Antibodies for the Treatment of AML

Abstract

The present invention relates to anti-CD25 antibodies for use in the treatment of acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL).

Description

[Technical field]

[0001] The present invention relates to anti-CD25 antibodies for use in the treatment of acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL). [Background technology]

[0002] CD25, also known as the alpha subunit of the interleukin-2 receptor (IL2RA), is a surface antigen that allows the binding of IL-2 with high affinity and the subsequent signaling cascade. CD25 is constitutively expressed on regulatory T cells (Tregs) that rely on IL-2 consumption for proliferation and is transiently upregulated on recently activated T cells. IL-2 is a major cytokine that plays a key role in the clonal expansion of antigen-specific T cells and the acquisition of their effector functions. The abundance of intratumoral Tregs, particularly the ratio of Tregs to effector T cells (Teff), has been shown to predict clinical outcomes in many solid tumors in humans (Non-Patent Document 1, Non-Patent Document 2). In fact, Tregs contribute to the immunosuppressive tumor microenvironment, and several strategies to deplete them have been evaluated.

[0003] CD25 Mab (RG6292) is a non-IL-2 inhibitory defucosylated IgG1 antibody that has been shown to efficiently deplete Tregs in human tumor explants and preclinical mouse models of cancer while allowing IL-2 redistribution to Teffs and the formation of antitumor adaptive immune responses (Non-Patent Document 3). CD25 Mab binds to CD25+ target cells and its crystallizable fragment (Fc) binds to Fc receptors expressed on the surface of effector cells such as natural killer (NK) cells, monocytes and macrophages. CD25 Mab mediates target cell killing through antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). CD25 Mab is currently being investigated in phase I monotherapy studies and in phase Ib clinical trials in combination with atezolizumab (anti-PD-L1 antibody).

[0004] In addition to its role in healthy T cells, expression of CD25 has also been described in many hematological malignancies, such as T-cell and B-cell lymphomas, as well as acute myeloid leukemia (AML) (Non-Patent Document 4). In particular, CD25 appears to be expressed on a subset of tumor cells in AML and diffuse large B-cell lymphoma (DLBCL), and its expression has been associated with reduced survival (Non-Patent Document 5, Non-Patent Document 6).

[0005] Moreover, there is some evidence that CD25 may be restricted to leukemic stem cells or cells with a progenitor phenotype in AML (Non-Patent Document 7, Non-Patent Document 8). These cells are thought to be involved in the spread of the disease and the high relapse rate despite recent advances in the treatment of AML (Non-Patent Document 9). Tregs were observed at a higher frequency in the bone marrow of AML patients compared to that of healthy volunteers, and the abundance of Tregs correlated with poor outcome in AML patients (Non-Patent Document 10). Therefore, there is a need for further treatment options for AML and DLBCL.

[0006] Current treatment options for AML include anthracycline-cytarabine regimens (such as daunorubicin and cytarabine), FLT3 inhibitors (e.g., gilteritinib, midostaurin, sorafenib), BCL-2 inhibitors (e.g., venetoclax), IDH inhibitors (e.g., enasidenib, ivosidenib), hypomethylating agents (e.g., azacitidine, decitabine) and antibody therapies (e.g., the CD33 antibody gemtuzumab ozogamicin), as well as combinations thereof.

[0007] The inventors have now discovered that anti-CD25 antibodies, such as CD25 Mab (RG6292), potentially have a dual mechanism of action, depleting suppressive Tregs as well as having a direct cytotoxic effect on CD25+ malignant cells, resulting in an antibody that may be an effective treatment for AML and DLBLC. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Nishikawa and Sakaguchi, 2014, Curr Opin Immunol 27, 1-7 [Non-Patent Document 2] Wing et al., 2019, Immunity 50, 302-316 [Non-Patent Document 3] Solomon et al., 2020, Nature Cancer, 1(12):1153-1166 [Non-Patent Document 4] Flynn and Hartley, 2017, Br J Haematol 179, 20-35 [Non-Patent Document 5] Fujiwara et al., 2013, Hematology 18, 14-19 [Non-Patent Document 6] Gonen et al., 2012, Blood 120, 2297-2306 [Non-Patent Document 7] Aref et al., 2020, Leuk Res Rep 13, 100203 [Non-Patent Document 8] Kageyama Y et al., 2018, PLOS One 13(12) e.0209295 [Non-Patent Document 9] Kantarjian et al., 2021, Blood Cancer J 11, 41 [Non-Patent Document 10] Dong et al., 2020, Front Immunol 11, 1710 Summary of the Invention

[0009] The present invention provides anti-CD25 antibodies for use in the treatment of acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL).

[0010] In a first aspect of the invention there is provided an anti-CD25 antibody for use in the treatment of acute myeloid leukemia or diffuse large B-cell lymphoma.

[0011] A second aspect of the invention provides an anti-CD25 antibody for use in the treatment of acute myeloid leukemia or diffuse large B-cell lymphoma, administered alone or in combination with one or more further therapeutic agents, wherein the anti-CD25 antibody and the further therapeutic agent are for separate, simultaneous or sequential administration.

[0012] A third aspect of the invention provides a combination of an anti-CD25 antibody and a further therapeutic agent for use in the treatment of acute myeloid leukemia or diffuse large B-cell lymphoma, wherein the anti-CD25 antibody and the further therapeutic agent are for separate, simultaneous or sequential administration.

[0013] A fourth aspect of the invention provides a method of treating acute myeloid leukemia or diffuse large B-cell lymphoma in a subject, comprising administering to the subject an effective amount of an anti-CD25 antibody.

[0014] A fifth aspect of the invention provides the use of an anti-CD25 antibody in the manufacture of a medicament for the treatment of acute myeloid leukemia or diffuse large B-cell lymphoma.

[0015] A sixth aspect of the invention provides the use of an anti-CD25 antibody and a further therapeutic agent in the manufacture of a medicament for the treatment of acute myeloid leukemia or diffuse large B-cell lymphoma.

[0016] A seventh aspect of the invention provides a method of selecting a patient suffering from acute myeloid leukemia for treatment with an anti-CD25 antibody, comprising measuring the level of CD25 expression in target cells in a sample from the patient, wherein if the cells have an expression level of more than about 900 CD25 molecules per cell then the patient is suitable for treatment with the antibody.

[0017] An eighth aspect of the invention provides a method for selecting a patient suffering from AML for treatment with an anti-CD25 antibody, the method comprising determining the presence or absence of a FLT3-ITD mutation in a sample from the patient, wherein if the mutation is present in the sample, the patient is suitable for treatment with the antibody.

[0018] A ninth aspect of the invention provides a method for predicting the response of an AML patient to treatment with an anti-CD25 antibody, comprising determining the presence or absence of an FLT3-ITD mutation in a sample from the patient, the presence of said mutation in said sample being indicative of the patient being likely to respond to treatment with an anti-CD25 antibody.

[0019] A tenth aspect of the invention provides a method of treating AML in a subject, comprising administering to the subject an effective amount of an anti-CD25 antibody, wherein the subject comprises the presence of a FLT3-ITD mutation.

[0020] An eleventh aspect of the invention provides a method of preventing or reducing the risk of relapse in an AML patient, comprising administering to the patient an anti-CD25 antibody.

[0021] A twelfth aspect of the invention provides a method of treating AML in a patient receiving combination therapy with a BCL-2 inhibitor and a hypomethylating agent, comprising administering to the patient an anti-CD25 antibody. [Brief description of the drawings]

[0022] [Figure 1]Figure 1 shows the expression of CD25 on target cells. The density of CD25 molecules on the surface of four target cells (iTreg, Pfeiffer, EOL-1 and AML-22) was determined using BD quantibrite™ beads after 17 hours of incubation in ADCC assay medium. The CD25 density of iTreg and Pfeiffer cells is shown on the left Y-axis, while that of EOL-1 and AML-22 is shown on the right Y-axis. The limit of detection (LOD) represents the minimum number of PE molecules present on the BD quantibrite™ beads, below which a linear relationship between the number of PE molecules per cell and the median fluorescence intensity (MFI) is not guaranteed. [Diagram 2] Figure 1 shows the ADCC potential and CD25 density on target cells induced by CD25 Mab treatment. (A) The ADCC activity of the tested compounds (CD25 Mab or isotype control antibody) is shown as the degree of killing. Calculations are based on the number of target cell events normalized to the number of target cells in the absence of effector NK cells and compound. Flow cytometry analysis was performed 17 hours after the start of the ADCC assay. (B) The density of CD25 molecules on the surface of the remaining viable target cells (EOL-1 and AML-22) is shown, determined 17 hours after the start of the ADCC assay using BD quantibrite™ beads. The limit of detection (LOD) represents the minimum number of PE molecules present on the BD quantibrite™ beads, below which a linear relationship between the number of PE molecules per cell and the median fluorescence intensity (MFI) is not guaranteed. [Diagram 3] Figure 1 shows CD16 expression on NK cells: (A) Percentage of NK cells expressing CD16 after 17 hours of co-incubation with EOL-1 or AML-22 cells and CD25 Mab or isotype control antibody; (B) Percentage of NK cells expressing CD16 after 17 hours of co-incubation with Pfeiffer cells or iTreg and CD25 Mab or isotype control antibody. [Figure 4]Figure 1 shows CD69 expression on NK cells: (A) Percentage of NK cells expressing CD69 after 17 hours of co-incubation with EOL-1 or AML-22 cells and CD25 Mab or isotype control antibody; (B) Percentage of NK cells expressing CD69 after 17 hours of co-incubation with Pfeiffer cells or iTreg and CD25 Mab or isotype control antibody. [Diagram 5] Figure 1 shows CD25 expression on NK cells: (A) Percentage of NK cells expressing CD25 after 17 hours of co-incubation with EOL-1 or AML-22 cells and CD25 Mab or isotype control antibody; (B) Percentage of NK cells expressing CD25 after 17 hours of co-incubation with Pfeiffer cells or iTreg and CD25 Mab or isotype control antibody. [Figure 6]Figure 1 shows the correlation between ADCC activity and CD25 density. (A) ADCC activity of the tested compounds (CD25 Mab or isotype control antibody) is shown as the relative luminescence count normalized to the maximum signal obtained for each cell line and NK cell donor pair. Results are obtained from two independent experiments with NK cells isolated from a total of four blood donors. Cytotoxicity assessment was performed by CytoTox-Glo™ luminescence reading 16-20 hours after the start of the ADCC assay. (B) EC50 values ​​derived from the ADCC assay by titration of CD25 Mab using CytoTox-Glo™ or flow cytometry as readout (three independent experiments with two NK cell donors each). EC50 values ​​were calculated using Prism 8 (GraphPad software) and its built-in nonlinear regression curve fit (log(agonist) vs. response, variable slope, 4 parameters). (C) The density of CD25 molecules on the surface of viable target cells (Pfeiffer, EOL-1 and AML-22) was determined using BD quantibrite™ beads 16-20 hours after the start of the ADCC assay. The limit of detection (LOD) represents the minimum number of PE molecules present on the BD quantibrite™ beads, below which a linear relationship between the number of PE molecules per cell and the median fluorescence intensity (MFI) is not guaranteed. Results were obtained from three independent experiments. [Figure 7]Figure 2: Killing activity of CD25+ AML cells and Tregs. (A) Killing activity of tested compounds (CD25 Mab or isotype control antibody) at 10 μg / ml is shown as the degree of killing of CD25+ AML cells. A positive control EOL-1 cell line and four AML patient samples were used as target cells. Flow cytometric analysis was performed 20 hours after the start of the ADCC assay. Mean ± SEM represents the results obtained with two NK cell donors and technical replicates. Multiple t-test, Holm-Sidak correction for multiple comparisons. (B): Killing activity of Tregs as above for (A). Mean ± SEM represents the results obtained with AML Tregs (n=2) or healthy BM Tregs (n=2). Multiple t-test, Holm-Sidak correction for multiple comparisons. Significance levels are indicated as follows: ns, not significant = P>0.05; *, P≦0.05; **, P≦0.01; ***, P≦0.001; ****, P≦0.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention provides anti-CD25 antibodies for use in treating acute myeloid leukemia (AML) or diffuse large B-cell lymphoma (DLBCL) in a subject, and methods of treating acute myeloid leukemia or diffuse large B-cell lymphoma in a subject with anti-CD25 antibodies.

