti-human IL-2 neutralizing antibody (clone: AB12-3G4) as a positive control. Cells were incubated with 10ug/ml antibody and then activated with CD3/CD28 beads for 72 hours before flow cytometry analysis. The results show the percentage of granzyme B positive proliferating CD4(a) or CD8(B) T cells.

FIG. 49: in the ADCC assay, antibody 4 was functionally characterized with respect to killing of CD25 positive cell line compared to human IgG1 isotype control. SU-DHL-1(A) or SR-786 (B) cells, which are high or low expressing CD25, were co-cultured with purified NK cells, respectively, in the presence of different concentrations of antibody (as shown). Target cell lysis was measured by the release of calcein into the supernatant 4 hours after calcein was added to NK cells. Data were normalized to saponin treated controls.

FIG. 50: antibody 4 was characterized for induction of ADCP as compared to the human IgG1 isotype control in the reporter bioassay. SU-DHL-1 cells expressing CD25 were co-cultured with Jurkat T cells genetically engineered to express Fc γ RIIA and an NFAT response element (NFAT-RE-luc2) that drives luciferase expression in the presence of various concentrations of antibody (as shown).

FIG. 51: characterization of antibody 2 binding 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 to human IgG1 isotype control.

FIG. 52: binding to CD25 expressed on human (A) and (B) or cynomolgus monkey (C) and (D) pan-T cells activated with CD3/CD28 beads followed by CD4 at increased antibody concentrations and compared to human IgG1 isotype control⁺And CD8⁺Gated onCharacterization of antibody 2.

FIG. 53: biolayer interferometry assays were performed on Octet Red384 by using standard sandwich format grouping assays, showing noncompetitive binding of antibody 2 and IL-2 (A) and competitive binding of IL-2 competing antibody to IL-2 (B). Anti-human CD25 antibody 2 was loaded onto the AHQ sensor. The sensor was then exposed to 100nM human CD25, followed by human IL-2. Additional binding of human IL2 following antigen association indicates an unoccupied epitope (non-competitor).

FIG. 54: biolayer interferometry was performed on Octet Red384 by using a standard sandwich format grouping assay, showing non-competitive binding of antibody 2 and dallizumab to CD 25. The reference monoclonal anti-human CD25 antibody darlizumab was loaded onto the AHQ sensor. The sensor was then exposed to 100nM human CD25 antigen, followed by anti-human CD25 antibody (antibody 2). Additional binding of the secondary antibody after antigen association indicates unoccupied epitopes (non-competitors), while no binding indicates epitope blocking (competitors).

FIG. 55: antibody 2 was characterized for blocking IL-2 signaling in a STAT5 phosphorylation assay using human-derived PBMCs, as compared to human IgG1 isotype control, daclizumab, or in the absence of primary antibody. Cells were incubated with 10. mu.g/ml antibody and the concentration of IL-2 was then increased (as shown). The analysis was limited to the percentage of CD3 positive cells that phosphorylated STAT 5.

FIG. 56: in the ADCC assay, antibody 2 function was characterized with respect to killing of CD25 positive cell line compared to human IgG1 isotype control. SU-DHL-1(A) or SR-786 (B) cells, which are high or low expressing CD25, were co-cultured with purified NK cells, respectively, in the presence of different concentrations of antibody (as shown). Target cell lysis was measured by the release of calcein into the supernatant 4 hours after calcein was added to NK cells. Data were normalized to saponin treated controls.

FIG. 57: antibody 2 function was characterized in the ADCP assay for phagocytosis of in vitro differentiated Treg cells compared to the human IgG1 isotype control. Tregs were co-cultured with MCSF differentiated macrophages in the presence of different concentrations of antibody (as shown). Staining with CD14+Macrophages and eFluor450 dye-labeled tregs were analyzed by two-color flow cytometry. Residual target cells are defined as being eFluor450-dye⁺/CD14^-A cell. Double labeled cells (eFluor 450-dye +/CD14+) are thought to represent phagocytosis of the target by macrophages. Phagocytosis of target cells was calculated using the following equation: % phagocytosis ═ 100 × [ (double positive%)/(double positive% + percent residual target)]。

FIG. 58: characterization of antibody 5 binding to CD25 expressed on Karpas299 cells at increased antibody concentrations and compared to human IgG1 isotype control.

FIG. 59: characterization of antibody 5 that blocks IL-2 signaling in the STAT5 phosphorylation assay using human PBMC. Mouse anti-human antibody MA-251 was used as a non-blocking control, while the clinical high-yield method of daclizumab (DAC HYP) was used as a blocking control, as opposed to the mouse IgG1 isotype control, the human IgG1 isotype control, or the absence of primary antibody, respectively. Cells were incubated with 10. mu.g/ml antibody and then with 10U/ml IL-2. The analysis was limited to the percentage of CD3 positive cells that phosphorylated STAT 5.

FIG. 60: competition assay in Octet. Antibody 1 was allowed to bind to immobilized rhCD25, then to the primary Ab (as a control) or secondary Ab, to an IL-2 competitor (e.g., the dallizumab and basiliximab studies) or an IL-2 non-competitor (e.g., 7G7B 6). Antibody 1 did not compete with IL-2 signal blocker study basiliximab (a) dallizumab (B), whereas it did compete with 7G7B6 (non-IL-2 blocker) (C).

FIG. 61: antibody 5 was characterized for induction of ADCC in a 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 Fc γ RIIA and an NFAT response element (NFAT-RE-luc2) that drives luciferase expression in the presence of various concentrations of antibody (as shown).

FIG. 62: antibody 5 function was characterized in the ADCP assay for phagocytosis of in vitro differentiated Treg cells compared to the human IgG1 isotype control. Tregs were co-cultured with MCSF differentiated macrophages in the presence of different concentrations of antibody (as shown). By usingCD14+ stained macrophages and eFluor450 dye-labeled tregs were analyzed by two-color flow cytometry. Residual target cells are defined as being eFluor450-dye⁺/CD14^-A cell. Double labeled cells (eFluor 450-dye +/CD14+) are thought to represent phagocytosis of the target by macrophages. Phagocytosis of target cells was calculated using the following equation: % phagocytosis ═ 100 × [ (double positive%)/(double positive% + percent residual target)]。

FIG. 63: characterization of antibody 6, antibody 7, antibody 8 and antibody 9 binding to CD25 expressed on Karpas299 cells at increased antibody concentrations and compared to human IgG1 isotype control.

FIG. 64: competition assay in Octet. The primary Ab (antibody 7) was bound to immobilized rhCD25 and then to the primary Ab (as a control) or the secondary Ab dallizumab (a) or basiliximab (B).

FIG. 65: characterization of antibody 7 for blocking IL-2 signaling in STAT5 phosphorylation assay, compared to human IgG1 isotype control, daclizumab-Hyp, or in the absence of primary antibody, using human-derived PBMCs. Cells were incubated with 10. mu.g/ml antibody and then with 10U/ml IL-2. The analysis was limited to the percentage of CD3 positive cells that phosphorylated STAT 5.

FIG. 66: antibody 7 was functionally characterized for induction of ADCC in a 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 Fc γ RIIA and an NFAT response element (NFAT-RE-luc2) that drives luciferase expression in the presence of various concentrations of antibody (as shown).

FIG. 67: antibody 7 function was characterized in the ADCP assay for phagocytosis of in vitro differentiated Treg cells compared to anti-human CD25 Fc silencing control antibodies. Tregs were co-cultured with MCSF differentiated macrophages in the presence of different concentrations of antibody (as shown). Two-color flow cytometry analysis was performed with CD14+ stained macrophages and eFluor450 dye labeled tregs. Residual target cells are defined as being eFluor450-dye⁺/CD14^-A cell. Double-labeled cells (eFluor 450-dye +/CD14+) were considered to representPhagocytosis of the target by macrophages. Phagocytosis of target cells was calculated using the following equation: % phagocytosis ═ 100 × [ (double positive%)/(double positive% + percent residual target)]。

FIG. 68: characterization of antibody 10, antibody 11, antibody 12, antibody 13, antibody 14, antibody 15, antibody 16, antibody 17, antibody 18, antibody 19, antibody 20 and antibody 21 that bound to CD25 expressed on Karpas299 cells at increased antibody concentrations and compared to human IgG1 isotype control.

FIG. 69: competition assay in Octet. The primary Ab (antibody 19) was bound to immobilized rhCD25 and then to the primary Ab (as a control) or the secondary Ab dallizumab (a) or basiliximab (B).

FIG. 70: characterization of antibody 10, antibody 11, antibody 12, antibody 13, antibody 14, antibody 15, antibody 16, antibody 17, antibody 18, antibody 19, antibody 20 and antibody 21 with respect to blocking IL-2 signaling in a STAT5 phosphorylation assay compared to a human IgG1 isotype control, daclizumab-Hyp, or in the absence of a primary antibody, using human-derived PBMC. Cells were incubated with 10. mu.g/ml antibody and then with 10U/ml IL-2. The analysis was limited to the percentage of CD3 positive cells that phosphorylated STAT 5.

FIG. 71: antibody 19 was functionally characterized for induction of ADCC in a 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 Fc γ RIIA and an NFAT response element (NFAT-RE-luc2) that drives luciferase expression in the presence of various concentrations of antibody (as shown).

FIG. 72: antibody 19 function was characterized in the ADCP assay for phagocytosis of in vitro differentiated Treg cells compared to anti-human CD25 Fc silencing control antibodies. Tregs were co-cultured with MCSF differentiated macrophages in the presence of different concentrations of antibody (as shown). Two-color flow cytometry analysis was performed with CD14+ stained macrophages and eFluor450 dye labeled tregs. Residual target cells are defined as being eFluor450-dye⁺/CD14^-A cell. Double-labeled cells (eFluor 450-dye +/CD14+) were coated withIt is thought to represent phagocytosis of the target by macrophages. Phagocytosis of target cells was calculated using the following equation: % phagocytosis ═ 100 × [ (double positive%)/(double positive% + percent residual target)]。

FIG. 73: antibody 19, antibody 12 and antibody 20 were functionally characterized for phagocytosis of in vitro differentiated Treg cells in the ADCP assay compared to an anti-human CD25 Fc silencing control antibody. Tregs were co-cultured with MCSF differentiated macrophages in the presence of different concentrations of antibody (as shown). Two-color flow cytometry analysis was performed with CD14+ stained macrophages and eFluor450 dye labeled tregs. Residual target cells are defined as being eFluor450-dye⁺/CD14^-A cell. Double labeled cells (eFluor 450-dye +/CD14+) are thought to represent phagocytosis of the target by macrophages. Phagocytosis of target cells was calculated using the following equation: % phagocytosis ═ 100 × [ (double positive%)/(double positive% + percent residual target)]。

FIG. 74: therapeutic activity of the non-IL-2 blocking anti-CD 25 antibody 7D4 mouse IgG2a in combination with BVAB16 immunotherapy resistance model in BVAB16 immunotherapy resistance model. Individual mice were treated with Gvax alone or in combination with 7D 4.

FIG. 75: therapeutic activity of non-IL-2 blocking anti-CD 25 antibodies (7D4 and 2E4) compared to IL-2 blocking antibody (PC61) in a CT26 tumor model using female BALB/c mice. The anti-CD 25 non-blocking antibodies 7D4 and 2E4 exert potent therapeutic activity against solid tumors. Both 7D4 and 2E4 were more effective than the IL-2 blocking antibody PC 61.

FIG. 76: the therapeutic activity of the non-IL-2 blocking anti-CD 25 antibody 7D4mIgG2a in the MCA205 model was evaluated under single and repeated injections in combination with anti-mouse PD-L1. Indicates mice that survived at the end of the experiment.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Example 1 in vitro characterization and preparation of non-IL-2-blocking or IL-2-blocking recombinant anti-mouse CD25 Treg depleted antibodies

Materials and methods

Origin of antibodies and recombinant production thereof

The heavy and light chain variable regions of the rat anti-murine CD25 antibody PC61 were sequenced from the PC-61.5.3 hybridoma (ATCC accession number TIB-222) by Rapid Amplification of CDNA Ends (RACE) and then cloned into the constant region and kappa chain of murine IgG2a (or the corresponding murine IgG1 sequence isolated from a commercial plasmid (Invivogen)).

Each antibody chain was then subcloned into a Murine Leukemia Virus (MLV) -derived retroviral vector. For preliminary experiments, antibodies were produced using K562 cells transduced with vectors encoding the heavy and light chains. The antibody was purified from the supernatant using a protein G hitrap sample column (GE healthcare), dialyzed against Phosphate Buffered Saline (PBS), concentrated, and filter sterilized.

