Combination therapy of cd70 and bcl-2 inhibitors for the treatment of acute myeloid leukemia

Abstract

The present invention relates to combination therapies, in particular combination therapies for the treatment of myeloid malignancies. The combination therapies are particularly useful in methods for the treatment of acute myeloid leukemia (AML). The combination therapies comprise an antibody or antigen-binding fragment thereof that binds to CD70 and a BCL-2 inhibitor, preferably venetoclax or a pharmaceutically acceptable salt thereof.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201980082377.7, filed in the Chinese National Phase of PCT International Patent Application No. PCT / EP2019 / 085982, having an international filing date of 18 December 2019, entitled “Combination therapy of CD70 and venetoclax as a BCL-2 inhibitor for the treatment of acute myeloid leukemia”. TECHNICAL FIELD

[0002] The present invention relates to combination therapies, in particular combination therapies for the treatment of myeloid malignancies. The combination therapies are particularly useful for the treatment of acute myeloid leukemia (AML). The combination therapies comprise an antibody or antigen-binding fragment thereof that binds to CD70 and a BCL-2 inhibitor, for example venetoclax or a pharmaceutically acceptable salt thereof. The combination therapies can also comprise an additional anti-cancer agent, for example an agent used to treat AML, such as azacitidine or decitabine. BACKGROUND

[0003] In recent years, the development of new cancer treatments has focused on molecular targets, in particular proteins, that are implicated in cancer progression. The list of molecular targets involved in tumor growth, invasion and metastasis is constantly expanding and includes proteins that are overexpressed by tumor cells as well as targets associated with systems that support tumor growth, such as the vasculature and the immune system. The number of therapeutic agents or anti-cancer agents designed to interact with these molecular targets is also constantly increasing. A large number of targeted cancer drugs are currently approved for clinical use, and many more are in development.

[0004] CD70 has been identified as a particularly interesting molecular target as it is constitutively expressed in many types of hematological malignancies and solid cancers (Junker et al., (2005) J Urol. 173:2150-3; Sloan et al., (2004) Am J Pathol. 164:315-23; Held-Feindt and Mentlein (2002) Int J Cancer 98:352-6; Hishima et al., (2000) Am J Surg Pathol. 24:742-6; Lens et al., (1999) Br J Haematol. 106:491-503; Boursalian et al., (2009) Adv Exp Med Biol. 647:108-119; Wajant H. (2016) Expert Opin Ther Targets 20(8):959-973). CD70 is a type II transmembrane glycoprotein belonging to the tumor necrosis factor (TNF) superfamily, which mediates its action through binding to its cognate cell surface receptor CD27. Both CD70 and CD27 are expressed by multiple cell types of the immune system, and the CD70-CD27 signaling pathway has been implicated in the regulation of several different aspects of the immune response. This is reflected in the fact that CD70 overexpression occurs in various autoimmune diseases including rheumatoid arthritis and psoriatic arthritis as well as lupus (Boursalian et al., (2009) Adv Exp Med Biol. 647:108-119; Han et al., (2005) Lupus 14(8):598-606; Lee et al., (2007) J Immunol. 179(4):2609-2615; Oelke et al., (2004) Arthritis Rheum. 50(6):1850-1860).

[0005] CD70 expression has been associated with poor prognosis in several cancers including B-cell lymphoma, renal cell carcinoma and breast cancer (Bertrand et al., (2013) Genes Chromosomes Cancer 52(8):764-774; Jilaveanu et al., (2012) Hum Pathol. 43(9): 1394-1399; Petrau et al., (2014) J Cancer 5(9):761-764). CD70 expression has also been found on metastatic tissue in a high proportion of cases, suggesting a key role of this molecule in cancer progression (Jacobs et al., (2015) Oncotarget 6(15): 13462-13475). Constitutive expression of CD70 and its receptor CD27 on tumor cells of the hematopoietic lineage has been associated with a role of the CD70-CD27 signaling axis in directly regulating proliferation and survival of tumor cells (Goto et al., (2012) Leuk Lymphoma 53(8): 1494-1500; Lens et al., (1999) Br J Haematol. 106(2); 491-503; Nilsson et al., (2005) Exp Hematol. 33(12): 1500-1507; van Doorn et al., (2004) Cancer Res. 64(16): 5578-5586).