[0024] The inventors have found that anti-CD25 antibodies can deplete CD25+ malignant cells with a wide range of CD25 expression levels. The inventors have shown direct killing of CD25+ AML and DLBCL cells, particularly CD25+ AML blast cells, by anti-CD25 antibodies. In particular, the inventors have found that anti-CD25 antibodies are effective in killing AML-associated tumor cells, despite the low CD25 expression levels in these tumor cells. The inventors have found that anti-CD25 antibodies can be used to target cancer cells with low CD25 expression, and are therefore also suitable for use in the treatment of cancers such as AML, where blast cells may have low CD25 expression levels.

[0025] CD25 is the alpha chain of the IL-2 receptor and is present on activated T cells, regulatory T cells, activated B cells, some NK T cells, some thymocytes, myeloid progenitor cells and oligodendrocytes. Low CD25 expression can also be seen on blast cells from AML patients. CD25 binds to CD122 and CD132 to form a heterotrimeric complex that acts as a high affinity receptor for IL-2. The consensus sequence of human CD25 is shown below and identified as SEQ ID NO:1 (Uniprot Accession No. P01589; the extracellular domain of mature human CD25 corresponding to amino acids 22-240 is underlined):

[0026] 10 20 30 40 50 MDSYLLMWGL LTFIMVPGCQ A ELCDDDPPE IPHATFKAMA YKEGTMLNCE 60 70 80 90 100 CKRGFRRIKS GSLYMLCTGN SSHSSWDNQC QCTSSATRNT TKQVTPQPEE 110 120 130 140 150 QKERKTTEMQ SPMQPVDQAS LPGHCREPPP WENEATERIY HFVVGQMVYY 160 170 180 190 200 QCVQGYRALH RGPAESVCKM THGKTRWTQP QLICTGEMET SQFPGEEKPQ 210 220 230 240 250 ASPEGRPESE TSCLVTTTDF QIQTEMAATM ETSIFTTEYQ VAVAGCVFLL 260 270 ISVLLLSGLT WQRRQRKSRR TI

[0027] As used herein, an "anti-CD25 antibody" or an "antibody that binds to CD25" refers to an antibody that can bind to the CD25 subunit of the IL-2 receptor, which is also known as the α subunit of the IL-2 receptor.

[0028] An anti-CD25 antibody is an antibody that can specifically bind to the CD25 subunit (antigen) of the IL-2 receptor. "Specific binding" and "bind specifically", and "specifically bind" refer to an antibody that binds to an antigen of interest with a specific binding affinity of about 10-6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10 -13 It is understood to mean having a dissociation constant (Kd) of less than 10 M. In a preferred embodiment, the dissociation constant is less than 10 -8 Less than M, for example 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10 -13 The range is M.

[0029] Anti-CD25 antibodies suitable for use in the present invention include, for example, those described in WO 2017 / 174331, WO 2018 / 167104, WO 2019 / 008386, WO 2019 / 175215, WO 2019 / 175216, WO 2019 / 175217, WO 2019 / 175220, WO 2019 / 17522, WO 2019 / 175223, WO 2019 / 175224, and WO 2019 / 175226, the contents of which are incorporated herein by reference.

[0030] As used herein, the term "antibody" refers to both intact immunoglobulin molecules and to fragments thereof that contain an antigen-binding site, including polyclonal, monoclonal, genetically engineered, or otherwise modified forms of antibodies, including, but not limited to, chimeric, humanized, heteroconjugate, and / or multispecific antibodies (e.g., bispecific antibodies, diabodies, tribodies, and tetrabodies), and antigen-binding fragments of antibodies including, for example, Fab', F(ab')2, Fab, Fv, rIgG, polypeptide-Fc fusions, single chain variants (scFv fragments, VHHs, Trans-bodies™, Affibodies™, shark single domain antibodies, single chain or tandem diabodies (TandAb™), VHHs, Anticalins™, Nanobodies™, minibodies, BiTE™, bicyclic peptides, and other alternative immunoglobulin protein scaffolds). In some embodiments, the antibody may lack the covalent modifications (e.g., attachment of glycans) that it would have if produced naturally. In some embodiments, the antibody may contain covalent modifications (e.g., attachment of glycans, detectable moieties, therapeutic moieties, catalytic moieties, or other chemical groups that improve the stability or administration of the antibody, such as polyethylene glycol). In some embodiments, the antibody may be in the form of a masked antibody (e.g., Probodies™). A masked antibody may contain a blocking or "masking" peptide that specifically binds to the antigen-binding surface of the antibody and prevents the antibody from binding to the antigen. The masking peptide is linked to the antibody by a cleavable linker (e.g., a protease). Selective cleavage of the linker in the desired environment, i.e., the tumor environment, can release the masking / blocking peptide, allowing antigen binding within the tumor, thereby limiting potential toxicity issues. "Antibody" can also refer to camelid antibodies (heavy chain only antibodies) and antibody-like molecules such as anticalins (Skerra (2008) FEBS J 275, 2677-83).In some embodiments, the antibodies are polyclonal or oligoclonal, which are generated as a panel of antibodies, each associated with a single antibody sequence and binding to more or less distinct epitopes within the antigen (e.g., different epitopes within the human CD25 extracellular domain associated with different reference anti-human CD25 antibodies). Polyclonal or oligoclonal antibodies can be provided as a single preparation for medical use, as described in the literature (Kearns JD et al., 2015. Mol Cancer Ther. 14:1625-36).

[0031] The antibodies used in the present invention may be monospecific, bispecific or multispecific. A "multispecific antibody" may be specific for different epitopes of one target antigen or polypeptide, or may contain antigen-binding domains specific for two or more target antigens or polypeptides. In some embodiments of the present invention, the antibody is monospecific. In some embodiments, the antibody binds to CD25 monovalently (i.e., in a ratio of one antibody to one CD25 molecule). In further embodiments, the antibody is a monospecific bivalent antibody, i.e., the antibody binds to CD25 in a ratio of one antibody to two CD25 molecules.

[0032] In some embodiments of the invention, the antibody is monoclonal. The antibody may additionally or alternatively be humanized or human. In further embodiments, the antibody is human, or in any case, is an antibody with a format and characteristics that allow for its use and administration in human subjects.

[0033] As used herein, "monoclonal antibody" is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0034] As used herein, a "human antibody" refers to an antibody having a variable region in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Additionally, if the antibody contains a constant region, the constant region also is derived from a human germline immunoglobulin sequence. The human antibodies of the invention may include 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).

[0035] Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins with the same structural characteristics. Immunoglobulins may be from any class, such as IgA, IgD, IgG, IgE or IgM. Immunoglobulins may be from any subclass, such as IgG1, IgG2, IgG3 or IgG4. In a preferred embodiment of the invention, the anti-CD25 antibody is from the IgG class, preferably the IgG1 subclass. In one embodiment, the anti-CD25 antibody is from the human IgG1 subclass.

[0036] In a preferred embodiment of the invention, the anti-CD25 antibody binds to FcγR with high affinity, preferably to an activating receptor with high affinity. Preferably, the antibody binds to FcγRI and / or FcγRIIa and / or FcγRIIIa with high affinity. In a specific embodiment, the antibody binds to FcγR with high affinity at about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 It binds to at least one activating Fcγ receptor with a dissociation constant less than M.

[0037] In some embodiments, the antibody is an IgG1 antibody, preferably a human IgG1 antibody, which can bind to at least one Fc activating receptor. For example, the antibody can bind to one or more receptors selected from FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the antibody can bind to FcγRIIIa. In some embodiments, the antibody can bind to FcγRIIIa and FcγRIIa and optionally FcγRI. In some embodiments, the antibody binds to these receptors with high affinity, for example, about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 It can bind with a dissociation constant less than M.

[0038] In some embodiments, the antibody binds to the inhibitory receptor FcγRIIb with low affinity. -7 Higher than M, about 10 -6 Higher than M or about 10 -5 It binds to FcγRIIb with a dissociation constant higher than M.

[0039] In some embodiments, the antibody may be defucosylated. The Fc region of the antibody may be modified to alter the glycosylation profile using techniques known in the art. Available techniques for producing antibodies with absent or reduced fucosylation profile include commercially available techniques such as GlyMAXX (ProBiogen) and methods such as those disclosed in WO 2011 / 035884.