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

METDTLLLWVLLLWVPGSTGEVQLQQSGAELVRPGTSVKLSCKVSGDTITAYYIHFVKQRPGQGLEWIGRIDPEDDSTEYAEKFKNKATITANTSSNTAHLKYSRLTSEDTATY FCTTDNMGATEFVYWGQGTLVTVSS

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

METDTLLLWVLLLWVPGSTGQVVLTQPKSVSASLESTVKLSCKLNSGNIGSYYMHWYQQREGRSPTNLIYRDDKRPDGAPDRFSGSIDISSNSAFLTINNVQTEDEAMYFCHSYDGRMYIFGGGTKLTV

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

Genes encoding the variable heavy chain domain and the variable light chain domain were codon optimized for expression in human cell lines and synthesized at NheI and AvaI restriction sites 5 'and 3' of the genes. Restriction digest cloning was performed to insert the 7D4 variable heavy domain gene into separate expression vectors containing mouse IgG1 and IgG2a constant domains. Restriction digest cloning was performed to insert the 7F4 variable light chain gene into an expression vector containing a mouse kappa constant domain. Suspended HEK293 cells cultured in serum-free medium were CO-transfected with heavy and light chain expression vectors and cultured at 37 ℃ for an additional 6 days in a 5% CO2 environment with shaking at 140 rpm. The culture was harvested by centrifugation at 4000rpm and clarified by further filtration through a 0.22 μ M filter. The supernatant was loaded onto a protein a column pre-equilibrated with PBS pH7.2, eluted with sodium citrate pH 3.5 and equilibrated with 10% (v/v)0.5MTris pH 9.0. The neutralized antibody solution was buffer-exchanged into PBS ph7.2 using a desalting column and concentrated using a centrifugal concentrator with a molecular weight cut-off of 30kDa as necessary. The protein concentration was determined by measuring the absorbance at 280nm and the purity was determined by SDS-PAGE.

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

EVQLQQSGAALVKPGASVKMSCKASGYSFPDSWVTWVKQSHGKSLEWIGDIFPNSGATNFNEKFKGKATLTVDKSTSTAYMELSRLTSEDSAIYYCTRLDYGYWGQGVMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLMISLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPILHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

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

EVQLQQSGAALVKPGASVKMSCKASGYSFPDSWVTWVKQSHGKSLEWIGDIFPNSGATNFNEKFKGKATLTVDKSTSTAYMELSRLTSEDSAIYYCTRLDYGYWGQGVMVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

the recloned anti-mouse CD25 kappa light chain DNA sequences of 7D4(migg1) and 7D4(migg2a) antibodies (mouse IgG2a) encode the following protein sequences:

DVVLTQTPPTLSATIGQSVSISCRSSQSLLHSNGNTYLNWLLQRPGQPPQLLIYLASRLESGVPNRFSGSGSGTDFTLKISGVEAEDLGVYYCVQSSHFPNTFGVGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

2E4 was produced by 2E4 hybridoma (gift from Dr. Shevach, Ethan M., national institutes of health). Hybridoma sequencing was performed by proprietary Next Generation Sequencing (NGS) based techniques. RNA samples were used to generate cDNA libraries. The library was sequenced on the Illumina platform. De novo assembly (De novo assembly) was used to reconstruct the sample transcriptome from the raw data. The variable domain sequence is identified by comparison to known sequences.

The variable heavy domain protein sequence of the anti-mouse CD 252E 4 antibody (mouse IgG1) has the following protein sequence:

EVQLVESGGGLVQPGRSLKLSCAASGFTFSDYGMAWVRQAPTKGLEWVASITNGGLNTYYRDSVKGRFTISRDNAKCTLYLQMDSLRSEDTATYYCATGGFSFWGQGTLVTVSS

the variable light domain protein sequence of anti-mouse CD 252E 4(migg1) has the following protein sequence:

DIVMTQSPTSMSISVGDRVTMNCKASQNVDSNVDWYQQKTGQSPKLLIYKASNRYTGVPDRFTGSGSGTDFTFTIRNMQAEDLAVYYCMQSNSYPLTFGSGTKLEIK

evaluation of the affinity of recombinant antibodies to mouse CD25

ForteBio affinity measurements are typically performed on Octet RED384 as described previously (see, e.g., Estep Pet al.,2013.mabs.5(2), 270-8). Briefly, ForteBio affinity measurements were performed by loading IgG onto AHQ sensors in-line. The sensor was equilibrated offline for 30 minutes in assay buffer and then monitored online for 60 seconds to establish a baseline. The sensor with IgG added was exposed to 100nM antigen for 3 min and then transferred to assay buffer for 3 min for off-rate measurement. All kinetics were analyzed using a 1:1 binding model.

Results

Two mouse hybridomas have been selected as reference antibodies for the evaluation of CD25 binding and Treg depletion properties of non-IL-2 blocking or IL-2 blocking anti-mouse CD25(7D4 (mouse IgM isotype) and PC61 (mouse IgG1 isotype), respectively). 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 (FIG. 1A). Recombinant variants of this antibody have been generated for testing antibodies, where the isotype is more active and relevant for functional studies (e.g. Treg depletion or effect on other immune cells). Moreover, in the case of 7D4, it is necessary to change the isotype, since the antibody aggregation properties of IgM antibodies may affect the results of the assay. Recombinant 7D4(mIgG1) as the original non-recombinant IgM isotype antibody still allowed binding of mouse IL-2 to mouse CD25 (fig. 1B). 7D4(mIgG1) also bound to mouse CD25 on the cell surface, similar to recombinant PC61(IgG2a), while the reference anti-human CD25 antibody did not bind (FIG. 1C).

The DNA sequence encoding the 7D4 heavy chain variable domain (and the PC61 heavy chain variable domain) has also been cloned into a vector that allows expression of a mouse CD25 binding domain (functionally corresponding to human IgG1) of the mouse IgG2a isotype. In this way, two recombinant anti-mouse CD25 antibodies with optimized ADCC activity can be compared, which can efficiently deplete intratumoral tregs, but have unique properties in the binding of mouse IL-2 to mouse CD 25. The resulting recombinant anti-mouse CD25 antibody has been tested for CD25 affinity. Different isotypes (mouse IgG2a or mouse IgG1) did not affect this property, since Kd between recombinants based on 7D4 was similar (about 1nM) and comparable to one of PC61(mIgG2a), measured at 4.6 nM.

The functional properties of these recombinant antibodies were also compared in an in vitro assay to determine their effect on granzyme B production in response to anti-CD 3 and anti-CD 28 stimulation (figure 2). Granzyme b (GnzB) is a serine protease expressed by memory T cells and NK cells, as well as activated CD4 and CD8T cells that strongly express and secrete GnzB during immune responses. The enzyme is an important mediator of cell death, histopathology and disease. In vitro stimulation and proliferation of T cells (> 80% are CD4T cells B that proliferate and express Gnz) with anti-CD 3 and anti-CD 28 antibodies can be affected by cytokines and antibodies. When this stimulation is performed in combination with neutralizing anti-IL-2 antibodies, the production, but not proliferation, of granzyme B will be inhibited: the frequency of cells proliferating and producing GnzB dropped from > 80% to < 1%, while the frequency of proliferating cells remained > 90%. This indicates that granzyme B production is dependent on IL-2 signaling, whereas cell proliferation is independent of IL-2 signaling. A similar decrease in granzyme B producing T cells was observed when PC61(mIgG1) was added to the stimulated T cells. However, 7D4(mIgG1) retained the ability of CD4T cells to respond to anti-CD 3 and anti-CD 28 stimulation, in large part by producing GnzB (> 65% are cells that still produce GnzB and proliferate). These results demonstrate that the PC 61-based antibody blocks IL-2 signaling, while 7D4 has only a small effect on this signaling, and therefore can be used as a surrogate antibody to assess 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 anti-tumor properties of non-IL-2 blocking or IL-2 blocking recombinant anti-mouse CD25 Treg depleting antibodies

Materials and methods

Mouse

In vivo studies were performed by Charles River Discovery Services, north carolina (CR Discovery Services). Female BALB/C mice (BALB/C AnNcr1, Charles River) and female C57BL/6 mice (C57BL/6Ncr1, Charles River) were between 7 and 9 weeks of age at the start of the study. CR discovery services specifically adhere to recommendations in "guidelines for laboratory animal care and use" regarding restrictions, feeding, surgery, feed and fluid regulation, and veterinary care. Animal care and use programs for CR Discovery Services have received approval from the international laboratory animal care assessment and certification institute to ensure compliance with accepted laboratory animal care and use standards.

Cell lines and tissue culture

MCA205 tumor cells (3-methylcholanthrene-induced weakly immunogenic fibrosarcoma cells; Lawsonia inermis C. cells)From g.kroemer, Gustave Roussy cancer institute) in dur's modified eagle's medium (DMEM, Sigma) supplemented with 10% fetal calf serum (FCS, Sigma), 100U/mL penicillin, 100 μ g/mL streptomycin and 2mM L-glutamine (all from Gibco). MC38 mouse colon cancer cells (CR discovery services) were grown to mid-log phase in Duchen Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, 2mM glutamine, 100 units/mL penicillin G, 100. mu.g/mL streptomycin sulfate and 25. mu.g/mL gentamicin. CT26 murine colon cancer cells (CR discovery services) were grown in RPMI-1640 medium containing 10% fetal bovine serum, 2mM glutamine, 100 units/mL penicillin G sodium, 100. mu.g/mL streptomycin sulfate, and 25. mu.g/mL gentamicin. All tumor cells were incubated in tissue culture flasks at 37 ℃ in a humidified incubator at 5% CO₂And an atmosphere of 95% air. K562 cells for antibody production were cultured in phenol red-free eiskoff modified duchenne medium (IMDM) supplemented with 10% IgG-free fcs (life technologies).

In vivo tumor experiments

Cultured tumor cells were trypsinized (MCA205) or not (MC38 and CT26), washed and resuspended in PBS and injected ventrally subcutaneously (s.c) (for the MCA205 and MC38 models in C57BL/6 mice, 5X 10 was injected⁵(ii) individual cells; for the CT26 model in BALB/c mice, 3X 10 was injected⁵Individual cells). Antibodies were injected intraperitoneally (i.p.) at the time points described in the figure legends. For functional experiments, tumors and draining lymph nodes were harvested 12 days after tumor implantation and processed for analysis by flow cytometry as described (Simpson et al (2013) J Exp Med 210, 1695-710). For the treatment experiments, tumors were measured twice weekly and the product of the 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-CD 3 (clone 145-2C11, eBiosciences, 25003182), anti-CD 4 (clone RM4-5, BD biosciences, 560782), anti-CD 8 (clone 53-6.7, Biolegend, 100750), anti-granzyme B (clone GB11, Invitrogen), anti-FoxP 3 (clone FJK-16s, eBiosciences) and Ki67(clone SolA15, eBiosciences, 48569882). Lymph nodes (groin, axilla, and brachium) and tumors from mice were excised and placed in serum-free RPMI. The lymph nodes were dispersed through a 70 μm filter, then the tumors were mechanically destroyed using genetlemecs acs (miltenyl biotech), and digested with a mixture of 0.33mg/ml dnase (Sigma-Aldrich) and 0.27mg/ml Liberase TL (Roche) in serum-free RPMI for 30 min at 37 ℃. The tumors were filtered through a 70 μm filter, and the resulting tumor single cell suspension was subjected to gradient passaging through Ficoll-paque (GE healthcare) to enrich leukocytes. Tumors and LN were washed in complete RPMI, resuspended in FACS buffer (500mL PBS, 2% FCS, 2mM EDTA) and plated in round bottom 96-well plates. The mixed reagent of surface antibody (mastermix) was prepared at the dilution recommended by the manufacturer: anti-CD 3 (clone 145-2C11, ebioscience, 25003182), anti-CD 4 (clone RM4-5, BD biosciences, 560782), anti-CD 8 (clone 53-6.7, Biolegend, 100750). Immobilizable vital dyes (efourr 780, eBioscience) are also included in the surface mixing reagents. After permeabilization for 20 minutes using the intracellular fixation and permeabilization buffer kit (eBioscience), the intracellular staining module consisting of the following antibodies for the manufacturer's recommended dilutions was applied: anti-granzyme B (clone GB11, Invitrogen), anti-FoxP 3 (clone FJK-16s, eBiosciences) and Ki67(CloneSolA15, eBiosciences, 48569882) antibodies.

Results

The MCA205 sarcoma mouse model allowed the generation of mice that could evaluate the immune response and overall efficacy against solid tumors in a short time against a panel of immunomodulatory compounds. In particular, recombinant anti-mouse CD25 antibodies based on mouse IgG2a were 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 of mice exposed to MCA 205. Another antibody (anti-mouse PD1) was included in the study as a negative control for the immunological effect on tregs.

Immunological analysis showed that the 7D4 antibody, when cloned into the mouse IgG2a backbone, showed a similar ability to deplete tregs as PC61 (mouse IgG2a) and subsequently increased the Teff to Treg ratio in the tumor and periphery, whereas anti-PD 1 was ineffective, either alone or in combination (fig. 3). Therefore, any further effects measured using 7D4(mIgG2a) as a surrogate antibody for non-IL-2 blocking anti-human CD25 antibody do not appear to be related to changes in Treg depletion properties.