[0006] Upregulation of CD70 expression on tumors, particularly solid tumors that do not co-express CD27, also contributes to immune suppression in the tumor microenvironment in multiple ways. For example, it has been demonstrated that CD70 binding to CD27 on regulatory T cells increases the frequency of regulatory T cells, reduces tumor-specific T cell responses, and promotes tumor growth in mice (Claus et al., (2012) Cancer Res. 72(14):3664-3676). CD70-CD27 signaling can also inhibit immune responses through tumor-induced T lymphocyte apoptosis, as demonstrated in renal cell carcinoma, glioma, and glioblastoma cells (Chahlavi et al., (2005) Cancer Res. 65(12):5428-5438; Diegmann et al., (2006) Neoplasia 8(11):933-938; Wischusen et al., (2002) Cancer Res 62(9):2592-2599). Finally, CD70 expression has also been associated with T cell exhaustion, whereby lymphocytes exhibit a more differentiated phenotype and are unable to kill tumor cells (Wang et al., (2012) Cancer Res 72(23):6119-6129; Yang et al., (2014) Leukemia 28(9): 1872-1884).

[0007] Given the importance of CD70 in cancer development, CD70 is an attractive target for anti-cancer therapies and antibodies targeting this cell surface protein are in clinical development (Jacob et al., (2015) Pharmacol Ther. 155:1-10; Silence et al., (2014) mAbs 6(2):523-532). SUMMARY

[0008] The present invention relates to combination therapies comprising an antibody or antigen-binding fragment thereof that binds to CD70. As noted above, the list of proteins implicated in tumor growth is ever expanding, and combination therapies that target two or more of these proteins as anti-cancer treatments are becoming increasingly attractive. In the combination therapies of the present invention, the antibody or antigen-binding fragment thereof that binds to CD70 is combined with a BCL-2 inhibitor, such as venetoclax or a pharmaceutically acceptable salt thereof. Overexpression of BCL-2 in cancer cells confers resistance to apoptosis, and thus, inhibition of this protein can promote tumor cell death. As explained elsewhere herein, venetoclax is an example of a potent, selective small molecule inhibitor of the BCL-2 protein. As reported herein, the combination of the antibody or antigen-binding fragment thereof that binds to CD70 and the BCL-2 inhibitor, such as venetoclax or a pharmaceutically acceptable salt thereof, provides an effective therapy for treating cancer, particularly myeloid malignancies, such as acute myeloid leukemia (AML).

[0009] In a first aspect, the present invention provides a composition comprising (i) an antibody or antigen-binding fragment thereof that binds to CD70; and (ii) a BCL-2 inhibitor. In certain preferred embodiments, the BCL-2 inhibitor is Compound (I) or a pharmaceutically acceptable salt thereof, as shown below.

[0010]

[0011] Compound (I) is also referred to herein as venetoclax.

[0012] In certain embodiments, the antibody or antigen-binding fragment that binds to CD70 is selected from the group consisting of: (i) an antibody or antigen-binding fragment comprising a variable heavy chain domain (VH) and a variable light chain domain (VL) comprising heavy chain CDRs (HCDR3, HCDR2, and HCDR1) and light chain CDRs (LCDR3, LCDR2, and LCDR1): HCDR3 comprising or consisting of SEQ ID NO: 3; HCDR2 comprising or consisting of SEQ ID NO: 2; HCDR1 comprising or consisting of SEQ ID NO: 1; LCDR3 comprising or consisting of SEQ ID NO: 7; LCDR2 comprising or consisting of SEQ ID NO: 6; and LCDR1 comprising or consisting of SEQ ID NO: 5; (ii) an antibody or antigen-binding fragment comprising: a VH domain comprising an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95% identical to SEQ ID NO: 4 and a VL domain comprising an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95% identical to SEQ ID NO: 8; or (iii) ARGX-110. In certain embodiments, the antibody is an IgG, preferably an IgG1.

[0013] The CD70 antibody or antigen-binding fragment of the composition can have one or more effector functions. In certain embodiments, the antibody or antigen-binding fragment has ADCC activity; and / or comprises afucosylated antibody domains; and / or has CDC activity; and / or has ADCP activity. In preferred embodiments, the CD70 antibody is ARGX-110.