[0040] In some embodiments, anti-CD25 antibodies induce ADCC activity. Anti-CD25 antibodies exhibit ADCC activity against CD25+ target cells. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize antibodies bound on target cells, thereby resulting in lysis of the target cells. In some embodiments, anti-CD25 antibodies induce ADCP activity. "Antibody-dependent cell-mediated phagocytosis" (ADCP) refers to a cell-mediated reaction in which phagocytes expressing Fc receptors (FcR), such as macrophages, recognize antibodies bound on target cells, thereby resulting in phagocytosis of the target cells.

[0041] The anti-CD25 antibodies used in the present invention may function through ADCC and ADCP activities. ADCC and ADCP can be measured using assays known and available in the art.

[0042] In some embodiments of the invention, the anti-CD25 antibody does not inhibit the binding of interleukin-2 to CD25. References herein to "do not inhibit the binding of interleukin-2 to CD25" may alternatively be expressed as the anti-CD25 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 the anti-CD25 antibody), i.e., the antibody does not block the binding of interleukin-2 to CD25, and in particular does not inhibit interleukin-2 signaling in CD25-expressing cells. References herein to a non-IL-2 blocking antibody may alternatively be expressed as an anti-CD25 antibody that "does not inhibit the binding of interleukin-2 to CD25" or an anti-CD25 antibody that "does not inhibit IL-2 signaling." References such as "non-blocking," "non-IL2 blocking," "non-blocking," or "non-blocking" (with respect to non-blocking of IL-2 binding to CD25 in the presence of an anti-CD25 antibody) include embodiments in which the anti-CD25 antibody of the invention does not block IL-2 signaling through CD25. That is, the anti-CD25 antibody inhibits IL-2 signaling by less than 50% compared to IL-2 signaling in the absence of the antibody. In certain embodiments of the invention described herein, the anti-CD25 antibody inhibits IL-2 signaling by less than about 50%, 40%, 35%, 30%, and preferably less than about 25% compared to IL-2 signaling in the absence of the antibody.

[0043] Some anti-CD25 antibodies can allow IL-2 to bind to CD25 but still block signaling through the CD25 receptor. Non-IL-2 blocking anti-CD25 antibodies allow IL-2 to bind to CD25 and promote at least 50% levels of signaling through the CD25 receptor compared to signaling in the absence of the anti-CD25 antibody.

[0044] IL-2 signaling through CD25 can be measured, for example, by methods discussed in WO 2018 / 167104 and known in the art. Comparison of IL-2 signaling in the presence and absence of an anti-CD25 antibody drug can be performed under the same or substantially the same conditions.

[0045] In some embodiments, IL-2 signaling can be measured by the level of phosphorylated STAT5 protein in cells using a standard Stat-5 phosphorylation assay. For example, a Stat-5 phosphorylation assay to measure IL-2 signaling can involve culturing PMBC cells in the presence of anti-CD25 antibody at a concentration of 10 μg / ml for 30 minutes, followed by the addition of various concentrations of IL-2 (e.g., 10 U / ml, or various concentrations of 0.25 U / ml, 0.74 U / ml, 2.22 U / ml, 6.66 U / ml, or 20 U / ml) for 10 minutes. Cells can then be permeabilized, after which the level of STAT5 protein can be measured using a fluorescently labeled antibody against phosphorylated STAT5 peptide analyzed by flow cytometry. The blocking percentage of IL-2 signaling can be calculated as follows: % blocking = 100 x [(% of Stat5+ cells without Ab group - % of Stat5+ cells with 10 μg / ml Ab group) / (% of Stat5+ cells without Ab group).

[0046] Examples of non-blocking anti-CD25 antibodies are described in WO 2018 / 167104, WO 2019 / 175215, WO 2019 / 175216, WO 2019 / 175217, WO 2019 / 175220, WO 2019 / 17522, WO 2019 / 175223, WO 2019 / 17524, WO 2019 / 17526, the entireties of which are incorporated by reference herein.

[0047] The anti-CD25 antibody may specifically bind to an epitope within the extracellular region of human CD25. In some embodiments, the antibody binds to an epitope that is distinct from the IL-2 binding site and does not block the binding of IL-2 to CD25.

[0048] As used herein, "epitope" refers to a portion of an antigen that is bound by an antibody or antigen-binding fragment. As is well known in the art, epitopes can be formed both from adjacent amino acids (linear epitopes) or non-adjacent amino acids juxtaposed by tertiary folding of a protein (conformational epitopes). Epitopes formed from adjacent amino acids typically remain on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.

[0049] Epitopes are conformational in the sense that they are composed of portions of an antigen that are not covalently adjacent in the antigen when the antigen is in the relevant conformation, but are adjacent to each other in three-dimensional space. For example, in the case of CD25, conformational epitopes are epitopes that are composed of non-adjacent amino acid residues in the CD25 extracellular domain, while linear epitopes are epitopes that are composed of adjacent amino acid residues in the CD25 extracellular domain. Means for determining the exact sequence and / or particular amino acid residues of an epitope for an anti-CD25 antibody are known in the literature and include competition with peptides derived from the antigen sequence, binding to truncated and / or mutagenized (e.g., by alanine scanning or other site-directed mutagenesis) CD25 sequences from various species, phage display-based screening, enzyme display techniques, or (co)crystallography techniques. Methods for determining the spatial conformation of epitopes are also well known in the art and include, for example, x-ray crystallography and 2D nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996). Thus, in some embodiments, an anti-CD25 antibody may recognize a conformational epitope.

[0050] In some embodiments, the anti-CD25 antibody binds to an epitope that includes one or more amino acid residues contained in one or more of the amino acid stretches selected from amino acids 150 to 163 of SEQ ID NO:1 (YQCVQGYRALHRGP) (SEQ ID NO:52), amino acids 166 to 186 of SEQ ID NO:1 (SVCKMTHGKTRWTQPQLICTG) (SEQ ID NO:53), amino acids 42 to 56 of SEQ ID NO:1 (KEGTMLNCECKRGFR) (SEQ ID NO:54), and amino acids 70 to 88 of SEQ ID NO:1 (NSSHSSWDNQCQCTSSATR) (SEQ ID NO:55). 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 contained in one or more amino acid stretches selected from amino acids 150 to 163 of SEQ ID NO: 1 (YQCVQGYRALHRGP) (SEQ ID NO: 52), amino acids 166 to 186 of SEQ ID NO: 1 (SVCKMTHGKTRWTQPQLICTG) (SEQ ID NO: 53), amino acids 42 to 56 of SEQ ID NO: 1 (KEGTMLNCECKRGFR) (SEQ ID NO: 54), and / or amino acids 70 to 88 of SEQ ID NO: 1 (NSSHSSWDNQCQCTSSATR) (SEQ ID NO: 55).

[0051] In some embodiments, the anti-CD25 antibody binds to an epitope on human CD25, the epitope comprising at least one sequence selected from amino acids 150 to 158 of SEQ ID NO: 1 (YQCVQGYRA) (SEQ ID NO: 56), amino acids 176 to 180 of SEQ ID NO: 1 (RWTQP) (SEQ ID NO: 57), amino acids 42 to 56 of SEQ ID NO: 1 (KEGTMLNCECKRGFR) (SEQ ID NO: 54), and amino acids 74 to 84 of SEQ ID NO: 1 (SSWDNQCQCTS) (SEQ ID NO: 58). Such antibodies do not inhibit binding of IL-2 to CD25.

[0052] In one embodiment, the anti-CD25 antibody binds to an epitope comprising the sequence of amino acids 70 to 84 of SEQ ID NO:1 (NSSHSSWDNQCQCTS) (SEQ ID NO:59).

[0053] Natural antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain contains a variable domain (V H ) followed by a number of constant domains. Each light chain has a variable domain (V L ) and a constant domain at the carboxy terminus.

[0054] The variable regions are capable of interacting with structurally complementary antigen targets and are characterized by differing amino acid sequences from antibodies with different antigen specificities. The variable regions of either the heavy or light chain contain amino acid sequences capable of specifically binding to antigen targets. Within these sequences are smaller sequences that are termed "hypervariable" due to their extreme variability between antibodies of different specificities. Such hypervariable regions are also called "complementarity determining regions" or "CDR" regions.

[0055] These CDR regions are the basis of the basic specificity of antibodies for a particular antigenic determinant. Although CDRs represent non-contiguous stretches of amino acids within the variable region, it has been found that, regardless of species, the positional locations of these important amino acid sequences within the variable heavy and light chain regions have similar positions within the amino acid sequences of the variable chains. There are three CDR regions in each of the variable heavy and light chains of all antibodies, each of which is non-contiguous with the others (designated H1, H2, H3, L1, L2, L3) for the respective heavy (H) and light (L) chains. The CDR regions designated herein are defined by Kabat (Kabat et al., 1977. J Biol Chem 252, 6609-6616).

[0056] In some embodiments, the anti-CD25 antibody is (a) a heavy chain variable region comprising a CDR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 2 to 5, a CDR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 6 to 11, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; A light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; an antibody or antigen-binding fragment thereof comprising (b) A heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 24, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25; A light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 26, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 27, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 28; and an antibody or antigen-binding fragment thereof comprising (c) a heavy chain variable region comprising a CDR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 31 to 33, a CDR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 34 to 38, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39; A light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42; an antibody or antigen-binding fragment thereof comprising is selected from the group consisting of:

[0057] In some embodiments, the anti-CD25 antibody is (a) A heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; A light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; an antibody or antigen-binding fragment thereof comprising b) A heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 7, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; A light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; an antibody or antigen-binding fragment thereof comprising c) A heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; A light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; an antibody or antigen-binding fragment thereof comprising d) A heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; A light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; an antibody or antigen-binding fragment thereof comprising e) A heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; A light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and an antibody or antigen-binding fragment thereof comprising f) A heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO:5, CDR-H2 comprising the amino acid sequence of SEQ ID NO:11, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:12; A light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; an antibody or antigen-binding fragment thereof comprising is selected from the group consisting of:

[0058] In some embodiments, the anti-CD25 antibody is a) an antibody comprising a heavy chain variable region comprising any one of the amino acid sequences of SEQ ID NOs: 16 to 21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; b) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; and c) an antibody comprising a heavy chain variable region comprising any one of the amino acid sequences of SEQ ID NOs: 43 to 48 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 49; is selected from the group consisting of:

[0059] In some embodiments, the anti-CD25 antibody is a) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; b) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; c) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; d) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; e) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; f) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; and g) an antibody comprising a heavy chain variable region comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 16 to 21, and a light chain variable region comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 22; is selected from the group consisting of:

[0060] SEQ ID NOs for the complementarity determining regions (HCDR1-3 and LCDR1-3) and heavy and light chain variable regions of the exemplified antibodies are provided in the table below.