MCA205 model mice treated with 7D4 also showed a higher percentage of GnzB-positive cells, such as proliferating CD 4-positive and CD 8-positive T cells, involved not only in anti-PD 1 treatment, but also in Il-2 (migg2a) blocking PC 61. In such treated mice, not only 7D4(migg2a), such as PC61(migg2a), did not affect Teff cells, but it also increased the frequency of Teff cells compared to PC61(migg2a), indicating an even higher anti-tumor activity of anti-human CD25 antibodies that did not block the IL-2/CD25 interaction (fig. 4).

Anti-human CD25, functionally equivalent to 7D4(migg2a), can be tested in the MCA205 murine model as well as in other models, such as CT26 and MC38 (colon cancer) or B16 (melanoma) models, for immunotherapy of cancer, in particular solid tumors. When administered in combination with the anti-PD 1 antibody, both IgG2a, anti-mouse CD25 antibodies showed therapeutic activity against the established CT26 tumor. Interestingly, the non-IL-2 blocking 7D4(migg2a) antibody showed significantly higher therapeutic activity than the PC 61-based antibody with the same isotype when used as monotherapy. At the end of the experiment, all mice treated with 7D4(mIg2a) showed only tumor growth control at volumes below 50mm³Whereas mice treated with PC61(mIg2a) did not show tumors smaller than 50mm³8 of 10 mice even reached 2000mm³The termination point of (1). This is also illustrated by the difference in survival rates, with all mice treated with 7D4(migg2a) still surviving at day 50, while only 2 out of 10 mice treated with PC61(migg2a) survived. Indeed, at least when the antibody was used at this concentration, the efficacy of 7D4(migg2a) was not further improved if PC61(migg2a) potency was greatly improved by combination with anti-PD 1.

Since these 7D4 and PC 61-based antibodies showed similar Treg depleting capacity (see fig. 3), this difference in potency can be explained at least in part by the lower effect of 7D4(migg2a) on the interaction between IL-2 and its receptor. This indicates that not only is the lack of IL-2/IL-2 receptor blocking activity not detrimental to therapeutic activity, but it may also provide therapeutic advantages. Thus, this data supports the selection of non-IL-2/IL-2 receptors that block CD 25-targeted antibodies for cancer therapy. These advantageous properties of the 7D4(migg2a) antibody were also confirmed when anti-mouse PD-L1 was used in the same CT26 murine model (fig. 6) or when MC38 murine model was used in the same combination with the antibody (fig. 7 and fig. 8).

These data indicate that Treg depletion, CD25 binding properties of antibodies based on 7D4 properties and with appropriate isotypes can be used in combination with other anti-cancer 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. The method can be achieved by generating and administering the two products as a novel mixture of monospecific antibodies or as a novel bispecific antibody. This approach, which involves constructing bispecific antibodies that bind to two antigen binding properties and a therapeutically relevant isotype (e.g., human IgG1), can be validated by using the Duobody technique that allows for efficient binding of single chain heavy and light chains from two different monospecific antibodies that were generated separately and contain a single matching point mutation in the CH3 domain, thus allowing Fab exchanges within a single heteromeric protein (Labrijn AF et al, Nat protoc.2014,9: 2450-63). The functional properties of such 7D 4-based Duobody products (e.g., including anti-PD 1 or anti-PD-L1) can be assessed by using a model for verifying cell interaction and depletion of 7D 4-based antibodies and antibody combinations as described above.

These results also indicate that the binding properties of 7D4 for mouse CD25 that do not interfere with the interaction of IL-2 with its receptor and IL-2 signaling in CD 25-expressing cells can be exploited in selecting anti-human CD25 of the isotype (e.g., human IgG1) consistent with this mechanism of action. Indeed, several other properties may be considered for screening anti-human CD25 antibody candidates with further improved properties in preparing, using and/or administering to treat cancer, in particular solid tumors.

These properties can also be defined according to known characteristics of anti-human CD25, such as Humax-TAC, basiliximab, or daclizumab, all of which have Kd in the nanomolar range for human CD25, but all block 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 of the present invention).

These functions may be one or more of the following:

affinity for recombinant, isolated monomeric human CD25, wherein K_DLess than 25nM, preferably less than 10nM, even more preferably less than 1nM (as established using techniques such as Octet, Kinexa, ELISA or others);

cross-reactivity to recombinant, isolated monomeric cynomolgus monkey CD25, wherein K_DLess than 75nM, preferably less than 30nM, and even more preferably less than 3nM (as established using Octet, Kinexa, ELISA, etc.);

affinity for recombinant monomeric human CD25 on the surface of CHO or MJ cells, where K_DLess than 100nM, preferably less than 10nM, even more preferably less than 1nM (established using techniques such as flow cytometry, cell-based ELISA, etc.);

affinity for recombinant monomeric rhesus CD25 on the surface of CHO cells, where K_DLess than 300nM, preferably less than 30nM, even more preferably less than 3nM (established using techniques such as flow cytometry, cell-based ELISA, etc.);

human Treg cells bind with a KD of less than 100nM, preferably less than 10nM, even more preferably less than 1nM (established using techniques such as flow cytometry, cell-based ELISA, etc.);

cynomolgus monkey Treg cells bind with a KD of less than 300nM, preferably less than 30nM, even more preferably less than 3nM (established using techniques such as flow cytometry, cell-based ELISA, etc.);

lack of inhibition of the interaction between human recombinant IL-2 and human recombinant CD25 in a biochemical assay (less than 25% of IL-2 binding to CD25 was blocked in the screen as described in example 1);

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 CD4 positive T cell assays upon activation granzyme B upregulation (less than 25% of baseline signal is inhibited as described in example 1); and/or

-a relevant potency assessment in a cell-based assay, such as ADCC, ADCP and/or CDC assay (EC50 below 10nM, preferably below 1nM, even more preferably below 0.1nM) in a cell line expressing human CD25 or primary Treg cells.

Example 3 further in vivo mouse model experiments with non-IL 2 blocking anti-mouse CD25 antibodies

Materials and methods

Therapeutic Activity of non-IL-2 blocking antibodies: 3X 10 in 0% Matrigel (Matrigel) injected subcutaneously on the flank of female BALB/c mice obtained from Charles River⁵CT26 tumor cells, each group n 15. Animals were randomized into treatment groups based on day 1 body weight. Treatment was started on day 6 and 200 μ g/animal was injected each time with each antibody (mouse IgG2a isotype, IL-2 neutralizing antibody, PC61mIgG 1(IL-2 signaling blocking anti-mouse CD25 antibody blocking mouse IgG1 isotype) and 7D4mIgG2a (IL-2 signaling blocking anti-mouse CD25 antibody blocking mouse IgG2a isotype). animals received monotherapy treatment of one group injected with each antibody, or received combination treatment of 7D4mIgG2a and IL-2 neutralizing antibody or 7D4mIgG2a and PC61mIgG1 antibody when tumor volume reached 2000mm³Or 50 days, mice were sacrificed based on first arrival.

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

Will be 3X 10⁵Individual CT26 cells were implanted subcutaneously in the flank. Matched pairs were performed on day 0 when tumors reached 30 to 60mm³And treatment is initiated. Treatment of 10mg/kg was given intraperitoneally once every two weeks on day 1 and thereafter. The group was treated or not treated with IL-2 neutralizing antibody PC61-m2a, non-IL-2 blocking antibody 7D4, non-IL-2 blocking antibody 2E 4.

Therapeutic Activity of non-IL-2 blocking antibodies in combination with aPDL1 therapy

As shown, mice were injected subcutaneously with 50000 MCA205 tumor cells, each group being either n-10 or n-5. Animals were randomized into treatment groups. Animals received monotherapy treatment of 7D4mIgG2a or aPD-L1 (clone 10F.9G2), combination treatment of 7D4mIgG2a with PD-L1 (clone 10F.9G2), or no treatment. Group acceptance: a7D4mIgG2a alone-day 10 (200ug), aPD-L1 rgig 2b (10f.9g2) -days 6, 9and 12 (200ug), aPD-L1+ a7D4 combination (aPDL-1 received on days 6, 9and 12, a7D4 received on day 10), or aPD-L1+ a7D4 combination (aPDL-1 received on days 6, 9and 12 and a7D4 received on day 10), -additional injections of a7D4 on day 15 + aPD-L1 on day 18 (only 5 mice).

Results

The anti-CD 25 depleting non-IL-2 blocking antibody 7D4mIgG2a induced tumor rejection in treated mice, while the other antibodies showed no effect as monotherapy compared to isotype control mouse IgG2 a. Combination with IL2 blocking antibody (PC61mIgG1 or IL2nAb) abolished the therapeutic activity of the non-IL-2 blocking antibody 7D4mIgG2a (fig. 13). This indicates that the non-IL-2 blocking characteristics of 7D4mIgG2a are critical for therapeutic activity. It also indicates that the therapeutic activity of the antibody is dependent on an anti-tumor immune response mediated by T effector cells that rely on IL-2 signaling for optimal activity. These results indicate that the optimal therapeutic activity of CD 25-targeted antibodies does not require IL-2/CD25 blocking activity and supports the use of anti-CD 25 non-IL-2 blocking antibodies as described herein in cancer therapy.

These results further indicate that the lack of IL-2/CD25 blocking activity is not detrimental to antibody therapeutic activity and supports the use of anti-CD 25 non-IL-2 blocking antibodies as described herein in the treatment of cancer.

These results further indicate that the non-IL-2 blocking antibodies 7D4 and 2E4 are more effective than the IL-2 blocking antibody PC 61. The anti-CD 25 non-blocking antibodies 7D4 and 2E4 exert potent therapeutic activity against solid tumors (fig. 75).

The results show that single or repeated injections of non-IL 2 blocking aacd 25 antibody 7D4 can enhance the anti-tumor response after initiating the aPDL1 therapy. Teff cell activation following treatment with aPDL1 was avoided by the aCD25 antibody (fig. 76).

Example 4 epitope characterization of anti-CD 25 non-IL-2 blocking antibodies

Epitope grouping (Epitope binding)

Epitope grouping of antibodies was performed on the Forte Bio Octet Red384 system (Pall Forte Biocorporation, Menlo Park, CA) using a standard sandwich format grouping assay. Anti-mouse CD25 PC61 antibody was loaded onto the AMC sensor and the unoccupied Fc binding site on the sensor was blocked with irrelevant mouse IgG1 antibody. The sensor was then exposed to 15nM of target antigen, followed by exposure to the 7D4 antibody. Data were processed using data analysis software 7.0 from ForteBio. Additional binding of the secondary antibody after antigen binding indicates unoccupied epitopes (non-competitors), while no binding indicates epitope blocking (competitors).

Epitope mapping of anti-CD 25 non-IL-2 blocking antibodies

Different sets of linear, monocyclic, β -turn mimetics, disulfide mimetics, discontinuous disulfide bonds, discontinuous epitope mimetic peptides (PepscanBV, The Netherlands; Timmermann P et al, 2007J. mol. Recognizt., 20,283-99; Langejk JP et al, 2011, Analytical biochemistry 417: 149. 155) representing human CD25 sequences (Uniprot record No. P01589) were synthesized using The solid phase Fmoc synthesis method, binding of The antibody to each synthetic peptide was tested in ELISA (Pepsn, The Netherlands). The peptide array was incubated with an anti-solution (overnight at 4 ℃), washed, and then The peptide array was incubated with a 1/1000 diluted suitable antibody peroxidase conjugate (2010-05; Southern Biotech) at 25 ℃ for 1 hour, followed by addition of peroxidase substrate 2,2' -azino-3-bis-thiozoline-sulfonate (ABH 3 ml/20. mu.H/20% of benzothiazole sulfonate)₂O₂. After 1 hour, color development was measured. The color rendering is quantified with a Charge Coupled Device (CCD) camera and an image processing system. Values obtained from the CCD camera ranged from 0 to 3000mAU, similar to a standard 96-well plate ELISA reader. To verify the quality of the synthetic peptides, a set of individual positive and negative control peptides were synthesized in parallel and screened with irrelevant control antibodies.

Results

Epitope grouping was performed to determine whether the antibodies bound to epitopes that overlap with the epitope of the commercially available mouse anti-human non-IL-2 blocking CD25 antibody 7G7B 6. The antibodies were further characterized to determine the epitope of non-IL-2 blocking antibodies. The epitope of the blocking antibody PC61 against mouse CD25 was determined for control comparison. The results of epitope mapping shown in table 1 for the anti-human CD25 antibody are shown in table 1, and those shown in table 2 for the anti-mouse CD25 antibody are as follows:

table 1-anti-human CD25 antibody:

minor epitopes

Amino acid (aa) sequence numbering is based on human CD25 taken from the sequence published under Uniprot accession number P01589.