[0014] In certain embodiments, the CD70 antigen-binding fragment of the composition is independently selected from the group consisting of: an antibody light chain variable domain (VL); an antibody heavy chain variable domain (VH); a single chain antibody (scFv); a F(ab')2 fragment; a Fab fragment; a Fd fragment; a Fv fragment; a one-armed (monovalent) antibody; a diabody, triabody, tetrabody, or any antigen-binding molecule formed by combination, assembly, or conjugation of such antigen-binding fragments.

[0015] In certain embodiments, the CD70 antibody or antigen-binding fragment thereof and the BCL-2 inhibitor are formulated as separate compositions. In certain embodiments, the CD70 antibody or antigen-binding fragment thereof and venetoclax or a pharmaceutically acceptable salt thereof are formulated as separate compositions.

[0016] The compositions of the application can comprise one or more additional therapeutic agents, such as at least one additional anti-cancer agent, preferably an agent for the treatment of myeloid malignancies. In certain embodiments, the additional anti-cancer agent is an agent for the treatment of acute myeloid leukemia (AML). In preferred embodiments, the compositions comprise a hypomethylating agent, preferably azacitidine or decitabine.

[0017] In another aspect, the present application provides a composition according to the first aspect of the application for use in therapy. In particular, the present application provides a composition according to the first aspect of the application for use in the treatment of a malignancy, preferably a myeloid malignancy, in a human subject. The present application also provides a method for the treatment of a malignancy, preferably a myeloid malignancy, in a human subject, the method comprising administering to the subject an effective amount of any of the compositions according to the first aspect of the application.

[0018] The present application also provides an antibody or antigen-binding fragment thereof that binds to CD70 for use in the treatment of a malignancy, preferably a myeloid malignancy, in a human subject, wherein the antibody molecule is administered in combination with a BCL-2 inhibitor, preferably Compound (I), or a pharmaceutically acceptable salt thereof. The present application also provides a BCL-2 inhibitor, preferably Compound (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of a myeloid malignancy in a human subject, wherein the BCL-2 inhibitor, preferably Compound (I), or a pharmaceutically acceptable salt thereof, is administered in combination with an antibody or antigen-binding fragment thereof that binds to CD70.

[0019] The compositions of the present application are particularly advantageous in that they exhibit synergistic efficacy. Thus, preferably, in embodiments of all aspects of the present application, the dose of the CD70 antibody or antigen-binding fragment thereof administered and / or provided in the composition and the dose of the BCL-2 inhibitor administered and / or provided in the composition are each selected so that the composition provides synergistic treatment.

[0020] In certain preferred embodiments of the compositions of the present application, the CD70 antibody or antigen-binding fragment thereof and the BCL-2 inhibitor are each present in the composition in an amount sufficient to provide synergistic cell killing when cultured with an AML cell line selected from the group consisting of: NOMO-1, MOLM-13, NB4 and MV4-11.

[0021] Regarding the malignant tumor to be treated with the composition of the present invention, the malignant tumor may be a newly diagnosed myeloid malignancy; a relapsed or refractory myeloid malignancy; or a myeloid malignancy selected from the following: acute myeloid leukemia (AML); myelodysplastic syndrome (MDS); myeloproliferative neoplasm (MPN); chronic myeloid leukemia (CML); and chronic myelomonocytic leukemia (CMML). In a particularly preferred embodiment, the composition of the present invention is used to treat acute myeloid leukemia (AML).

[0022] In some embodiments, the subjects or patients treated according to the method of the invention are newly diagnosed AML patients who are unsuitable for standard intensive chemotherapy. The subjects may be newly diagnosed AML patients aged 75 years or older, or newly diagnosed AML patients with comorbidities that preclude the use of standard intensive chemotherapy.

[0023] In some embodiments, the CD70 antibody or its antigen-binding fragment is administered at a dose ranging from 0.1 mg / kg to 25 mg / kg, preferably 10 mg / kg. Optionally or additionally, the BCL-2 inhibitor, preferably veneclade or a pharmaceutically acceptable salt thereof, may be administered at a dose ranging from 100 mg to 600 mg. In a preferred embodiment, the method described herein comprises administering a composition further comprising azacitidine, wherein the azacitidine is administered at 75 mg / kg. 2 The dosage is administered. In another preferred embodiment, the method herein includes administering a composition further comprising decitabine, wherein the decitabine is administered at a dose of 20 mg / m². 2 Dosage administration.