[0061] [Table 1]

[0062] Such antibodies are further described in WO 2019 / 175216, WO 2019 / 175217 and WO 2019 / 1175222, the contents of which are incorporated herein by reference.

[0063] The antibody referred to herein as aCD25-a-686 may also be referred to as RG6292. In a preferred embodiment, the anti-CD25 antibody is RG6292. The anti-CD25 antibody referred to as "RG6292" is a defucosylated human IgG1 monoclonal antibody. RG6292 has a heavy chain sequence having the sequence of SEQ ID NO:50 and a light chain sequence having the sequence of SEQ ID NO:51.

[0064] Such antibodies are known as "non-IL-2 blocking" antibodies and do not inhibit the binding of IL-2 to CD25.

[0065] Variants of the antibodies defined above may also be used. The variants of the antibodies have the following sequence for each CDR sequence: (i) at least 85% identity; and / or (ii) one, two or three amino acid substitutions compared to SEQ ID NOs: 2-15, 23-28, or 31-42; The antibody includes an antibody comprising an amino acid sequence having the formula:

[0066] The antibody variant has the following sequences for the light and heavy chains: (i) at least 80% identity; and / or (ii) one, two, three, four or five amino acid substitutions compared to SEQ ID NOs: 16-22, 29, 30 or 43-51; Also included is an antibody comprising an amino acid sequence having the formula:

[0067] For example, one embodiment of the present invention is an anti-CD25 antibody for use in the treatment of AML, comprising: a) a heavy chain variable region comprising: CDR-H1 comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 2 to 5; CDR-H2 comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 6 to 11; and CDR-H3 comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12; A light chain variable region comprising: CDR-L1 comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13; CDR-L2 comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 14; and CDR-L3 comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 15; an antibody or antigen-binding fragment thereof comprising b) a heavy chain variable region comprising: CDR-H1 comprising an amino acid sequence having one, two or three amino acid substitutions compared to any one of SEQ ID NOs: 2 to 5; CDR-H2 comprising an amino acid sequence having one, two or three amino acid substitutions compared to any one of SEQ ID NOs: 6 to 11; and CDR-H3 comprising an amino acid sequence having one, two or three amino acid substitutions compared to SEQ ID NO: 12; A light chain variable region comprising: CDR-L1 comprising an amino acid sequence having one, two or three amino acid substitutions compared to SEQ ID NO: 13; CDR-L2 comprising an amino acid sequence having one, two or three amino acid substitutions compared to SEQ ID NO: 14; and CDR-L3 comprising an amino acid sequence having one, two or three amino acid substitutions compared to SEQ ID NO: 15; and an antibody or antigen-binding fragment thereof comprising c) i) an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 16 to 21, or ii) an amino acid sequence having one, two, three, four or five amino acid substitutions compared to SEQ ID NOs: 16 to 21; and a heavy chain variable region comprising i) an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 22, or ii) an amino acid sequence which has one, two, three, four or five amino acid substitutions compared to SEQ ID NO: 22; and a light chain variable region comprising an antibody or antigen-binding fragment thereof comprising The present invention provides an anti-CD25 antibody selected from the group comprising:

[0068] Percent identity (%), as known in the art, is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, determined by the match between strings of such sequences. Although there are many methods to measure the identity between two polypeptide or two polynucleotide sequences, the commonly used methods to determine identity are codified in computer programs. Preferred computer programs for determining the identity between two sequences include, but are not limited to, the GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)). The percent identity of two amino acid sequences or two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps may be introduced in the first sequence for best alignment of the sequences) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is the alignment of two sequences that results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e., % identity = number of identical positions / total number of positions x 100). In general, the statement of % identity in this specification refers to the % identity along the entire length of the molecule, unless otherwise specified or implied by the context.

[0069] In some embodiments, the anti-CD25 antibodies kill cancer cells, Treg cells, AML blast cells and / or PMBC cells. In some embodiments, the antibodies kill Treg cells and blast cells with CD25 expression levels greater than about 900 CD25 molecules per cell. Preferably, the antibodies kill Treg cells and blast cells with CD25 expression levels greater than about 1000 CD25 molecules per cell, preferably in the range of about 1000-40000, about 1000-5000, or about 1000-2500 CD25 molecules per cell.

[0070] The CD25 expression level in a particular cell is also referred to as CD25 density, and is a measure of the number of CD25 molecules per cell.The CD25 expression level or density in a cell can be determined, for example, by flow cytometry, as discussed in the Examples and as known in the art.A cell having about 1000 CD25 molecules per cell is considered to be a low-expressing CD25 cell, compared to cells such as Treg cells that have high CD25 expression.Such low-expressing CD25 cells include AML blasts.

[0071] In some embodiments of the invention, anti-CD25 antibodies are used to kill low expressing CD25 cells, such as CD25+ AML blast cells.

[0072] In some embodiments, the anti-CD25 antibody induces a reduction in CD16 expression in NK cells by up to 25% when the antibody and NK cells are co-incubated with cells expressing 900-5000 CD25 molecules per cell. Preferably, the NK cells are CD56dim NK cells. The reduction in CD16 expression can be measured, for example, by methods discussed in the Examples and methods known in the art.

[0073] The present invention relates to the treatment of acute myeloid leukemia (AML) or diffuse large B-cell lymphoma (DLBCL). Preferably, the present invention relates to the treatment of acute myeloid leukemia (AML). Acute myeloid leukemia (AML) is a cancer of the blood in which abnormal cells of the myeloid population, such as myeloblasts, red blood cells and platelets, grow and accumulate in the bone marrow and spread to the blood. Classification schemes for AML are known in the art, such as the WHO Classification of AML (2008) and the French-American-British (FAB) classification. Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of non-Hodgkin's lymphoma.

[0074] Anti-CD25 antibodies can be used to target AML blast cells in patients. In some embodiments, the CD25 expression level on tumor cells from a subject to be treated is at least about 900 CD25 molecules per cell. In some embodiments, the CD25 expression level on tumor cells from a subject to be treated is in the range of about 900 to about 5000 CD25 molecules per cell.

[0075] As used herein, the terms "treatment," "treat," or "treating" of AML or DLBCL refer to any administration of a substance (e.g., an anti-CD25 antibody) that partially or completely relieves, alleviates, ameliorates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms. A positive therapeutic effect can be, for example, a reduction in the number of cancer cells, i.e., a reduction in AML blasts.

[0076] The subject of any of the embodiments of the invention described herein is preferably 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. The subject may also be referred to herein as a patient.

[0077] The dosage regimen of the therapeutics described herein that are effective for treating cancer patients may vary depending on factors such as the patient's condition, age, and weight, and the ability of the therapy to induce an anti-cancer response in the subject. The anti-CD25 antibody may be used in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" refers to an amount (e.g., an amount of an agent or pharmaceutical composition) that is sufficient to treat a disease and / or condition when administered to a population suffering from or susceptible to the disease and / or condition according to a therapeutic administration regimen. A therapeutically effective amount is an amount that reduces the incidence and / or severity of, stabilizes and / or delays the onset of one or more symptoms of a disease, disorder, and / or condition. Those skilled in the art will understand that a "therapeutically effective amount" does not actually require therapeutic success to be achieved in a particular subject.

[0078] Selection of an appropriate dosage is considered to be within the ability of one of ordinary skill in the art, e.g., 0.01 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 or 50 mg / kg. In some embodiments, such amounts are unit dosages (or whole fractions thereof) appropriate for administration according to a dosing regimen (i.e., a therapeutic dosing regimen) determined to correlate with a desired or beneficial outcome when administered to a relevant population. Dosages may also vary with respect to route of administration, treatment cycles, or dose escalation protocols that can be used to determine maximum tolerated doses and dose-limiting toxicities (if any) associated with administration of the antibody at increasing doses.

[0079] In some embodiments, the dosing regimen includes multiple doses, each spaced apart by the same length of time. Alternatively, the dosing regimen includes multiple doses and at least two different time periods between each dose. In some embodiments, all doses in the dosing regimen are the same unit dosage. Alternatively, the various doses in the dosing regimen are of different amounts. In some embodiments, the dosing regimen includes a first dose at a first dosage amount, followed by one or more additional doses at a second dosage amount different from the first dosage amount. The dosing regimen may include a first dose at a first dosage amount, followed by one or more additional doses at a second dosage amount equal to the first dosage amount. In some embodiments, the dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0080] The anti-CD25 antibodies according to any aspect of the invention described herein may be in the form of a pharmaceutical composition additionally comprising a pharma- ceutically acceptable carrier, diluent or excipient. These compositions include, for example, liquid, semi-solid and solid dosage formulations, 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. Pharmaceutical compositions containing the antibodies 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 types of administration involving physical disruption of a subject's tissue, and administration of pharmaceutical compositions via disruption of tissue). Such formulations may be in the form of injectable or infusible solutions suitable for, for example, intradermal, intratumoral or subcutaneous administration, or intravenous infusion. Administration may involve intermittent administration. Alternatively, administration may involve continuous administration (e.g., perfusion) for at least a selected period of time, either simultaneously with or between administrations of other compounds. In some embodiments, the antibodies may be prepared with carriers that protect against rapid release and / or degradation, such as controlled release formulations, e.g., implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used.