Table 2-anti-mouse CD25 antibody:

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

Epitope mapping studies using the Pepscan technique showed that anti-human antibodies bind to human CD25 at an epitope that does not overlap with the IL-2 binding site on CD 25. The epitope bound by anti-human antibodies is different from basiliximab and daclizumab. The epitope to which basiliximab and dallizumab bind comprises residues in the region of amino acids 137 to 143 (of SEQ ID NO: 1) that overlap laterally with the interaction of CD25 with IL-2 (Binder M et al, Cancer Res 2007vol 67(8): 3518-23). The anti-mouse CD25 non-blocking antibodies 2E4 and 7D4 recognize different epitopes from PC 61.

Example 5: characterization of mouse anti-CD 25 antibody

Antibody binding to CHO cells expressing mouse CD25

Binding to CHO cells expressing CD25 was checked by staining the test article with 30mg/ml antibody (anti-CD 25 primary antibody, 7D1, PC61 and 2E4) followed by a semilog serial dilution (7 points) on ice for 30 minutes. Then stained with a secondary antibody (Alexa Fluor 647-AffiniPure Fab fragment goat anti-human IgG (H + L) - (Jackson ImmunoResearch)) at a concentration of 1mg/ml on ice for 30 minutes. All samples were stained in duplicate. Live cells were gated during sample collection by flow cytometry using FSC versus SSC parameters. Mean Fluorescence Intensity (MFI) of stained cells was plotted on an XY plot, MFI was plotted against log concentration, and the data was fitted to a non-linear regression curve from which EC50 was calculated. The results are shown in fig. 11, demonstrating that the anti-mouse CD25 antibody binds to CHO cells expressing mouse CD 25.

Affinity measurement of anti-mCD 25 antibodies

K of anti-mouse CD25 antibodies 7D4, PC61 and 2E4 was measured by SPR in Biacore 2000 at an ambient experimental temperature of 25 ℃ using a CM-5 sensor chip_DTo determine the affinity of the anti-mouse CD25 antibodies 7D4, PC61, and 2E 4. Initially, anti-mouse antibodies were fixed to RUs 16000 to 18000 in assay buffer (pH7.4,10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% tween 20) for 10 minutes, spanning all flow cells. The ligand (antibody test article) was sequentially loaded to capture levels between 119 and 163 RU. The analyte (his-labeled recombinant mouse CD25) was then associated with a two-fold dilution of assay buffer (minimum concentration of 3.13nM) starting at 800nM for 6 minutes. Dissociation was performed in assay buffer within 10 minutes. The regeneration step between sample concentrations was performed in 10mM glycine pH 1.7 for 10 minutes. The whole process was maintained at a flow rate of 25. mu.l/min. Kinetic data were fitted using a global model bivalent analyte analysis software provided by Biacore with a reference difference set (reference subset). The SPR-based analysis is shown in figure 12. The Kd values established for the anti-mouse CD25 antibody in this assay are as follows: for 7D4, it is 2.6X 10^-9M; for 2E4, 114X 10^-9M; for PC61, it is 3.6X 10^-9M (results not shown).

Anti-mouse antibody competition in Octet

Antibody competition was performed on a Forte Bio Octet Red96 system (Pall Forte Bio corp., USA) using a standard sandwich packet assay. Anti-mouse CD25 antibody at 10nM was loaded onto the MC sensor for 900 seconds, and the unoccupied Fc binding site on the sensor was blocked with irrelevant mouse IgG2a antibody. The sensor was exposed to 15nM of the target antigen (his-tag labeled mouse CD25) for 600 seconds, followed by exposure to a secondary anti-CD 25 antibody (also 10 nM). Data were processed using ForteBio data analysis software 9.0. Additional binding of the second antibody after antigen binding indicates that the epitope is unoccupied, while no binding indicates that the epitope is blocked.

Competitive binding to 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:

using InvitrogenFlowComp mouse Pan T (CD90.2) kit (Cat: 11465D) isolated Pan T cells from splenocytes. 200000 cells were plated and left to stand at 37 ℃ for 2 hours. 50ug/ml antibody was added and incubated with cells for 30 min at 37 ℃ and then cells were stimulated with IL-2(50U/ml) for 10 min at 37 ℃.

IL-2 induced phosphorylation of STAT5 was stopped when cells were fixed and permeabilized with the eBioscience TM Foxp 3/transcription factor staining buffer kit (Invitrogen) and treated with BD Phosflow Perm buffer III (BD biosciences). The cells were then stained simultaneously with surface and intracellular fluorochrome-labeled antibodies (STAT5-Alexa Fluor 647clone 47/STAT5/pY694 BD Bioscience, CD3-PerCP-Cy5.5 clone 17A2 Bioscience, CD4-PE clone RM4-5 Bioscience, FoxP3-AF488clone FJK-16sEbioscience), samples were obtained on a Fortessa LSR X20 flow cytometer (BD Bioscience) and analyzed using BDFACSDIVA software. Doublets were excluded using FCS-H vs FCS-A, and lymphocytes were defined using SSC-A vs FCS-A parameters. CD3 was defined using CD3 PerCP-Cy5.5-A vs FCS-A plot⁺T cells, and gates are plotted on a histogram showing the count of comparative STAT5 Alexa Fluor 647-A to determine STAT5⁺CD3⁺A population of T cells. The percentage of blockade of IL-2 signaling was calculated as follows: % blockade 100 × [ (% Stat5 in Ab-free group)⁺These% Stat5 of cell-50 ug/ml Ab group⁺cell)/(Ab-free group% Stat5⁺Cell)]. By different subsets of cells (CD 4)⁺、CD8⁺、CD4⁺Further analysis of STAT5 phosphorylation by FoxP3-) was also assessed by gating the corresponding subsets and analyzed as described above. Images and statistical analysis were performed using GraphPad Prism v7 (results not shown). The results are shown in FIG. 14.

Results：

Anti-mouse antibodies 7D4 and 2E4 were further evaluated for their ability to bind CD25 and not interfere with IL-2 signaling by CD 25-expressing target cells, anti-mouse antibodies 7D4 and 2E 4. non-IL-2 blockers 7D4 and 2E4 competed for binding to CD25, whereas PC61(IL-2 signaling blocker) did not compete with either 2E4 or 7D4 for binding to CD25 (fig. 12).

The STAT5 assay demonstrated that 7D4 and 2E4 did not block IL-2 signaling, whereas IL-2 signaling was blocked by the "blocking" antibody PC61 (fig. 14).

Example 6: in vivo depletion of tregs

200. mu.l of 1X 10 in RPMI1640 medium⁵A second thoracic fat pad tissue from Balb/c mice was implanted with individual 4T1 cells. When the tumor reaches 50 to 100mm³At this time, mice were randomly assigned and administered to each mouse in a single intraperitoneal gentle dose of 2 μ g,20 μ g, or 200 μ g mouse anti-mouse CD25(7D4) antibody. On days 3 and 9, tumor tissue and whole blood were isolated for immunophenotyping.

Results：

Antibody 7D4 showed Treg depleting activity in whole blood and tumor tissues based on post-dose analysis by immunophenotypic analysis on day 3 and day 9 (fig. 15).

Example 7: characterization of anti-CD 25 antibody 7G76B

Binding of anti-CD 25 antibodies to human CD25 expressing cells:

7G76B was assessed by binding to lymphoma human cell lines Karpas299, SU-DHL-1 and SR-786 and in vitro differentiated Treg cells. Binding to human cell lines expressing CD25 (SU-DHL-1 and SR-786) was detected by first blocking the cells with Trustain (Biolegend), followed by incubation with anti-CD 25 antibody titrated in a semilog dilution series from the highest concentration of 20. mu.g/ml at 4 ℃ for 30 minutes, followed by washing and incubation with PE conjugated anti-human IgG Fc antibody (Biolegend). The cells were washed again and resuspended in FACS buffer containing DAPI and harvested on intellichyt iquee. Live cells were gated during sample collection by flow cytometry using FSC versus SSC parameters. The mean intensity of Geo for stained cells was plotted on an XY plot, plotted against log concentration, and the data was fitted to a non-linear regression curve from which EC50 was calculated.

Binding to CD25 expressing Karpas299 cells and in vitro differentiated tregs was detected by: the test article (anti-CD 25 primary antibody) was stained with 30mg/ml antibody and then half-log serially diluted (7 points) on ice for 30 minutes. Then stained with a secondary antibody (Alexa Fluor 647-AffiniPure Fab fragment goat anti-human IgG (H + L) - (Jackson ImmunoResearch)) at a concentration of 1mg/ml on ice for 30 minutes. All samples were stained in duplicate. Live cells were gated during sample collection by flow cytometry using FSC versus SSC parameters. Mean Fluorescence Intensity (MFI) of stained cells was plotted on an XY plot, MFI was plotted against log concentration, and the data was fitted to a non-linear regression curve from which EC50 was calculated. The results shown in fig. 16 and fig. 21 demonstrate that the anti-CD 25 antibody binds to cells expressing CD 25.

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

IL-2 blockade was characterized using a STAT5 phosphorylation assay in which IL-2 signaling was examined. Previously frozen PBMC (stemcell Technologies) were incubated in 96-U bottom well plates for 30 minutes in the presence of 10U/ml anti-CD 25 antibody, followed by addition of different concentrations of IL-2(Peprotech) of 0.1, 1 or 10U/ml in RPMI1640 (Life Technologies) containing 10% FBS (Sigma), 2mM L-glutamine (Life Technologies) and 10000U/ml Pen-strep (Sigma). IL-2 induced phosphorylation of STAT5 was stopped when cells were fixed and permeabilized with the eBioscience TM Foxp 3/transcription factor staining buffer kit (Invitrogen) and treated with BD Phosflow Perm buffer III (BD biosciences). When the cells were fixed and permeabilized with the eBioscience TM Foxp 3/transcription factor staining buffer set (Invitrogen) and BD PhosflowIL-2 induced phosphorylation of STAT5 was stopped upon treatment with Perm buffer III (BD biosciences). The cells were then stained simultaneously with surface and intracellular fluorochrome-labeled antibodies (STAT5-Alexa Fluor 647clone 47/STAT5/pY694 BD Bioscience, CD3-PerCP-Cy5.5 clone UCHT1 Biolegene, CD4-BV510 clone SK3 BD Bioscience, CD8-Alexa Fluor 700clone RPA-T8Invitrogen, CD45RA-PE-Cy7clone HI100Invitrogen, FoxP3-Alexa Fluor 488clone236A/E7 Invitrogen), samples were obtained on a Fortessa LSR X20 flow cytometer (BD Bioscience) and analyzed using the FACSDVA software. Doublets were excluded using FCS-H vs FCS-A, and lymphocytes were defined using SSC-A vs FCS-A parameters. CD3 was defined using CD3 PerCP-Cy5.5-A vs FCS-A plot⁺T cells, and gates are plotted on a histogram showing the count of comparative STAT5 Alexa Fluor 647-A to determine STAT5⁺CD3⁺A population of T cells. The percentage of blockade of IL-2 signaling was calculated as follows: % blockade 100 × [ (% Stat5 in Ab-free group)⁺These% Stat5 of cell-10 ug/ml Ab group⁺cell)/(Ab-free group% Stat5⁺Cell)]. By different subsets of cells (CD 4)⁺、CD8⁺、CD4⁺FoxP3+, naive and memory T cells) further analysis of STAT5 phosphorylation was also assessed by gating the corresponding subsets and analyzed as described above. Images and statistical analysis were performed using GraphPad Prism v7 (results not shown). The results are shown in FIGS. 17 and 22.

In vitro T cell activation assay:

the effect of IL-2 signaling on the Teff response was characterized in a T cell activation assay in which intracellular granzyme b (grb) up-regulation and proliferation was examined. Primary human pan-T cells (Stemcell technologies) previously frozen were labeled with eFluor450 cell proliferation dye (Invitrogen) according to manufacturer's recommendations and labeled at 1X 10⁵Cells/well were added to RPMI1640 (Life Technologies) containing 10% FBS (Sigma), 2mM L-glutamine (Life Technologies) and 10000U/ml Pen-strep (Sigma) in 96-U bottom well plates. Then, the cells were treated with 10. mu.g/ml anti-CD 25 antibody or control antibody, followed by human T activator CD3/CD28 (cell to bead ratio 20: 1; Gibco) at 37 ℃ in 5% CO₂Incubate in humidified incubator for 72 hours. To assess T cell activation, cells were stained with eBioscience FixableViabilitydye efluor780(Invitrogen), followed by fluorochrome-labeled antibodies against surface T cell markers (CD3-PerCP-Cy5.5 clone UCHT1Biolegend, CD4-BV510 clone SK3 BD Bioscience, CD8-Alexa Fluor 700clone RPA-T8Invitrogen, CD45RA-PE-Cy7clone HI100Invitrogen, CD25-BUV737 clone 2A3 BD Bioscience), then fixed and permeabilized with eBioscience TM Foxp 3/transcription factor staining buffer kit (Invitrogen), then cloned against intracellular GrB and nuclear xP3 (GB enzyme B-PE clone 11, FoxP clone 3884/APC 38236). Samples were obtained on a Fortessa LSR X20 flow cytometer (BD Bioscience) and analyzed using BD FACSDIVA software. Doublets were excluded using FCS-H vs FCS-A, and lymphocytes were defined using SSC-A vs FCS-A parameters. Samples were obtained on a Fortessa LSR X20 flow cytometer (BD Bioscience) and analyzed using BD FACSDIVA software. Doublets were excluded using FCS-H and FCS-A, and lymphocytes were defined using SSC-A and FCS-A parameters. Evaluation of viable CD3 from GrB-PE-A and proliferative eFluor450-A profiles⁺Lymphocyte-gated CD4⁺And CD8⁺T cell subsets. The result was that the proliferation of GrB-positive cells accounted for the total CD4⁺Percentage of T cell population. Mapping and statistical analysis was performed using GraphPad Prism v 7. The results are shown in FIG. 18.

In vitro ADCC assay:

antibody-dependent cell-mediated cytotoxicity assays (ADCC assays) were performed using SU-DHL-1 or SR-786(CD25 positive) human cell lines as target cells and human NK cells as effector cell sources to characterize anti-human CD25 antibodies. NK cells were isolated from PBMC of healthy donors using NK cell negative isolation kit (Stemcell Technologies). NK cells were cultured overnight in the presence of 2ng/mL IL-2 (Peprotech). In the presence of anti-CD 25 or isotype antibodies, SU-DHL-1 or SR-786 target cells were loaded and plated with calcein-AM (thermolysis), 4 replicates per condition, 5% CO at 37 deg.C₂Incubate for 30 minutes. After incubation, NK cells were added at 37 ℃ with a target to effect (T: E) ratio of 1:10 (10000 target cells and 100000 effector cells)Lower 5% CO₂Incubate for 4 hours. Readings of calcein fluorescence in the supernatant were performed on 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). Dose response curves were generated using Graphpad Prism v7 to generate raw data plots. The percent target cell lysis was plotted on an XY plot, normalized calcein AM percent release was plotted against log concentration, and the data was fitted to a non-linear regression curve from which EC50 was calculated. The results are shown in FIG. 19.

In vitro ADCP assay:

antibody-dependent cell-mediated phagocytosis (ADCP) assays were performed using in vitro differentiated tregs as target cells and monocyte-derived macrophages as effector cells. PBMC were isolated from white blood cell cones (leucocytes) by Ficoll gradient centrifugation. Monocytes (CD14+ cells) were isolated using CD14 microbeads (Miltenyi Biotec). Monocytes were cultured for 5 days under 50ng/ml M-CSF in RPMI1640 (Life Technologies) containing 10% FBS (Sigma), 2mM L-glutamine (Life Technologies) and 10000U/ml penicillin-streptomycin (Sigma), and 3 days later fresh medium containing M-CSF was added. Human Treg cell differentiation kit (R)&D Systems) to isolate regulatory T cells (tregs). These cells were incubated at 37 ℃ with 5% CO₂Incubate for 5 days in a humidified incubator and label with eFluor450 dye (Invitrogen) according to the manufacturer's recommendations. On day 5, macrophages and tregs labeled with eFluor450 dye were co-cultured at an effective target ratio of 10:1 for 4 hours in the presence of anti-CD 25 antibody or control, as described below. At 1 × 10⁴Target cells (Tregs) were added at 1X 10 per well⁵Effector cells (macrophages) were added per cell/well, i.e. at an effective target ratio of 10: 1. anti-CD 25 antibody was then added at the highest concentration of 1. mu.g/ml, followed by log-serial dilution in duplicate (7 points). Cells and antibodies were incubated at 37 ℃ with 5% CO₂Incubate for 4 hours. To evaluate ADCP, cells were placed on ice, stained with the cell surface marker CD14 (clone MFP9 BD Biosciences, CD 14-PerCP-Cy5.5), and fixed with eBioscience fixing buffer. Carried out using Fortessa LSR X20Two-color flow cytometry analysis. Residual target cells were defined as eFluor450-dye⁺/CD14^-The cell of (1). Macrophage as CD14⁺. Double labeled cells (eFluor 450-dye +/CD14+) are thought to represent phagocytosis of the target by macrophages. Phagocytosis of target cells was calculated using the following equation: % phagocytosis ═ 100 × [ (double positive%)/(double positive% + percent residual target)]. The results are shown in FIG. 20.

Counting:

curve fitting was performed using Prism software (GraphPad) to determine EC50 values and maximal activity.

Human antibodies do not block IL2-CD25 interaction

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

Results：

The 7G7B6 antibody was further evaluated for its ability to not interfere with IL-2 signaling and its ability to kill target cells expressing CD25 against 7G7B6 antibody. In the STAT5 assay, 7G7B6 did not block IL-2 signaling regardless of the IL-2 concentration tested, whereas IL-2 signaling was completely blocked by the reference antibody darlizumab (fig. 17). It has been shown that dallizumab, which blocks the interaction of CD25 with IL-2 by a so-called "Tac" epitope (Queen C et al,1989, PNAS.86(24):10029-10033, andDielekova B,2013, Neurothelitherapeutics, 10(1): 55-67), binds to an epitope different from 7G7B6 (FIGS. 10 and 24B), which may explain why dallizumab blocks IL-2 signaling while 7G7B6 does not block IL-2 signaling in the STAT5 phosphorylation assay (FIG. 17). In addition, dallizumab reduced the effector response of activated T cells, probably because dallizumab blocked IL-2 signaling, while 7G7B6, which did not block IL-2 signaling, had no negative effect on T cell response (fig. 18). Finally, the 7G7B6 chimeric antibody kills CD25 expressing cells, i.e., tumor cells or regulatory T cells, by ADCC (fig. 19) and ADCP (fig. 20) when compared to IgG1 isotype antibody.

In summary, 7G7B6 has been characterized as a chimeric antibody and demonstrated effective killing of CD25 positive cells (tregs or cancer cell lines) and did not interfere with IL-2 signaling and therefore did not inhibit T-effector responses. Thus, 7G7B6 is a Treg depleting antibody that can be used as a monotherapy or in combination to treat cancer.

The MA-251 antibody was further evaluated for its ability to not interfere with IL-2 signaling. MA251 antibody was evaluated in an IL2-CD25 Octet competition assay. Simultaneous IL2 binding and MA251 binding to CD25 was observed (fig. 23), indicating that MA251 binds in a non-competitive manner. In the STAT5 assay, MA-251 did not block IL-2 signaling, regardless of the IL-2 concentration tested, whereas IL-2 signaling was completely blocked by the reference antibody darlizumab (FIG. 22). It has been demonstrated that daclizumab, which blocks the interaction of CD25 with IL-2 by the so-called "Tac" epitope (Queen C et al,1989and Bielekova B,2013), binds to an epitope different from MA-251 (fig. 10 and fig. 24(E)), which may explain why daclizumab blocks IL-2 signaling while MA251 does not block IL-2 signaling in the STAT5 phosphorylation assay (fig. 22).

Example 8: anti-human CD25 Ab competition assay

Antibody competition was performed on a Forte Bio Octet Red96 system (Pall Forte Bio corp., USA) using a standard sandwich packet assay. 26.8nM his-labeled recombinant human CD25 was loaded on the Ni-NTA sensor for 200 seconds. After the baseline step, the kinetic buffer sensor was exposed to 66.6nM of the primary antibody for 600 seconds or 1800 seconds, followed by exposure to the secondary anti-CD 25 antibody (also 66.6nM, 600 seconds or 1800 seconds exposure). Data were processed using Forte Bio data analysis software 9.0. Additional binding of the secondary antibody indicates unoccupied epitopes (no epitope competition), while no binding indicates epitope blocking (epitope competition).

Results

The mabs that were not IL-2 signal blockers (antibody 1 and antibody 3) competed with each other or with 7G7B6 and MA251, while they did not compete with either study dallizumab or study basiliximab (examples (a) to (N), fig. 24). The IL-2 signaling blocker (i.e., TSK031) did compete with study dallizumab and study basiliximab, and did not compete with 7G7B6 (examples (O) to (Q), fig. 24).

Example 9: therapeutic analysis of non-blocking antibodies

On day 0, 200. mu.l of 1X 10 in RPMI1640⁷Individual SU-DHL-1 cells were implanted in the right flank. On day 12, mice with palpable tumors were randomly assigned to treatment with vehicle or with antibody 1 at 2mg/kg twice weekly. On day 15, tumors were sized from 100 to 200mm³Mice in (2) were randomly assigned and dosed with vehicle, 2mg/kg antibody 1 twice weekly or a single dose of 10mg/kg antibody 1.

Results

Antibody 1 administered at 2mg/kg twice weekly prevented the growth of 9/10 mice with palpable tumors (fig. 25(a) - (B)). In the case of tumors of 100 to 200mm in size³Of the mice (2 mg/kg twice weekly and a single dose of 10mg/kg) also prevented tumor growth (FIGS. 25(C) - (E)).

Example 10: affinity measurement of anti-human CD25 antibodies

K of anti-human CD25 antibody was measured by SPR in Biacore 2000 using a CM-5 sensor chip at an ambient experimental temperature of 25 deg.C_DTo determine the affinity of anti-human CD25 antibodies. Initially, anti-human antibodies in assay buffer (pH7.4,10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% Tween 20) across all flow cells fixed to RU 12000 to 14000 for 10 minutes. The ligands (antibody test articles) were sequentially loaded to capture levels between 145 to 190 RU. The analyte (his-labeled recombinant human CD25) was then associated with a two-fold dilution of assay buffer (minimum concentration of 3.13nM) starting at 400nM for 6 minutes. Dissociation was performed in assay buffer within 10 minutes. The regeneration step between sample concentrations was performed in 10mM glycine pH 1.7 for 10 minutes. The whole process was maintained at a flow rate of 25. mu.l/min. FIGS. 26(C) and 26(D) use the global two-state reaction conformational change score provided by Biacore with reference difference setThe analysis software fits the kinetic data. Fig. 26(a), 26(B), and 26(E) use the 1:1 langmuir model with reference difference sets.

ForteBio affinity measurements are typically performed on Octet RED384 as previously described (see, e.g., Estep P et al, 2013.mabs.5(2), 270-8).

Alternatively, K of anti-human CD25 antibody was measured by biolayer interferometry on an Octet Red96 system (Pall Forte Bio Corp., USA)_DTo determine the affinity of anti-human CD25 antibodies. The sensor was equilibrated offline for 10 minutes in kinetic buffer and then monitored online for 60 seconds to establish a baseline. 13.32nM antibody was loaded into the AHC biosensor for 200 seconds, then different concentrations of his-labeled rhCD25(1:3 serial dilution, 50nM to 0.54nM) were added for 600 seconds and allowed to dissociate in kinetic buffer for 400 seconds. The kinetic data were fitted using global 1:1 analysis software supplied by Pall Forte Bio with a reference difference set. The results are shown in FIG. 26 (F).

As a result:

the results are shown in FIG. 26. The Kd values established for the anti-CD 25 antibody in this assay are as follows: for antibody 1, 3.2X 10^-9M; for antibody 3, 3.8X 10^-9M, for daclizumab, 0.61X 10^-9M。

Example 11: characterization of anti-CD 25 antibodies-antibody 1 to antibody 21

Binding of anti-CD 25 antibodies to CD25 expressing cells:

candidates were evaluated by binding to lymphoma human cell lines such as Karpas299, SU-DHL-1 and SR-786 cells, in vitro differentiated Treg cells, activated human or cynomolgus PBMC, HSC-F cynomolgus T cell lines and CHO cells.

Binding to human cell lines expressing CD25 (SU-DHL-1 and SR-786) was detected by: cells were first blocked with trustain (Biolegend) and then incubated with anti-CD 25 antibody titrated in a semilog dilution series from the highest concentration of 20 μ g/ml for 30 minutes at 4 ℃, followed by washing and incubation with PE conjugated anti-human IgG Fc antibody (Biolegend). The cells were washed again and resuspended in FACS buffer containing DAPI and harvested on intellichyt iquee. Live cells were gated during sample collection by flow cytometry using FSC versus SSC parameters. The mean intensity of Geo for stained cells was plotted on an XY plot, plotted against log concentration, and the data was fitted to a non-linear regression curve from which EC50 was calculated.

Binding to CD25 expressing Karpas299 cells and in vitro differentiated tregs was detected by: the test article (anti-CD 25 primary antibody) was stained with 30mg/ml antibody and then half-log serially diluted (7 points) on ice for 30 minutes. Then stained with a 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 Fc gamma fragment- (Jackson ImmunoResearch)) at a concentration of 1mg/ml on ice for 30 minutes. All samples were stained in duplicate. Live cells were gated during sample collection by flow cytometry using FSC versus SSC parameters. Mean Fluorescence Intensity (MFI) of stained cells was plotted on an XY plot, MFI was plotted against log concentration, and the data was fitted to a non-linear regression curve from which EC50 was calculated.