[0081] Those skilled in the art will understand that, for example, the route of delivery (e.g., oral, intravenous, subcutaneous, etc.) may affect the dosage and / or the required dosage may affect the route of delivery. For example, concentrated delivery may be desirable and / or useful when a particularly high concentration of the agent in a particular site or location is targeted. Other factors to be considered when optimizing the route and / or administration schedule for a given treatment regimen include, for example, the particular cancer being treated (e.g., type, stage, location, etc.), the clinical condition of the subject (e.g., age, overall health, etc.), the presence or absence of concomitant therapy, and other factors known to medical practitioners. Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentration. Sterile injectable solutions can be prepared by incorporating the required amount of the antibody in an appropriate solvent containing one or a combination of the ingredients listed above, as needed, 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 discussed herein. Sterile injectable formulations may be prepared using non-toxic parenterally acceptable diluents or solvents. Each pharmaceutical composition used according to the present invention may contain pharma- ceutical acceptable dispersing agents, wetting agents, suspending agents, isotonic agents, coatings, antibacterial and antifungal agents, carriers, excipients, salts, or stabilizers, which are non-toxic to subjects at the dosages and concentrations used. Preferably, such compositions may further contain pharma- ceutical acceptable carriers or excipients used in the treatment of cancer, compatible with a given method and / or site of administration, e.g., parenteral (e.g., subcutaneous, intradermal, or intravenous injection), intratumoral or peritumoral administration. As used herein, the term "pharmaceutically acceptable" applied to a carrier, diluent, or excipient used to formulate a composition disclosed herein means that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0082] In some embodiments, the anti-CD25 antibody may be part of a combination therapy with other therapeutic agents. Thus, a second aspect of the invention provides an anti-CD25 antibody for use in the treatment of AML or DLBCL, the anti-CD25 antibody being administered in combination with one or more additional therapeutic agents. A third aspect of the invention provides a combination of an anti-CD25 antibody for use in the treatment of AML or DLBCL with one or more additional therapeutic agents. The anti-CD25 antibody and the additional therapeutic agent are for separate, simultaneous or sequential administration.

[0083] The anti-CD25 antibody may be administered in combination with a costimulatory antibody, chemotherapy and / or radiation therapy (by irradiation from outside the body or by administration of a radioconjugated compound), cytokine-based therapy, targeted therapy, vaccine or adjuvant, or any combination thereof. "In combination" may refer to administration of an additional therapy before, simultaneously with, or after administration of the anti-CD25 antibody. The anti-CD25 antibody and the additional therapeutic agent may be for separate, simultaneous, or sequential administration.

[0084] The anti-CD25 antibody and other therapeutic agent may be administered via the same or different delivery routes and / or according to various schedules. Alternatively or additionally, in some embodiments, one or more doses of a first active agent are administered substantially simultaneously with one or more other active agents, and in some embodiments via a common route and / or as part of a single composition.

[0085] In some embodiments, the other therapeutic agent may be selected from one or more FLT3 inhibitors (e.g., gilteritinib, midostaurin, sorafenib, quizartinib, crenolanib), BCL-2 inhibitors (e.g., venetoclax), IDH inhibitors (e.g., enasidenib, ivosidenib), hypomethylating agents (e.g., azacitidine, decitabine), additional antibodies (e.g., CD33 antibodies, such as gemtuzumab ozogamicin), and combinations thereof. In some embodiments, the anti-CD25 antibody may be used in combination with an anthracycline-cytarabine regimen (such as a regimen of daunorubicin and cytarabine), optionally in combination with additional therapeutic agents. In some embodiments, the antibody is intended to be used in combination with a BCL-2 inhibitor and a hypomethylating agent. In some embodiments, the anti-CD25 antibody is used in combination with venetoclax, optionally in combination with additional therapeutic agents, such as azacitidine.

[0086] Other therapeutic agents include, but are not limited to, other chemotherapeutic agents such as cytotoxic agents, such as alkylating agents, anthracyclines, epothilones, nitrosoureas, ethylenimines / methylmelamines, alkyl sulfonates, alkylating agents, antimetabolites, pyrimidine analogs, epipodophylloxines, enzymes such as L-asparaginase, biological response modifiers such as IFNα, IFN-γ, IL-2, IL-12, G-CSF and GM-CSF, platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin, anthracenediones, substituted ureas such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenal cortex suppressants such as mitotane (o,p'-DDD) and aminoglutethimide, adrenal cortex inhibitors such as cytotoxic agents, and the like. These include, but are not limited to, hormones and antagonists, including steroid antagonists, such as prednisone and equivalents, dexamethasone, and aminoglutethimide; progestins, such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate; estrogens, such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens, such as tamoxifen; androgens, including testosterone propionate and fluoxymesterone / equivalents; antiandrogens, such as flutamide, gonadotropin releasing hormone analogs, and leuprolide; and nonsteroidal antiandrogens, such as flutamide.

[0087] In some embodiments, the other therapeutic agent may be an immune checkpoint inhibitor. In some embodiments, the present invention also provides for treating AML with an anti-CD25 antibody in combination with at least one immune checkpoint inhibitor. As used herein, "immune checkpoint" or "immune checkpoint protein" refers to a protein that belongs to an inhibitory pathway in the immune system, particularly for regulating T cell responses. Under normal physiological conditions, immune checkpoints are very important for preventing autoimmunity, particularly during responses to pathogens. Cancer cells may alter the expression control of immune checkpoint proteins to evade immune surveillance.

[0088] 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 also OX40, GITR, ICOS, 4-1BB and HVEM. Immune checkpoint proteins may also refer to proteins that bind to other immune checkpoint proteins. Such proteins include PD-L1, PD-L2, CD80, CD86, HVEM, LLT1 and GAL9. Immune checkpoint inhibitors can inhibit immune checkpoint proteins. For example, the immune checkpoint inhibitor may be an antibody that specifically binds to the immune checkpoint inhibitor, or may be other antagonists of the immune checkpoint protein.

[0089] In some embodiments of the invention, the immune checkpoint protein is PD-1 or PD-L1, and the immune checkpoint inhibitor may be an inhibitor of PD-1 or PD-L1, i.e., an antagonist of PD-1 or PD-L1. In further embodiments, the immune checkpoint inhibitor disrupts PD-1 / PD-L1 interaction via an anti-PD-1 antibody or an anti-PD-L1 antibody. Anti-PD-1 antibodies known in the art include nivolumab and pembrolizumab. Anti-PD-L1 antibodies include antibodies such as atezolizumab (MPDL3280A).

[0090] In some embodiments, the other therapeutic agent may be a cancer vaccine. A further embodiment of the present invention provides for treating AML with anti-CD25 antibodies in combination with a cancer vaccine. As used herein, "cancer vaccine" refers to a therapeutic cancer vaccine that is administered to a cancer patient and is designed to eradicate cancer cells by enhancing the patient's own immune response. Cancer vaccines include tumor cell vaccines (autologous and allogeneic), dendritic cell vaccines (ex vivo generated and peptide activated), protein / peptide-based cancer vaccines and genetic vaccines (DNA, RNA and virus-based vaccines). Thus, therapeutic cancer vaccines can in principle be utilized to inhibit further growth of advanced cancers and / or recurrent tumors that are resistant to conventional therapies such as surgery, radiation therapy and chemotherapy. Tumor cell-based vaccines (autologous and allogeneic) include vaccines genetically engineered to secrete soluble immunostimulants, such as cytokines (IL-2, IFN-g, IL12, GMCSF, FLT3L), single chain Fv antibodies against immunomodulatory receptors (PD-1, CTLA-4, GITR, ICOS, OX40, 4-1BB), and / or to express ligands of immunostimulatory receptors, such as ICOS-ligand, 4-1BB-ligand, GITR-ligand and / or OX40-ligand, among others, on the cell membrane. In some embodiments, the cancer vaccine may be the GVAX antitumor vaccine.

[0091] In some embodiments, the combination therapy does not include administration of a cancer vaccine in combination with an anti-CD25 antibody.

[0092] In some embodiments, the anti-CD25 antibody is not conjugated to another therapeutic agent, e.g., the anti-CD25 antibody is not in the form of an antibody-drug conjugate. In some embodiments, the anti-CD25 antibody is not Kamidanlumabtesirin (ADCT-301). Kamidanlumabtesirin is an anti-CD25 antibody known as HuMax™-TAC conjugated to a pyrrolobenzodiazepine (PBD) dimer via a dipeptide cleavable linker.

[0093] In some embodiments of the invention, the anti-CD25 antibody is administered as a monotherapy, e.g., when the anti-CD25 antibody is administered as a monotherapy, the anti-CD25 antibody is the only therapeutically active agent administered, e.g., the only therapeutically active agent administered to treat AML or DLBCL.

[0094] A fourth aspect of the invention provides a method of treating AML or DLBCL in a subject, comprising administering to the subject an effective amount of an anti-CD25 antibody.

[0095] A fifth aspect of the invention provides the use of an anti-CD25 antibody in the manufacture of a medicament for the treatment of AML or DLBCL. The anti-CD25 antibody according to these further aspects of the invention may be an anti-CD25 antibody as described for the first aspect.

[0096] The method of treating AML may further comprise administering one or more additional therapeutic agents. In some embodiments, the method further comprises administering one or more immune checkpoint inhibitors, cancer vaccines, FLT3 inhibitors, BCL-2 inhibitors, IDH inhibitors, hypomethylating agents, additional antibodies, and combinations thereof, as described above, or in combination with an anthracycline-cytarabine regimen. The additional therapeutic agents may be administered individually, simultaneously, or sequentially.

[0097] A sixth aspect of the invention provides the use of an anti-CD25 antibody and a further therapeutic agent in the manufacture of a medicament for the treatment of AML or DLBCL, wherein the anti-CD25 antibody and the further therapeutic agent are for separate, simultaneous or sequential administration.

[0098] Further therapeutic agents according to the fourth, fifth and sixth aspects of the invention may be as defined for the first, second and third aspects of the invention.

[0099] The present invention also relates to selecting patients suffering from acute myeloid leukemia (AML) for treatment with an anti-CD25 antibody. A seventh aspect of the present invention provides a method for selecting a patient suffering from AML for treatment with an anti-CD25 antibody, comprising measuring the CD25 expression level in target cells in a sample from the patient, wherein the patient is suitable for treatment with the antibody if the cells have an expression level of more than about 900 CD25 molecules per cell.

[0100] The method can be used to determine whether a patient is suitable for treatment with an anti-CD25 antibody. If target cells from the patient have an expression level of more than about 900, preferably more than 1000, CD25 molecules per cell, the patient is deemed suitable for treatment with an anti-CD25 antibody. The method can then further comprise administering an anti-CD25 antibody to the patient.

[0101] The method may further comprise the step of determining whether functional FcR+ effector cells are present in the sample.