Binding to activated human or cynomolgus PMBC expressing CD25 was detected by: the test article (anti-CD 25 primary antibody) was stained with 20mg/ml antibody and then half-log serially diluted (7 points) on ice for 30 minutes. Then stained with a secondary antibody (rabbit anti-human Fcg F (ab')2- (Jackson ImmunoResearch)) at a concentration of 1mg/ml for 30 minutes on ice. 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 staining cohort of 4 test articles at a time. Live lymphocytes were gated during sample collection by flow cytometry using FSC versus SSC parameters. CD4 to be gated⁺And CD8⁺The Mean Fluorescence Intensity (MFI) of the T cell subsets was plotted on an XY plot, MFI plotted against log concentration, and the data were fitted to a non-linear regression curve from which EC50 was calculated.

Binding to cynomolgus monkey T cell line expressing HSC-F of CD25 was detected by: the test article was stained with 20mg/ml antibody (anti-CD 25 primary antibody) and then half-log serially diluted (7 points) 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 staining cohort of 4 test articles at a time. Live lymphocytes were gated during sample collection by flow cytometry using FSC versus SSC parameters. Mean Fluorescence Intensity (MFI) of live cells was plotted on an XY plot, MFI was plotted against log concentration, and the data was fitted to a non-linear regression curve from which EC50 was calculated.

Binding to CHO cells expressing CD25 was also detected. Approximately 100000 antigen-overexpressing cells were washed with wash buffer and incubated with 100 μ l of 100nM IgG for 15 min at room temperature. Cells were then washed twice with wash buffer and incubated with 100. mu.l of 1:100 human PE on ice for 15 minutes. Cells were then washed twice with wash buffer and analyzed on a facscan II analyzer (BD Biosciences). Unmodified CHO cell lines were also used as negative controls.

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

IL-2 blockade was characterized using a STAT5 phosphorylation assay in which IL-2 signaling was examined. Previously frozen PBMC (stemcell Technologies) were incubated in 96-U bottom well plates for 30 min in the presence of 10U/ml anti-CD 25 antibody, followed by different concentrations of IL-2(PeProtech) of 10U/ml or 0.25U/ml, 0.74U/ml, 2.22U/ml, 6.66U/ml or 20U/ml in RPMI1640 (Life Technologies) containing 10% FBS (Sigma), 2mM L-glutamine (Life Technologies) and 10000U/ml Pen-strep (Sigma). IL-2 induced phosphorylation of STAT5 was stopped when cells were fixed and permeabilized with the eBioscience TMFoxp 3/transcription factor staining buffer kit (Invitrogen) and treated with BD Phosflow Perm buffer III (BDbiosciences). IL-2 induced phosphorylation of STAT5 was stopped when cells were fixed and permeabilized with the eBioscience TMFoxp 3/transcription factor staining buffer set (Invitrogen) and treated with BD Phosflow Perm buffer III (BDbiosciences). The cells were then stained simultaneously with surface and intracellular fluorochrome-labeled antibodies (STAT5-Alexa Fluor 647clone 47/STAT5/pY694 BD Bioscience, CD3-PerCP-Cy5.5 clone UCHT1 biosegend, CD4-BV510 clone SK3 BD Bioscience, CD8-Alexa Fluor 700clone RPA-T8Invitrogen, CD45RA-PE-Cy7clone HI100Invitrogen, FoxP3-Alexa Fluor 488clone236A/E7 Invitrogen) samples were obtained on a Fortessa LSR X20 flow cytometer (BD Bioscience) and analyzed using BD FACSDIVA software. Doublets were excluded using FCS-H vs FCS-A, and lymphocytes were defined using SSC-A vs FCS-A parameters. CD3 was defined using CD3 PerCP-Cy5.5-A vs FCS-A plot⁺T cells, and gates are plotted on a histogram showing the count of comparative STAT5 Alexa Fluor 647-A to determine STAT5⁺CD3⁺A population of T cells. The percentage of blockade of IL-2 signaling was calculated as follows: % blockade 100 × [ (% Stat5 in Ab-free group)⁺These% Stat5 of cell-10 ug/ml Ab group⁺cell)/(Ab-free group% Stat5⁺Cell)]. By different subsets of cells (CD 4)⁺、CD8⁺、CD4⁺FoxP3+, naive and memory T cells) further analysis of STAT5 phosphorylation was also assessed by gating the corresponding subsets and analyzed as described above. Images and statistical analysis were performed using GraphPad Prism v 7.

In vitro T cell activation assay:

the effect of IL-2 signaling on the Teff response was characterized in a T cell activation assay in which intracellular granzyme b (grb) up-regulation and proliferation was examined. Primary human pan-T cells (Stemcell technologies) previously frozen were labeled with eFluor450 cell proliferation dye (Invitrogen) according to manufacturer's recommendations and labeled at 1X 10⁵Cells/well were added to RPMI1640 (Life Technologies) containing 10% FBS (Sigma), 2mM L-glutamine (Life Technologies) and 10000U/ml Pen-strep (Sigma) in 96-U bottom well plates. Then, the cells were treated with 10. mu.g/ml anti-CD 25 antibody or control antibody, followed by human T activator CD3/CD28 (cell to bead ratio 20: 1; Gibco) at 37 ℃ in 5% CO₂Incubate in humidified incubator for 72 hours. To assess T cell activation, cells were stained with eBioscience FixableViabilitydye effector 780(Invitrogen), followed by fluorochrome-labeled antibodies to surface T cell markers (CD3-PerCP-Cy5.5 clone UCHT1Biolegend, CD4-BV510 clone SK3 BD Bioscience, CD8-Alexa Fluor 700clone RPA-T8Invitrogen, CD45RA-PE-Cy7clone HI100Invitrogen, CD25-BUV737 clone 2A3 BD Bioscience), and then stainedCells were fixed and permeabilized with the eBioscience TM Foxp 3/transcription factor staining buffer kit (Invitrogen) and then stained for intracellular GrB and nuclear Foxp3 (granzyme B-PE clone GB11 BD Bioscience, Foxp3-APC clone 236A/E7). Samples were obtained on a Fortessa LSR X20 flow cytometer (BD Bioscience) and analyzed using BD FACSDIVA software. Doublets were excluded using FCS-H vs FCS-A, and lymphocytes were defined using SSC-A vs FCS-A parameters. Samples were obtained on a Fortessa LSR X20 flow cytometer (BD Bioscience) and analyzed using BD FACSDIVA software. Doublets were excluded using FCS-H and FCS-A, and lymphocytes were defined using SSC-A and FCS-A parameters. Evaluation of viable CD3 from GrB-PE-A and proliferative eFluor450-A profiles⁺Lymphocyte-gated CD4⁺And CD8⁺T cell subsets. The results showed that the proliferating GrB-positive cells accounted for all CD4⁺Or CD8⁺Percentage of T cell population. Mapping and statistical analysis was performed using GraphPad Prism v 7.

In vitro ADCC assay:

antibody-dependent cell-mediated cytotoxicity assays (ADCC assays) were performed using SU-DHL-1 or SR-786(CD25 positive) human cell lines as target cells and human NK cells as effector cell sources to characterize anti-human CD25 antibodies. NK cells were isolated from PBMC of healthy donors using NK cell negative isolation kit (Stemcell Technologies). NK cells were cultured overnight in the presence of 2ng/mL IL-2 (Peprotech). In the presence of anti-CD 25 or isotype antibodies, SU-DHL-1 or SR-786 target cells were loaded and plated with calcein-AM (thermolysis), 4 replicates per condition, 5% CO at 37 deg.C₂Incubate for 30 minutes. After incubation, NK cells were added at a target-to-effect (T: E) ratio of 1:10 (10000 target cells and 100000 effector cells) and 5% CO at 37 deg.C₂Incubate for 4 hours. Readings of calcein fluorescence in the supernatant were performed on 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). Dose response curves were generated using Graphpad Prism v7 to generate raw data plots. Plotting percent target cell lysis on an XY plot, normalized% calcein AM Release versusThe log concentration was plotted and the data was fitted to a non-linear regression curve from which EC50 was calculated.

ADDC was also determined in luciferase reporter system assays. SR786 cells expressing CD25 (referred to herein as target (T) cells) were incubated with different concentrations of anti-CD 25mAb (or control IgG) at 37 ℃ for 20 minutes in low IgG FBS supplemented medium (RPMI with 4% FBS). ADCC effector cells (E) were then added to the cell-mAb mixture at an E: T ratio of 1: 1. The effector cells were Jurkat cells stably transfected with the luciferase reporter system and overexpressing CD16/Fc γ riiia (promega). After overnight incubation at 37 ℃, cells were lysed and luciferase activity was measured by hydrolysis of the luminescent release of specific luciferase substrates according to the manufacturer's instructions (Promega Bio-Glow protocol).

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

antibody-dependent cell-mediated phagocytosis (ADCP) assays were performed using in vitro differentiated tregs as target cells and monocyte-derived macrophages as effector cells. PBMC were isolated from white blood cell cones (leucocytes) by Ficoll gradient centrifugation. Monocytes (CD14+ cells) were isolated using CD14 microbeads (Miltenyi Biotec). Monocytes were cultured for 5 days under 50ng/ml M-CSF in RPMI1640 (Life Technologies) containing 10% FBS (Sigma), 2mM L-glutamine (Life Technologies) and 10000U/ml penicillin-streptomycin (Sigma), and 3 days later fresh medium containing M-CSF was added. Human Treg cell differentiation kit (R)&D Systems) to isolate regulatory T cells (tregs). These cells were incubated at 37 ℃ with 5% CO₂Incubate for 5 days in a humidified incubator and label with eFluor450 dye (Invitrogen) according to the manufacturer's recommendations. On day 5, macrophages and tregs labeled with eFluor450 dye were co-cultured at an effective target ratio of 10:1 for 4 hours in the presence of anti-CD 25 antibody or control, as described below. At 1 × 10⁴Target cells (Tregs) were added at 1X 10 per well⁵Effector cells (macrophages) were added per cell/well, i.e. at an effective target ratio of 10: 1. anti-CD 25 antibody was then added at the highest concentration of 1. mu.g/ml, followed by log-serial dilution in duplicate (7 points). Cells and antibodies were combined at 35% CO at 7 ℃₂Incubate for 4 hours. To evaluate ADCP, cells were placed on ice, stained with the cell surface marker CD14 (clone MFP9 BD Biosciences, CD 14-PerCP-Cy5.5), and fixed with eBioscience fixing buffer. Two-color flow cytometry analysis was performed using Fortessa LSR X20. Residual target cells were defined as eFluor450-dye⁺/CD14^-The cell of (1). Macrophage as CD14⁺. Double labeled cells (eFluor 450-dye +/CD14+) are thought to represent phagocytosis of the target by macrophages. Phagocytosis of target cells was calculated using the following equation: % phagocytosis ═ 100 × [ (double positive%)/(double positive% + percent residual target)]。

In vitro ADCP assay using Fc γ RIIa-H Reporter assay

ADCP bioassay Effector cells (Fc. gamma. RIIa-H) were obtained from Promega (Cat # G9881/5; Lot # 0000261099). SUDHL-1 target cells (25. mu.l/well) were plated at 5000 cells/well using 96-well white polystyrene plates (Costar; Cat # 3917). Test antibodies were serially diluted using 3-fold dilutions and 25 μ Ι was added to the cells. Add 50,000. mu.l of effector cells per well in a volume of 25. mu.l to give a ratio of effector cells to target cells of 10: 1. All target cells, antibodies and effector cells were plated using cell culture media. The plates were incubated at 37 ℃ for 18 hours. The plate was then removed from the incubator and kept at room temperature for 20 minutes. Mu.l Bio-Glo luciferase assay substrate buffer was added to each well, followed by incubation for 30 minutes and measurement of luminescence using the GloMax Multi detection System (Promega).

Counting:

EC50 values and maximal activity were determined by curve fitting using Prism software (GraphPad).

Results

Antibody 1 antibodies were evaluated for their ability to not interfere with IL-2 signaling and to kill target cells expressing CD 25. The results of binding to rhCD25 and the IL-2 competitive binding assay are shown in fig. 27 and 28. Ligand binding assays using Octet showed that antibody 1 did not affect the binding of IL-2 to CD25 (fig. 29). This was confirmed in the STAT5 assay, where antibody 1 did not block IL-2 signaling, regardless of the IL-2 concentration tested, whereas IL-2 signaling was completely blocked by the reference antibody dallizumab (fig. 31). It has been demonstrated that dallizumab, which blocks the interaction of CD25 with IL-2 by the so-called "Tac" epitope (Queen C et al,1989and Bielekova B,2013), binds an epitope different from that of antibody 1 (fig. 30), which may explain why dallizumab blocks IL-2 signaling but antibody 1 does not block IL-2 signaling (fig. 31). Furthermore, daclizumab reduced the effector response of activated T cells, probably because it blocked IL-2 signaling, while antibody 1, which did not block IL-2 signaling, had no negative effect on T cell response (fig. 32). Finally, antibody 1 kills CD25 expressing cells, i.e., tumor cells or regulatory T cells, by ADCC (fig. 33) and ADCP (fig. 34) when compared to IgG1 isotype antibody.