[0102] The method may further include obtaining a sample from the subject. The sample may be a biological tissue or fluid sample from the subject. In some embodiments, the sample is a bone marrow sample from the patient.

[0103] The level of CD25 expression on target cells can be measured by methods known in the art, including, for example, flow cytometry, as discussed in the Examples.

[0104] AML patients with FLT3 internal tandem duplication (FLT3-ITD) mutations are associated with poor prognosis and increased risk of relapse (Dohner et al, 2017, Blood 129(4) 424-447). FLT-ITD mutations involve a tandem duplication of at least 3-1000 more nucleotides within the juxtamembrane domain of FLT3. The inventors have further surprisingly found that AML patients with FLT3-ITD mutations, compared to wild-type FLT3, exhibit a high prevalence of CD25-expressing AML cells. Thus, patients with FLT3-ITD mutations may be candidates for anti-CD25 antibody therapy. Additionally, the presence of FLT3-ITD mutations can be used as a biomarker to identify, diagnose and / or predict whether an AML patient would benefit from anti-CD25 antibody therapy alone or in combination with other therapeutic agents to treat AML.

[0105] Thus, an eighth aspect of the invention includes a method for selecting a patient suffering from acute myeloid leukemia for treatment with an anti-CD25 antibody, comprising determining the presence or absence of a FLT3-ITD mutation in a sample from the patient, whereby if the mutation is present in the sample, the patient is suitable for treatment with the antibody. The method can be used to identify whether a patient is particularly suitable for anti-CD25 antibody therapy.

[0106] If the patient is determined to have a FLT3-IRD mutation, the method may further comprise administering to the patient an anti-CD25 antibody. The anti-CD25 antibody may be as defined above for other aspects of the invention.

[0107] A ninth aspect of the invention provides a method for predicting the response of an AML patient to treatment with an anti-CD25 antibody, comprising determining the presence or absence of an FLT3-ITD mutation in a sample from the patient, the presence of said mutation in said sample being indicative of the patient being likely to respond to treatment with an anti-CD25 antibody.

[0108] A tenth aspect of the present invention provides a method of treating acute myeloid leukemia in a subject, comprising administering to the subject an effective amount of an anti-CD25 antibody, wherein the subject comprises the presence of a FLT3-ITD mutation. The method may further comprise determining the presence of a FLT3-ITD mutation in a sample from a patient suffering from AML.

[0109] If the patient is diagnosed with AML, the patient may be diagnosed with AML characterized by the presence of a FLT3-ITD mutation. Dohner et al, 2017, Blood 129(4) 424-447. If the patient is diagnosed with AML characterized by the presence of a FLT3-ITD mutation, the anti-CD25 antibodies described herein may be particularly useful for treating AML.

[0110] The presence or absence of FLT3-ITD mutation can be determined by methods known in the art. For example, see Spencer DH et al, 2013 Journal Molecular Diagnostics, vol. 15(1), 81-83; Engen C et al, 2021, Molecular Oncology, vol. 15, 2300-2317. For example, the presence or absence of mutation can be determined by a method selected from the group of DNA sequencing and mutation screening techniques.

[0111] The sample can be a blood sample or a bone marrow sample from the patient. In some embodiments, the predictive or selective method is an in vitro method.

[0112] An eleventh aspect of the invention provides a method of preventing or reducing the risk of relapse in an AML patient, comprising administering to the patient an anti-CD25 antibody. The anti-CD25 antibody may be as defined above for the other aspects of the invention.

[0113] Anti-CD25 antibodies can target LSCs (leukemic stem cells) and / or immature AML blasts, or cells with a progenitor phenotype, helping to prevent or reduce the risk of relapse.

[0114] The method may further comprise administering one or more further therapeutic agents. The one or more further therapeutic agents may be as defined for the first, second and third aspects of the invention. In one embodiment, the further therapeutic agent is a FLT3 inhibitor. In a further embodiment, the one or more further therapeutic agents is a combination of a BCL-2 inhibitor and a hypomethylating agent, such as a combination of venetoclax and azacitidine. The anti-CD25 antibody and the further therapeutic agent may be for separate, simultaneous or sequential administration.

[0115] A twelfth aspect of the invention includes a method of treating AML in a patient receiving combination therapy with a BCL-2 inhibitor and a hypomethylating agent, comprising administering to the patient an anti-CD25 antibody. The anti-CD25 antibody may be as described above. In one embodiment, the combination therapy with a BCL-2 inhibitor and a hypomethylating agent comprises a venetoclax-azacitidine combination.

[0116] The inventors have found that AML patients treated with a combination of a BCL-2 inhibitor and a hypomethylating agent, such as venetoclax and azacitidine, still exhibit detectable levels of CD25+ AML cells. Thus, further treatment of AML patients with an anti-CD25 antibody, administered separately, simultaneously or sequentially, may help prevent relapse and disease progression.

[0117] The present invention also provides an anti-CD25 antibody for use in the method of the above further aspect of the invention.

[0118] Aspects and embodiments described herein using the term "comprising" may include other features or steps within their scope. It is also understood that aspects and embodiments described as "comprising" also describe aspects and embodiments in which the term "comprising" is replaced with the term "consisting essentially of" or "consisting of."

[0119] The phrase "selected from the group comprising," when these occur in this specification, may be replaced with the phrase "selected from the group consisting of," and vice versa.

[0120] It is also understood that the present application discloses all combinations of any of the above aspects and embodiments with each other unless the context requires otherwise.Similarly, the present application discloses all combinations of preferred and / or optional features alone or together with any of the other aspects unless the context requires otherwise.

[0121] The present invention will now be further described by the following examples with reference to the drawings, which are intended to serve to assist those skilled in the art in practicing the invention and are not intended to limit the scope of the invention in any way. EXAMPLES

[0122] Example 1 Cells and antibodies Pfeiffer tumor cells (ATCC), an established large B-cell lymphoma cell line, were cultured in RPMI 1640 medium (Gibco) containing 20% ​​FBS (Gibco), 1x Glutamax (Gibco), 1x non-essential amino acids (Gibco) and 1% sodium pyruvate (Gibco). EOL-1 tumor cells (DSMZ), an established acute myeloid leukemia cell line, were cultured in RPMI 1640 medium (Gibco) containing 10% FBS (Gibco), 1x Glutamax (Gibco), 1x non-essential amino acids (Gibco) and 1% sodium pyruvate (Gibco).

[0123] AML22 cells were derived from an AML patient and expanded by transplantation into NSG mice. These patient-derived cells cannot be cultured in vitro, so they were thawed and used directly on the day of the assay.

[0124] In vitro induced Treg (iTreg) cells were generated using Dynabeads Human T-Activator CD3 / CD28 (1 × 10 6iTregs were differentiated from naive CD4+ T cells by activation with 10% heat-inactivated human AB serum (Sigma), 1× Glutamax (Gibco), N-acetylcysteine ​​(2 mg / mL, Sigma), 1% sodium pyruvate (Gibco), 1× HEPES (Gibco), 1× non-essential amino acids (Gibco), 50 μM 2-mercaptoethanol (Thermo Fisher Scientific), proleukin / aldesleukin (300 U / mL, Novartis), 10 ng / mL recombinant human transforming growth factor-β1 (R&D Systems), and 100 ng / mL rapamycin (Sigma) in X-Vivo 15 (Lonza). Naive CD4+ T cells were isolated from human PBMCs using the Human Naive CD4+ T Cell Isolation Kit II (Miltenyi Biotec). The purity of iTregs was confirmed by flow cytometry analysis of human CD3, CD4, CD25 and FoxP3 co-expression (>90%). Cryopreserved iTregs were thawed and used directly on the day of the assay.

[0125] CD25 Mab, also called RG6292, is a defucosylated human IgG1 mAb produced using GlymaxX technology and enhances the ADCC potential of CD25-expressing target cells. Human IgG1 isotype control antibody was purchased from Biolegend.

[0126] Isolation of human PBMCs and NK cells Human PBMCs from healthy donors were isolated from buffy coats (Zurich Blood Donation Centre in accordance with the Declaration of Helsinki) using standard density gradient separation on Histopaque-1077 (Sigma-Aldrich). NK cells were isolated using a human NK cell isolation kit (Miltenyi Biotec) and activated overnight in RPMI 1640 medium (Gibco) containing 10% FcS (Gibco), 1x Glutamax (Gibco) and proleukin / aldesleukin (100 U / mL, Novartis).

[0127] ADCC assay with flow cytometry readout Target cells (Pfeiffer, EOL-1, AML22, iTreg) were mixed with activated primary NK cells at a 2:1 effector to target cell ratio (80,000 NK cells and 40,000 target cells per well) in assay medium (RPMI 1640 (Gibco) containing 2% FBS (Gibco) and 1x Glutamax (Gibco)). Compounds (CD25 Mab or isotype control antibody) were added to U-bottom 96-well plates (TPP) in a 7-fold dilution series with a starting final concentration of 21 μg / mL. The assay plate was placed on an orbital shaker at 300 rpm for 5 minutes to allow mixing of cells and antibodies. After pre-incubation at room temperature for 15 minutes, the plate was incubated at 37°C / 5% CO2 for 17 hours.

[0128] Cells were stained with FSV440UV viability dye diluted in PBS (BD) for 10 min at room temperature. Cells were washed with FACS buffer (PBS containing 0.1% BSA) and stained with fluorochrome-conjugated antibodies against human CD16 (3G8), CD45 (HI30), CD34 (8G12), CD56 (5.1H11), CD3 (UCHT1), CD20 (2H7), CD117 (YB5.B8), CD25 (24212), CD123 (9F5), CD69 (FN50), CD4 (A161A1), CD127 (A019D5) for 30 min at 4° C. in FACS buffer. After two washing steps with FACS buffer, cells were fixed and permeabilized with FoxP3 transcription factor staining set (eBioscience) for 60 min at room temperature. For nuclear staining, cells were then stained for human FoxP3 (259D), Bcl-2 (Bcl-2 / 100) and Ki-67 (Ki67) in 1×PERM buffer for 45 min at room temperature. Samples were washed twice with 1×PERM buffer and resuspended in FACS buffer for acquisition. All antibodies used for flow cytometry were from Biolegend, BD or R&D Systems.

[0129] Samples were acquired on a 5-laser A5 Symphony instrument (BD) and data analysis was performed with FlowJo v10.6.2 and Prism 8 (GraphPad software).

[0130] ADCC assay with luminescent readout Target cells (Pfeiffer, EOL-1, AML22) were mixed with activated primary NK cells at a 2:1 effector to target cell ratio (20,000 NK cells and 10,000 target cells per well) in assay medium (RPMI 1640 (Gibco) containing 2% FBS (Gibco) and 1x Glutamax (Gibco)). Compounds (CD25 Mab or isotype control antibody) were added to white 384-well flat-bottom tissue culture plates (Falcon) in a 7-fold dilution series with a starting final concentration of 21 μg / mL. The assay plate was placed on an orbital shaker at 300 rpm for 5 min to allow mixing of cells and antibodies. After preincubation at room temperature for 15 min, the plate was incubated at 37°C / 5% CO2 for 16-20 h.

[0131] Assay plates were equilibrated uncovered at room temperature for approximately 15 minutes. Cytotoxicity was measured using the CytoTox-Glo™ Cytotoxicity Assay (Promega) according to the manufacturer's instructions. Briefly, reconstituted AAF-Glo™ Reagent was added to each well at 1-4 dilutions (final volume of 40 μl / well). Assay plates were incubated at room temperature for 15-60 minutes, followed by luminescence measurements using a Tecan Spark 10M luminescence plate reader (integration time of 500 ms). Data analysis was performed with Prism 8 (GraphPad software).

[0132] Quantification of CD25 density by flow cytometry BD quantibrite™ beads were used to estimate the antibody bound per cell (ABC), which corresponds to the number of PE molecules per cell if the PE:mAb ratio is 1:1. If monovalent binding of the PE-conjugated anti-human CD25 antibody is assumed, the number of CD25 molecules is equivalent to the number of PE molecules on the cell surface.

[0133] Cell assay samples and beads were acquired on a 5-laser Symphony instrument (BD) with similar instrument settings. Analysis of flow cytometry data was performed in FlowJo v10.6 (Tree Star) and Prism 8 (GraphPad software). Linear regression of Log10 PE molecules per bead against Log10 fluorescence and interpolation of the number of PE molecules per cell were performed as per the manufacturer's instructions.

[0134] CD25 density was quantified on four CD25-expressing target cells.

[0135] result To evaluate the ability of CD25 Mabs to kill AML cells with low CD25 expression levels, antibody-dependent cellular cytotoxicity (ADCC) assays were performed comparing target cells with various CD25 expression levels. CD25 density (number of molecules per cell) was quantified using BD quantibrite™ beads. If monovalent binding of the antibody is assumed, the number of CD25 molecules is equivalent to the number of PE molecules on the cell surface.

[0136] As shown in Figure 1, iTreg and the DLBCL cell line Pfeiffer express relatively high levels of CD25, whereas the AML cell line EOL-1 and patient-derived AML22 cells retain only low numbers of CD25 receptors on their surface. These results were obtained after 17 hours of incubation of target cells alone in ADCC assay medium.

[0137] Despite the fact that CD25 Mabs are characterized by their avidity in binding to CD25, which results in preferential killing of cells with high CD25 expression levels (Non-Patent Document 3), the ADCC capacity of CD25 Mabs targeting EOL-1 and AML-22 cells was comparable to that of CD25-high target cells. After 17 hours of co-incubation with CD25 Mabs, more than 80% of the target cells were killed by NK cells (Figure 2A), whereas no specific killing was observed with the isotype control antibody. The increased killing of the EOL-1 cell line observed at the highest isotype control antibody concentration (21 μg / mL) is likely due to non-specific activity. Although the EC50 values ​​for the two cell lines tested were similar (approximately 0.03 μg / mL), the baseline killing in the absence of compound was different. In fact, 40% to 50% of AML-22 cells were killed by activated NK cells compared to 20% of EOL-1 cells. AML-22 cells are primary cells that exhibit a rapid decline in viability when cultured in vitro, and we hypothesize that AML-22 cells may express molecules that render them susceptible to NK cell-mediated killing.

[0138] CD25 density on target cells that survived at the end of the 17-hour ADCC assay (Fig. 2B) was also evaluated. At CD25 Mab concentrations above 0.4 μg / mL, target cells were found to express 1000-1500 CD25 molecules per cell. Moreover, conventional CD4 and CD8 T cells in humans in the periphery and in tumors, with CD25 expression levels of approximately 800 and 400 receptors per cell, respectively, do not appear to be targets for depletion by CD25 Mabs (data not shown). This therefore indicates that the threshold of CD25 expression on target cells required to trigger killing by CD25 Mabs is likely to be approximately 1000 receptors per cell. As CD25 densities in primary human AML bone marrow samples (CD25+ AML blasts and Tregs) are 1000-4000 (data not shown), depletion of these cells would be expected.

[0139] We investigated the expression of functional markers in NK cells after target engagement by binding of antibody Fc portions to the FcγRIIIa (CD16) receptor expressed on CD56dim NK cells. This subset of NK cells represents the most prevalent population (90% of NK cells) in human peripheral blood and, in contrast to the CD56brightCD16neg NK cell subset, is a potent ADCC mediator.

[0140] Moderate downregulation of CD16 was observed on NK cells after binding to low CD25 density EOL-1 and AML-22 target cells (Figure 3A). In contrast, CD16 expression was strongly reduced in a CD25 Mab dose-dependent manner after co-incubation of effector cells with high CD25 density Pfeiffer and iTreg target cells (Figure 3B). Thus, the results indicate that the magnitude of CD16 downregulation correlates with the CD25 density on target cells.

[0141] In addition, NK cell functionality can be measured by upregulation of the activation markers CD69 and CD25. Expression of CD69 was increased in a dose-dependent manner after treatment with CD25 Mab in all target cells tested. Similar to the observation of high baseline killing of AML22 target cells, baseline expression of CD69 was also increased. In fact, when co-incubated with AML22 in the absence of drug compounds, 45%-60% of NK cells expressed CD69 (Figure 4A). Taken together, these results suggest that AML22 target cells express receptors or secrete factors that induce strong NK cell baseline activation levels. On the other hand, when contacted with the other three target cells, only 20% of NK cells expressed CD69 at baseline, and after treatment with CD25 Mab, expression increased up to 80% (Figure 4B).

[0142] We also observed upregulation of CD25 on NK cells, the strength of which was proportional to the CD25 density on the target cells. As shown in Figure 5A, CD25 expression on NK cells co-incubated with low density CD25+ target cells (AML22 and EOL-1) showed a modest 20% increase. In contrast, up to 60% of NK cells treated with CD25 Mab expressed CD25 when exposed to target cells with high CD25 density (Figure 5B). Notably, despite the increased expression of CD25, NK cells were unaffected, indicating no signs of fratricide (data not shown).

[0143] To increase the reliability of the results obtained by flow cytometry, ADCC assays were performed using four NK cell donors and luminescence readouts (CytoTox-Glo™ cytotoxicity assay). As shown in Figure 6A, consistent and comparable killing activity was observed for the three target cells (AML22, Pfeiffer, EOL-1). EC50 values ​​calculated from dose-response curves demonstrated that similar results were obtained between flow cytometry-based and luminescence-based ADCC readouts. For example, the EC50 value for EOL-1 target cells was 0.035 μg / mL (±0.011 SEM) using CytoTox-Glo™ and 0.030 (±0.007 SEM) μg / mL by flow cytometry (Figure 6B). CD25 Mabs demonstrated potent killing activity across target cells at CD25 densities ranging from 1500 to 38000 receptors per well (Figure 6C). Since CD25 density in human samples is within this expression range (data not shown), we expect efficient killing of target cells in human PBMCs and tumors of AML and DLBCL patients, as long as functional FcR+ effector cells are present.

[0144] These experiments show direct killing of CD25+ AML and DLBCL cells. The antibody was able to deplete CD25+ malignant cells at a wide range of CD25 expression levels. Since the expression levels in human samples are within this range and the indirect effect of CD25 Mabs by depleting Tregs has already been demonstrated, we expect that the dual mechanism of action of CD25 Mabs is involved in the treatment of AML and DLBCL. CD25 Mabs can deplete suppressive Tregs and have a direct cytotoxic effect on CD25+ malignant cells of AML and DLBCL.

[0145] Example 2 Primary human samples Human peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) from AML patients were purchased from Discovery Life Sciences. Samples were collected under the approval of the appropriate Institutional Review Board (IRB) or Ethics Committee. PBMCs from healthy donors (HDs) were isolated from buffy coats (Zurich Blood Donation Center) using standard density gradient centrifugation. All human samples were collected in accordance with the Declaration of Helsinki from HDs or patients who provided written informed consent.

[0146] Isolation of NK cells NK cells were isolated using a human NK cell isolation kit (Miltenyi Biotec) and activated overnight in RPMI 1640 medium (Gibco) containing 10% FcS (Gibco), 1x Glutamax (Gibco) and proleukin / aldesleukin (100 U / mL, Novartis).

[0147] ADCC assay EOL-1 positive control cell line or AML patient samples containing CD25 expressing target cells were mixed with activated primary NK cells at a 2:1 effector to target cell ratio (80000 NK cells and 40000 target cells per well) in assay medium (RPMI 1640 (Gibco) containing 2% FBS (Gibco) and 1x Glutamax (Gibco)). Compounds (CD25 Mab (RG6292) or isotype control antibody (human IgG1 isotype control antibody, Biolegend, QA16A12)) were added to U-bottom 96-well plates (TPP) at a concentration of 10 μg / mL. The assay plate was placed on an orbital shaker at 300 rpm for 5 minutes to allow mixing of cells and antibodies. After pre-incubation at room temperature for 15 minutes, samples were incubated at 37°C / 5% CO2 for 20 hours. Flow cytometry readings were performed as described in the following section. Precision Count Beads™ (Biolegend) were added before sample acquisition, and killing activity was calculated based on absolute numbers (cells / μl) normalized to the number of target cells in the absence of allogeneic effector NK cells and compound.