In summary, antibody 1 has been characterized and demonstrated effective killing of CD25 positive cells (tregs or cancer cell lines) and did not interfere with IL-2 signaling and therefore did not inhibit T-effector responses. Thus, antibody 1 is a Treg depleting antibody that can be used as a monotherapy or in combination to treat cancer.

Antibody 3 antibodies were evaluated for their ability to not interfere with IL-2 signaling and to kill target cells expressing CD 25. The results of binding to rhCD25 and the IL-2 competitive binding assay are shown in fig. 35 and fig. 36. Ligand binding assays using Octet showed that antibody 3 did not affect the binding of IL-2 to CD25 (fig. 37). This was confirmed in the STAT5 assay, where antibody 3 did not block IL-2 signaling regardless of the IL-2 concentration tested, whereas IL-2 signaling was completely blocked by the reference antibody dallizumab (fig. 39). It has been demonstrated that daclizumab, which blocks the interaction of CD25 with IL-2 by a so-called "Tac" epitope, binds an epitope different from that of antibody 3 (fig. 38), which may explain why daclizumab blocks IL-2 signaling while antibody 3 does not block IL-2 signaling (fig. 39). Furthermore, daclizumab reduced the effector response of activated T cells, probably because it blocked IL-2 signaling, while antibody 3, which did not block IL-2 signaling, had only minimal, if any, effect on T cell responses when compared to the case without antibody or with isotype control (figure 40). Finally, antibody 3 kills CD25 expressing cells, i.e., tumor cells or regulatory T cells, by ADCC (fig. 41) and ADCP (fig. 42) when compared to IgG1 isotype antibodies.

In summary, antibody 3 has been characterized and demonstrated effective killing of CD25 positive cells (tregs or cancer cell lines) and did not interfere with IL-2 signaling and therefore did not inhibit T-effector responses. Thus, antibody 3 is a Treg depleting antibody that can be used as a monotherapy or in combination to treat cancer.

Antibody 4 antibodies were evaluated for their ability to not interfere with IL-2 signaling and to kill target cells expressing CD 25. The results of binding to rhCD25 and the IL-2 competitive binding assay are shown in fig. 43 and fig. 44. Ligand binding assays using Octet showed that antibody 4 did not affect the binding of IL-2 to CD25 (fig. 45). This was confirmed in the STAT5 assay, where antibody 4 did not block IL-2 signaling regardless of the IL-2 concentration tested, whereas IL-2 signaling was completely blocked by the reference antibody dallizumab (fig. 47). It has been demonstrated that daclizumab, which blocks the interaction of CD25 with IL-2 by a so-called "Tac" epitope, binds an epitope different from antibody 4 (fig. 46), which may explain why daclizumab blocks IL-2 signaling while antibody 4 does not block IL-2 signaling (fig. 47). Furthermore, daclizumab reduced the effector response of activated T cells, probably because it blocked IL-2 signaling, while antibody 4, which did not block IL-2 signaling, had no negative effect on T cell response (fig. 48). Finally, antibody 4 killed cells expressing CD25, i.e., tumor cells or regulatory T cells, by ADCC (fig. 49) and ADCP (fig. 50) when compared to IgG1 isotype antibody.

In summary, antibody 4 has been characterized and demonstrated effective killing of CD25 positive cells (tregs or cancer cell lines) and did not interfere with IL-2 signaling and therefore did not inhibit T-effector responses. Thus, antibody 4 is a Treg depleting antibody, which can be used as a monotherapy or in combination to treat cancer.

Antibody 2 antibodies were evaluated for their ability to not interfere with IL-2 signaling and to kill target cells expressing CD 25. The results of binding to rhCD25 and the IL-2 competitive binding assay are shown in fig. 51 and fig. 52. Ligand binding assays using Octet showed that antibody 2 did not affect the binding of IL-2 to CD25 (fig. 53). This was confirmed in the STAT5 assay, where antibody 2 did not block IL-2 signaling regardless of the IL-2 concentration tested, whereas IL-2 signaling was completely blocked by the reference antibody dallizumab (fig. 55). It has been demonstrated that daclizumab, which blocks the interaction of CD25 with IL-2 by a so-called "Tac" epitope, binds an epitope different from that of antibody 2 (fig. 54), which may explain why daclizumab blocks IL-2 signaling while antibody 2 does not block IL-2 signaling (fig. 55). Finally, antibody 2 kills CD25 expressing cells, i.e., tumor cells or regulatory T cells, by ADCC (fig. 56) and ADCP (fig. 57) when compared to IgG1 isotype antibody.

In summary, antibody 2 has been characterized and demonstrated effective killing of CD25 positive cells (tregs or cancer cell lines) and did not interfere with IL-2 signaling and therefore did not inhibit T-effector responses. Thus, antibody 2 is a Treg depleting antibody, which can be used as a monotherapy or in combination to treat cancer.

The antibody 5 antibody is characterized by comprising a heavy chain variable region comprising the following sequence:

EVQLVESGGGLIQPGGSLRLSCAASGFTLDSYGVSWVRQAPGKGLEWVGVTSSGGSAYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRYVYTGGYLYHYGMDLWGQGTLVTVSS(SEQ ID NO:10)；

the light chain variable region comprises the following sequence:

DIQMTQSPSSLSASVGDRVTITCRASQSISDYLAWYQQKPGKVPKLLIYAASTLPFGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQGTYDSSDWYWAFGGGTKVEIK(SEQ ID NO:14)。

as described above, the sequences of the complementarity determining regions (CDRs, i.e., CDR1, CDR2, and CDR3) and the Framework Regions (FRs) are defined according to the Kabat numbering scheme.

Antibody 5 antibodies were evaluated for their ability to not interfere with IL-2 signaling and to kill target cells expressing CD 25. The results of binding to rhCD25 are shown in fig. 58. The STAT5 assay showed that antibody 5 did not block the IL-2 signaling tested, whereas IL-2 signaling was completely blocked by the antibody darlizumab (fig. 59). Competition assays showed that antibody 5 did not compete with the IL-2 signal blocker, daclizumab or basiliximab (fig. 60(a) and (B)), while it did compete with 7G7B6 (non-IL-2 blocker) (fig. 60 (C)). Finally, antibody 5 killed cells expressing CD25, i.e., tumor cells or regulatory T cells, by ADCC (fig. 61) and ADCP (fig. 61) when compared to anti-human CD25 Fc silencing control antibodies.

In summary, antibody 5 has been characterized and demonstrated effective killing of CD25 positive cells (tregs or cancer cell lines) and did not interfere with IL-2 signaling and therefore did not inhibit T-effector responses. Thus, antibody 5 is a Treg depleting antibody that can be used as a monotherapy or in combination to treat cancer.

Antibody 6, antibody 7, antibody 8 and antibody 9 the antibodies are characterized by comprising the following sequences:

antibody 6 comprises a heavy chain variable region comprising the sequence:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRDYGNSYYYALDYWGQGTLVTVSS(SEQ ID NO:23)；

the light chain variable region comprises the following sequence:

EIVLTQSPGTLSLSPGERATLSCRASSSVSFMHWLQQKPGQAPRPLIYATSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCQQWSSNPPAFGQGTKLEIK(SEQ ID NO:25)。

antibody 7 comprises a heavy chain variable region comprising the sequence:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRDYGNSYYYALDYWGQGTLVTVSS(SEQ ID NO:23)；

the light chain variable region comprises the following sequence:

QIVLTQSPGTLSLSPGERATLSCRASSSVSFMHWLQQKPGQSPRPLIYATSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCQQWSSNPPAFGQGTKLEIK(SEQ ID NO:26)。

antibody 8 comprises a heavy chain variable region comprising the sequence:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYFCARDRDYGNSYYYALDYWGQGTLVTVSS(SEQ ID NO:24)；

the light chain variable region comprises the following sequence:

EIVLTQSPGTLSLSPGERATLSCRASSSVSFMHWLQQKPGQAPRPLIYATSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCQQWSSNPPAFGQGTKLEIK(SEQ ID NO:25)。

antibody 9 comprises a heavy chain variable region comprising the sequence:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYFCARDRDYGNSYYYALDYWGQGTLVTVSS(SEQ ID NO:24)；

the light chain variable region comprises the following sequence:

QIVLTQSPGTLSLSPGERATLSCRASSSVSFMHWLQQKPGQSPRPLIYATSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCQQWSSNPPAFGQGTKLEIK(SEQ ID NO:26)。

as described above, the sequences of the complementarity determining regions (CDRs, i.e., CDR1, CDR2, and CDR3) and the Framework Regions (FRs) are defined according to the Kabat numbering scheme.

The results of epitope mapping showed that antibody 6, antibody 7, antibody 8 and antibody 9 bound to human CD25 in the region of amino acids 150 to 163 (YQCVQGYRALHRGP) and 166 to 180 (SVCKMTHGKTRWTQP) of SEQ ID NO:1 and to the human CD25 extracellular protein sequence at 10^-8M to 10^-10The Kd value of M binds.

Antibody 6, antibody 7, antibody 8 and antibody 9 were evaluated for their ability to not interfere with IL-2 signaling and to kill target cells expressing CD 25. The results of binding to rhCD25 are shown in fig. 63. The STAT5 assay showed that the antibody did not block the IL-2 signaling tested, whereas IL-2 signaling was completely blocked by the antibody darlizumab (fig. 65). Competition assays showed that antibody 7 did not compete with IL-2 signal blocker daclizumab or basiliximab (fig. 64(a) and (B)). Finally, antibody 7 killed cells expressing CD25, i.e., tumor cells or regulatory T cells, by ADCC (fig. 66) and ADCP (fig. 67) when compared to anti-human CD25 Fc silencing control antibodies.

In summary, antibody 6, antibody 7, antibody 8 and antibody 9 have been characterized and demonstrated effective killing of CD25 positive cells (tregs or cancer cell lines) and did not interfere with IL-2 signaling and therefore did not inhibit T-effector responses. Thus, the antibody is a Treg depleting antibody, which can be used for monotherapy or in combination for the treatment of cancer.

Antibody 10, antibody 11, antibody 12, antibody 13, antibody 14, antibody 15, antibody 16, antibody 17, antibody 18, antibody 19, antibody 20, antibody 21, the antibody being characterized in that it comprises a heavy chain variable region comprising the following sequence:

as described above, the sequences of the complementarity determining regions (CDRs, i.e., CDR1, CDR2, and CDR3) and the Framework Regions (FRs) are defined according to the Kabat numbering scheme.

The results of epitope mapping showed that antibody 10, antibody 11, antibody 12, antibody 13, antibody 14, antibody 15, antibody 16, antibody 17, antibody 18, antibody 19, antibody 20, antibody 21 bound to human CD25 in the region of amino acids 150 to 163 (YQCVQGYRALHRGP) and 166 to 180 (SVCKMTHGKTRWTQP) of SEQ ID NO:1 and bound to human CD25 extracellular protein sequence at 10^-8M to 10^-10The Kd value of M binds.

Antibodies were evaluated for their ability to not interfere with IL-2 signaling and to kill target cells expressing CD 25. The results of binding to rhCD25 are shown in fig. 68. The STAT5 assay showed that the antibody did not block the IL-2 signaling tested, whereas the IL-2 signaling was completely blocked by the antibody darlizumab (fig. 70). Competition assays showed that antibody 19 did not compete with IL-2 signal blocker daclizumab or basiliximab (fig. 69(a) and (B)). Finally, antibody 12, antibody 19 and antibody 20 kill CD25 expressing cells, tumor cells or regulatory T cells by ADCC (figure 71) and ADCP (figures 72 and 73) when compared to an anti-human CD25 Fc silencing control antibody.

In summary, antibody 10, antibody 11, antibody 12, antibody 13, antibody 14, antibody 15, antibody 16, antibody 17, antibody 18, antibody 19, antibody 20, antibody 21 have been characterized and demonstrated effective killing of CD25 positive cells (tregs or cancer cell lines) and did not interfere with IL-2 signaling and therefore did not inhibit T-effect responses. Thus, the antibody is a Treg depleting antibody, which can be used as a monotherapy or in combination to treat cancer.

Example 12: treatment analysis in combination with cancer vaccines

The therapeutic activity of the non-IL-2 blocking anti-CD 25 antibody 7D4 mouse IgG2a in combination with GVAX in a BVAB16 immunotherapy-resistant mouse model was determined. On day 0, 50X 10 intradermal implantation³And B16BL6 cells. On day 5, 200 μ g of non-IL-2 blocking anti-CD 25 antibody was administered intraperitoneally, with or without administration. On days 6, 9and 12, mice were treated with 1X 10⁶Irradiated (150Gy) B16B16 cells were adjuvanted with GM-CSF (GVAX) treatment or no treatment. Tumor growth and mouse survival were monitored until day 33. The results are shown in FIG. 74.

A synergistic effect of the combination of GVAX and 7D4 non-blocking anti-CD 25 antibodies was observed in the B16B16 model. Thus, administration of 7D4 with cancer vaccine enhanced the vaccine-induced anti-tumor response. These results indicate that non-IL 2 blocking depleting anti-CD 25 antibodies can be used in combination with cancer vaccines for the treatment of human cancer. In addition, the data show that non-IL 2 blocking depleting antibodies are able to enhance vaccine-induced immune responses, with broader applications than cancer.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in connection with certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology, cellular immunology or related fields are intended to be within the scope of the following claims.

Claims (63)

1. A method of treating a human subject having cancer, the method comprising the step of administering an anti-CD 25 antibody to the subject, wherein the subject has a solid tumor, wherein the antibody does not inhibit binding of interleukin-2 (IL-2) to CD 25.

2. The method of claim 1, wherein the anti-CD 25 antibody competes with antibody 7G7B6 for binding to human CD 25; and/or competes with antibody MA251 for binding to human CD 25.

3. The method of claim 1 or 2, wherein the anti-CD 25 antibody binds to the same epitope recognized by antibody 7G7B6 and/or an epitope recognized by antibody MA 251.

4. The method of any one of claims 1 to 3, wherein the anti-CD 25 antibody specifically binds to an epitope of human CD25, wherein the epitope comprises one or more amino acid residues comprised in one or more amino acid segments selected from the group consisting of amino acid 150 to 163 (YQCVQGYRALHRGP) of SEQ ID NO:1, amino acid 166 to 186 (SVCKMTHGKTRWTQPQLICTG) of SEQ ID NO:1, amino acid 42 to 56 (KEGTMLNCECKRGFR) of SEQ ID NO:1, amino acid 70 to 88 (NSSHSSWDNQCQCTSSATR) of SEQ ID NO: 1.

5. The method of claim 4, wherein the anti-CD 25 antibody specifically binds to an epitope of human CD25, wherein the epitope comprises at least one sequence selected from the group consisting of: amino acids 150 to 158 of SEQ ID NO:1 (YQCVQGYRA), amino acids 176 to 180 of SEQ ID NO:1 (RQTQP), 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 method of claim 4, wherein the anti-CD 25 antibody specifically binds to an epitope of human CD25, wherein the epitope comprises at least one sequence selected from the group consisting of: 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 method of claim 4, wherein the anti-CD 25 antibody specifically binds to an epitope of human CD25, wherein the epitope comprises at least one sequence selected from the group consisting of: 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 method of claim 4, wherein the anti-CD 25 antibody binds to an epitope comprising the sequence of amino acids 42 to 56 (KEGTMLNCECKRGFR) of SEQ ID NO: 1.

9. The method of claim 4, wherein the anti-CD 25 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 (YQCVQGYRALH) of SEQ ID NO: 1.

10. The method of claim 4, wherein the anti-CD 25 antibody binds to an epitope comprising the sequence of amino acids 42 to 56 of SEQ ID NO:1(KEGTMLNCECKRGFR) and amino acids 74 to 84 of SEQ ID NO:1 (SSWDNQCQCTSSATR).

11. The method of claim 4, wherein the anti-CD 25 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 84 of SEQ ID NO:1 (SSWDNQCQCTSSATR).

12. The method of claim 4, wherein the anti-CD 25 antibody binds to an epitope comprising the sequence of amino acids 150 to 158 (YQCVQGYRA) and 176 to 180 (RWTQP) of SEQ ID NO 1.

13. The method of claim 4, wherein the anti-CD 25 antibody binds to an epitope comprising the sequence of amino acids 150 to 158 of SEQ ID NO:1 (YQCVQGYRA) and amino acids 176 to 186 of SEQ ID NO:1 (RWTQPQLICTG).

14. The method of claim 4, wherein the anti-CD 25 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 method of claim 4, wherein the anti-CD 25 antibody binds to an epitope comprising the sequence of amino acids 74 to 84 (SSWDNQCQCTSSATR) of SEQ ID NO: 1.

16. The method of claim 4, wherein the anti-CD 25 antibody binds to an epitope comprising the sequence of amino acids 70 to 84 (NSSHSSWDNQCQCTS) of SEQ ID NO: 1.

17. The method of claim 4, wherein the anti-CD 25 antibody binds to an epitope comprising the sequence of amino acids 176 to 180 (RWDQP) of SEQ ID NO. 1.

18. The method of claim 4, wherein the anti-CD 25 antibody binds to an epitope comprising the sequence of amino acids 166 to 180 (SVCKMTHGKTRWTQP) of SEQ ID NO: 1.

19. The method of claim 4, wherein the anti-CD 25 antibody binds to an epitope comprising the sequence of amino acids 176 to 186 of SEQ ID NO. 1 (RWTQPQLICTG).

20. The method of any one of claims 1 to 19, wherein said anti-CD 25 antibody is an IgG1 antibody that binds with high affinity to at least one activating fcgamma receptor selected from fcyri, fcyriic, and/or fcyriiia and depletes tumor infiltrating regulatory T cells.

21. The method of any one of claims 1 to 19, wherein the anti-CD 25 antibody:

(a) binds to Fc γ receptor with an activation inhibition rate (a/I) superior to 1; and/or

(b) Binds Fc γ RIIa with a higher affinity than to Fc γ RI, Fc γ RIIc and/or Fc γ RIIb.

22. The method of any one of claims 1 to 21, wherein the antibody is selected from the group consisting of:

(a) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO. 3 and a light chain comprising the amino acid sequence of SEQ ID NO. 4;

(b) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO. 5 and a light chain comprising the amino acid sequence of SEQ ID NO. 6;

(c) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO. 10 and a light chain comprising the amino acid sequence of SEQ ID NO. 14;

(d) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO. 18 and a light chain comprising the amino acid sequence of SEQ ID NO. 22;

(d) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO. 23 and a light chain comprising the amino acid sequence of SEQ ID NO. 25;

(e) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO. 23 and a light chain comprising the amino acid sequence of SEQ ID NO. 26;

(f) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO. 24 and a light chain comprising the amino acid sequence of SEQ ID NO. 25;

(g) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO. 24 and a light chain comprising the amino acid sequence of SEQ ID NO. 26;

(h) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO. 27 and a light chain comprising the amino acid sequence of SEQ ID NO. 30;

(i) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO. 27 and a light chain comprising the amino acid sequence of SEQ ID NO. 31;

(j) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO 27 and a light chain comprising the amino acid sequence of SEQ ID NO 32;

(k) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO. 27 and a light chain comprising the amino acid sequence of SEQ ID NO. 33;

(1) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO 28 and a light chain comprising the amino acid sequence of SEQ ID NO 30;

(m) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:28 and a light chain comprising the amino acid sequence of SEQ ID NO: 31;

(n) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:28 and a light chain comprising the amino acid sequence of SEQ ID NO: 32;

(o) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:28 and a light chain comprising the amino acid sequence of SEQ ID NO: 33;

(p) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:29 and a light chain comprising the amino acid sequence of SEQ ID NO: 30;

(q) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:29 and a light chain comprising the amino acid sequence of SEQ ID NO: 31;

(r) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:29 and a light chain comprising the amino acid sequence of SEQ ID NO: 32; and

(s) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:29 and a light chain comprising the amino acid sequence of SEQ ID NO: 33.

23. The method of any one of claims 1 to 22, wherein the anti-CD 25 antibody is a human IgG2 antibody.

24. The method of any one of claims 1 to 23, wherein the anti-CD 25 antibody has a dissociation constant (Kd) for CD25 of less than 10^-7M。

25. The method of any one of claims 1 to 24, wherein said anti-CD 25 antibody inhibits IL-2 signaling by less than 50%.

26. The method of any one of claims 1 to 25, wherein the anti-CD 25 antibody is a monoclonal antibody.

27. The method of any one of claims 1 to 26, wherein the anti-CD 25 antibody is a human antibody, a chimeric antibody, or a humanized antibody.

28. The method of any one of claims 1 to 27, wherein the antibody is an affinity matured variant of the antibody.

29. The method of any one of claims 1 to 28, wherein the antibody is a humanized variant and/or an affinity matured variant of 7G7B6 or MA 251.

30. The method according to any one of claims 1 to 29, wherein the anti-CD 25 antibody elicits an enhanced CDC, ADCC and/or ADCP reaction, preferably an increased ADCC and/or ADCP reaction, more preferably an increased ADCC reaction.

31. The method of any one of claims 1 to 30, wherein the anti-CD 25 antibody is administered to a subject having an established tumor.

32. The method of any one of claims 1 to 31, further comprising the step of identifying a subject having a solid tumor.

33. The method of any one of claims 1 to 32, further comprising administering an immune checkpoint inhibitor to the subject.

34. The method of claim 33, wherein the immune checkpoint inhibitor is a PD-1 antagonist.

35. The method of claim 34, wherein the PD-1 antagonist is an anti-PD-1 antibody or an anti-PD-L1 antibody.

36. The method of any one of claims 1 to 32, further comprising administering a cancer vaccine.

37. The method of claim 36, wherein the cancer vaccine is a GVAX cancer vaccine.

38. An anti-CD 25 antibody as defined in any one of claims 1 to 30.

39. The anti-CD 25 antibody according to claim 38, wherein the anti-CD 25 antibody is for use in medicine.

40. Use of an anti-CD 25 antibody as defined in any one of claims 1 to 30 for the treatment of cancer in a human subject, wherein the subject has a solid tumor.

41. Use of an anti-CD 25 antibody as defined in any one of claims 1 to 30 in the manufacture of a medicament for the treatment of cancer in a human subject, wherein the subject has a solid tumor.

42. The anti-CD 25 antibody according to claim 40 or the anti-CD 25 antibody according to claim 41, for use in combination with an additional therapeutic agent.

43. The anti-CD 25 antibody according to claim 42 or the use of an anti-CD 25 antibody according to claim 42, wherein the additional therapeutic agent is an immune checkpoint inhibitor.

44. The anti-CD 25 antibody according to claim 43 or the use according to claim 43, wherein the immune checkpoint inhibitor is a PD-1 antagonist.

45. The anti-CD 25 antibody according to claim 42 or the anti-CD 25 antibody according to claim 41, for use wherein the additional therapeutic agent is a cancer vaccine.

46. A combination of an anti-CD 25 antibody as defined in any one of claims 1 to 30 and a further therapeutic agent for use in the treatment of cancer in a human subject, wherein the subject has a solid tumor and the anti-CD 25 antibody and the further therapeutic agent are administered simultaneously, separately or sequentially.

47. A kit for the treatment of cancer comprising an anti-CD 25 antibody as defined in any one of claims 1 to 30 and an additional therapeutic agent.

48. A pharmaceutical composition comprising an anti-CD 25 antibody as defined in any one of claims 1 to 30 in a pharmaceutically acceptable medium.

49. The pharmaceutical composition of claim 48, further comprising an additional therapeutic agent.

50. The combination according to claim 46, kit according to claim 47, or pharmaceutical composition according to claim 49, wherein the additional therapeutic agent is an immune checkpoint inhibitor.

51. The combination according to claim 46, kit according to claim 47, or pharmaceutical composition according to claim 49, wherein the immune checkpoint inhibitor is a PD-1 antagonist.

52. The combination according to claim 46, kit according to claim 47, or pharmaceutical composition according to claim 49, wherein the additional therapeutic agent is a cancer vaccine.

53. A bispecific antibody comprising:

(a) a first antigen-binding moiety that binds to CD 25; and

(b) a second antigen-binding moiety that binds to an immune checkpoint protein;

wherein the CD25 binding moiety does not inhibit binding of interleukin-2 (IL-2) to CD25, and preferably the bispecific antibody is an IgG1 antibody that binds with high affinity to at least one activating Fc γ receptor selected from Fc γ RI, Fc γ RIIa, Fc γ RIII and depletes tumor infiltrating regulatory T cells.

54. The bispecific antibody of claim 53, wherein the immune checkpoint protein is 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.

55. The bispecific antibody of claim 53 or 54, wherein said immune checkpoint protein is expressed on a tumor cell.

56. The bispecific antibody of claim 53 or 54, wherein the immune checkpoint protein is PD-L1.

57. The bispecific antibody of claim 53, wherein the second antigen-binding moiety that binds PD-L1 is comprised in atezumab.

58. A method of treating cancer comprising the step of administering to a subject a bispecific antibody as defined in any one of claims 53 to 57.

59. The method of claim 57, wherein the subject has a solid tumor.

60. A bispecific antibody as defined in any one of claims 53 to 57 for use in the treatment of cancer in a subject.

61. The bispecific antibody of claim 60, wherein the subject has a solid tumor.

62. A method of depleting regulatory T cells in a subject comprising the step of administering an anti-CD 25 antibody to the subject, wherein the antibody is as defined in any one of claims 1 to 30.

63. The method of claim 62, wherein the subject has a solid tumor.