[0148] result Having demonstrated the cytotoxicity of CD25 Mab against EOL-1 cell line and AML22 cells (Example 1), we sought to evaluate its functional activity with AML patient material. To this end, we selected the four patients with the highest frequency of CD25+ AML cells and performed ex vivo ADCC assays. We showed specific killing of CD25+ AML cells in all samples at saturating antibody concentrations (Figure 7A). Moreover, we evaluated killing of Tregs in the same experimental setup and found that both AML and HD Tregs were efficiently depleted by CD25 Mab treatment (Figure 7B). Taken together, these results provide a proof-of-concept of the dual mechanism of action of CD25 Mab in AML patient samples using HD NK cells.

[0149] Example 3 Flow cytometry panel design, staining, and acquisition We proceeded according to recent guidelines for panel design and optimal validation (Liechti et al., 2021, Nat Immunol 22, 1190-1197). Of note, all antibodies were titrated and the panel was validated using a combination of fluorescence minus one (FMO) controls and biological controls (cell populations lacking one or a set of markers).

[0150] Cryopreserved AML patient and HD samples were thawed in DMEM / F-12 medium (Gibco) containing 10% FBS (Gibco). Cells were incubated with Human TruStain FcX (Biolegend) and stained with Zombie NIR (Biolegend) viability dye diluted in PBS. Cells were then stained for surface antigens (HLA-DR (G46-6), CD16 (3G8), CD45 (HI30), CD33 (P67.6), CD45RA (HI100), CD34 (8G12), CD56 (5.1H11), CD3 (UCHT1), CD19 (HIB19), CD117 (YB5.B8), CD25 (24212)) in staining buffer containing FACS buffer (PBS containing 0.1% BSA) and Brilliant Stain buffer (BD). Cells were stained with fluorochrome-conjugated antibodies against CD123 (9F5), CD69 (FN50), CD4 (A161A1), CD8a (RPA-T8), CD71 (M-A712), CD127 (A019D5), CD14, CD38 (HIT2), CD235A (HIR2), CLEC12A (50C1), PD-1 (EH12.1), and TIM3 (7D3). Cells were fixed and permeabilized using FoxP3 transcription factor staining set (eBioscience) and subsequently stained for intracellular antigens (FoxP3 (259D), Bcl-2 (Bcl-2 / 100), and Ki-67 (Ki67)) in 1× PERM buffer. Samples were acquired on a 5-laser Aurora Spectrocytometer (Cytek).

[0151] Pre-processing steps (application of compensation matrix, gating on each live single cell) were performed using SpectroFlo™ software. Unmixed files were checked individually and manual spillover correction was adjusted if necessary using the integrated software. Notably, only minor modifications were required. Events in FCS files that passed all quality control steps were exported for further computational analysis with R.

[0152] Computational flow cytometry data analysis We used a patient-centric approach to identify CD25+AML clusters and Tregs, applying the computational analysis workflow described below to each patient individually. FCS files were loaded into R and processed as described in the vignette for the flowCore R package. Logicle transformation was applied to the expression matrix. Density plots for each marker were visually inspected to define thresholds that allowed quantification of positive and negative expression, as well as positive cutoff values ​​assigned by conventional manual gating.

[0153] Dimensionality reduction was performed using the Uniform Manifold Approximation and Projection (UMAP) algorithm as part of the uwot R package. Unsupervised clustering was performed in the high-dimensional space using PhenoGraph (Levine et al., 2015, Cell 162, 184-197), and the results were visualized as color overlays on UMAP plots. To reach biologically meaningful cell populations, clusters generated by the algorithm were merged and manually annotated based on marker expression. Downstream analyses reported cell abundance, marker expression, and, where appropriate, percentage of positive cells. Cells negative for CD45 expression and all other markers present in the panel (non-immune cells) as well as very small clusters (<0.05%) were filtered out.

[0154] The CD25+ cell population identified by computer analysis was verified by manual gating. Figures were generated using Prism v8.4.2 (GraphPad Software).

[0155] result Internal tandem duplications (ITDs) in the FLT3 gene are present in approximately 25% of AML patients and are associated with poor prognosis and increased risk of relapse (Dohner et al., 2010, Blood 115, 453-474).

[0156] As previously reported by others (Angelini et al., 2015, Clin Cancer Res 21, 3977-3985), using high-dimensional flow cytometry analysis of AML patient samples, the inventors demonstrated that the presence of FLT3-ITD mutations results in a strong increase in the prevalence of CD25+ AML cells (data not shown).

[0157] Moreover, we observed that BCL-2 was highly expressed in CD25+ AML clusters with immature phenotype (data not shown). This finding is consistent with previous reports that showed high BCL-2 expression in the leukemia stem cell compartment (Lagadinou et al., 2013, Cell Stem Cell 12, 329-341; Renders et al., 2021, Blood 138, 3469-3469). Interestingly, CD25+ AML clusters were detected in all four patients treated with hypomethylating agents combined with venetoclax (HMA-VEN).

[0158] Thus, these results support the treatment of AML patients with FLT3-ITD mutations with anti-CD25 antibodies. These results also support the use of anti-CD25 antibodies in the treatment of AML, particularly to reduce the risk of relapse, e.g., as combination therapy with FLT3 inhibitors or BCL-2 inhibitors, e.g., venetoclax.

[0159] All publications cited in the above specification are incorporated herein by reference. It will be apparent to those skilled in the art that various modifications and variations can be made in the described methods and systems of the invention without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific 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 that are obvious to those skilled in molecular biology, cellular immunology, or related fields are intended to be within the scope of the following claims.

[0160] A summary of the sequences referenced in this application is provided in the table below.

[0161] [Table 2] TIFF2024533119000004.tif254170TIFF2024533119000005.tif253170TIFF2024533119000006.tif185170

Claims

1. A pharmaceutical composition for treating acute myeloid leukemia (AML) or diffuse large B-cell lymphoma (DLBCL) in a subject, comprising an anti-CD25 antibody.

2. The pharmaceutical composition of claim 1, wherein the anti-CD25 antibody inhibits IL-2 signaling via CD25 by less than 50% compared to IL-2 signaling in the absence of the antibody, preferably inhibits IL-2 signaling via CD25 by less than 25% compared to IL-2 signaling in the absence of the antibody.

3. The antibody (a) a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 2 to 5, CDR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 6 to 11, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; an antibody comprising (b) a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 24, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25; a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 26, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 27, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 28; and an antibody comprising (c) a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 31 to 33, CDR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 34 to 38, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39; a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42; an antibody comprising 2. The pharmaceutical composition of claim 1, selected from the group consisting of:

4. The pharmaceutical composition of claim 1, wherein the antibody is RG6292.

5. The pharmaceutical composition of claim 1, wherein the antibody binds to an epitope comprising at least one sequence selected from amino acids 150 to 158 of SEQ ID NO: 1, amino acids 176 to 180 of SEQ ID NO: 1, amino acids 42 to 56 of SEQ ID NO: 1, and amino acids 74 to 84 of SEQ ID NO: 1, preferably an epitope comprising amino acids 70 to 84 of SEQ ID NO:

1.

6. The pharmaceutical composition of claim 1, wherein the antibody kills cancer cells, Treg cells, AML blast cells and / or PMBC cells.

7. The pharmaceutical composition of claim 6, wherein the antibody kills Treg cells and blast cells having a CD25 expression level of greater than about 900 CD25 molecules per cell.

8. The pharmaceutical composition of claim 7, wherein the antibody kills Treg cells and blast cells having a CD25 expression level of more than about 1,000 CD25 molecules per cell, kills Treg cells and blast cells having a CD25 expression level in the range of about 1,000 to about 40,000 CD25 molecules per cell, or kills Treg cells and blast cells having a CD25 expression level in the range of about 1,000 to about 5,000 CD25 molecules per cell.

9. The antibody Induce ADCC activity; It is a monoclonal antibody; an IgG antibody, preferably an IgG1 antibody; is a monospecific antibody, preferably a bivalent monospecific antibody; defucosylated; and / or is a human or humanized antibody, The pharmaceutical composition of claim 1.

10. 2. The pharmaceutical composition of claim 1, wherein the antibody induces a decrease in CD16 expression in NK cells by up to 25% when the antibody and NK cells are co-incubated with cells expressing 900 to 5000 CD25 molecules per cell, and optionally the NK cells are CD56dim NK cells.

11. The pharmaceutical composition of claim 1, wherein the antibody is not conjugated to an additional therapeutic agent.

12. The pharmaceutical composition of claim 1, wherein the antibody is administered in combination with one or more additional therapeutic agents, preferably wherein the one or more additional therapeutic agents are selected from immune checkpoint inhibitors, cancer vaccines, FLT3 inhibitors, BCL-2 inhibitors, IDH inhibitors, hypomethylating agents, anthracyclines, and combinations thereof.

13. The antibody comprising: an immune checkpoint inhibitor that is a PD-1 antagonist, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody; a BCL-2 inhibitor, preferably wherein said BCL-2 inhibitor is venetoclax; and / or a hypomethylating agent, preferably wherein said hypomethylating agent is azacitidine; administered in combination with The pharmaceutical composition of claim 12.

14. The pharmaceutical composition of claim 1, wherein the antibody is used as a monotherapy.

15. 2. The pharmaceutical composition of claim 1, wherein the CD25 expression level in tumor cells from the subject is at least about 900 CD25 molecules per cell, preferably in the range of about 900 to about 5000 CD25 molecules per cell.

16. A combination of an anti-CD25 antibody as defined in any one of claims 1 to 11 and one or more further therapeutic agents for the treatment of acute myeloid leukemia or diffuse large B-cell lymphoma in a subject, wherein the anti-CD25 antibody and the further therapeutic agents are for separate, simultaneous or sequential administration.

17. 17. The combination of claim 16, wherein the one or more further therapeutic agents are as defined in claim 12.

18. 1. A method for selecting a patient suffering from acute myeloid leukemia for treatment with an anti-CD25 antibody, comprising determining the CD25 expression level on target cells in a sample from the patient, wherein the patient is suitable for treatment with the antibody if the cells have an expression level of greater than about 900 CD25 molecules per cell.

19. the sample is a bone marrow sample from the patient; the target cells are blast cells and / or Treg cells; and / or wherein the CD25 expression level is measured by flow cytometry.

20. The method of claim 18.

20. The method of claim 18, wherein the treatment comprises administering to the patient an anti-CD25 antibody, preferably wherein the anti-CD25 antibody is as defined in any one of claims 1 to 11.

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