[0024] In some embodiments, the method further includes monitoring the patient's myeloblast count. The patient's peripheral blood and / or myeloblast count may be reduced, for example, to less than 25%, to less than 5%, to minimal residual disease levels, or to undetectable levels. In some embodiments, the myeloblast count is reduced to 5% to 25%, and the percentage of myeloblasts is reduced by more than 50% compared to pre-treatment levels.

[0025] In some embodiments, the method induces a partial response. In some embodiments, the method induces a complete response, optionally accompanied by platelet recovery and / or neutrophil recovery. The method can induce transfusion-independent red blood cell or platelet count, or both, for 8 weeks or longer, 10 weeks or longer, or 12 weeks or longer. In some embodiments, the method reduces mortality after a 30-day or 60-day time period.

[0026] In certain embodiments, the method increases survival. For example, the method can increase survival relative to one or more standard of care agents used to treat the particular myeloid malignancy being treated with the composition. The method can induce a negative minimal residual disease state.

[0027] In certain embodiments, the method further comprises the step of subjecting the subject to a bone marrow transplant. Alternatively or additionally, the method can further comprise the step of administering one or more additional anti-cancer agents. The one or more additional anti-cancer agents can be selected from any agent useful in treating myeloid malignancies, preferably AML. Preferred agents can be selected from a selectin inhibitor (e.g., GMI-1271); a FMS-like tyrosine kinase receptor 3 (FLT3) inhibitor (e.g., midostaurin); a cyclin-dependent kinase inhibitor; an aminopeptidase inhibitor; a JAK / STAT inhibitor; cytarabine; an anthracycline (e.g., daunorubicin, idarubicin); doxorubicin; hydroxyurea; Vyxeos (daunorubicin + cytarabine); an IDH1 or IDH2 inhibitor, such as Idhifa (or Enasidenib) or Tibsovo (or ivosidenib); a Smoothened inhibitor, such as Glasdegib; a BET bromodomain inhibitor; a CD123 or CD33 targeting agent; an HDAC inhibitor; an LSC targeting agent; an AML bone marrow niche targeting agent; and a NEDD8 activating enzyme inhibitor, such as Pevonedistat. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1: Cusatuzumab and decitabine co-treatment synergistically eliminated NOMO-1 AML cells. NOMO-1 AML cells were treated with vehicle, cusatuzumab, venetoclax or decitabine alone or in constant ratios combinations in the presence of CFSE-labeled NK cells (ratio 1 : 1). The number of NOMO-1 AML cells per well was counted after 72 hours, the extent of viable cells was determined by annexin V staining and the effect of drug treatment was calculated as the ratio of surviving cells to vehicle-treated cells. Combination index (CI) values were calculated and a plot of values between 0 and 10 versus the fraction affected (Fa) values was drawn. Fa-CI plots [Chou-Talalay plots] assessing synergy and / or antagonism are illustrated. Fa values of 0, 0.5 and 1 correspond to 0%, 50% and 100% of cells killed. CI values of <1, 1, >1 indicate synergy, additivity and antagonism, respectively. 50 s represent the Fa values reached by the Ve / Cusa (lower Fa) and Ve / De / Cusa (upper Fa) combinations at IC 50 concentrations. Ve / De: venetoclax and decitabine; De / Cusa: decitabine and cusatuzumab; Ve / Cusa: venetoclax and cusatuzumab; Ve / De / Cusa: venetoclax and decitabine and cusatuzumab.

[0029] Figure 2 : Cusatuzumab and decitabine co-treatment synergistically eliminated NOMO-1 AML cells. Individual CI-Fa plots for the data of each combination are illustrated in Figure 1 (A) venetoclax and decitabine; (B) decitabine and cusatuzumab; (C) venetoclax and cusatuzumab; (D) venetoclax, decitabine and cusatuzumab.

[0030] Figure 3: Combination of gemtuzumab ozogamicin and decitabine synergistically eliminated NB4 AML cells. NB4 AML cells were treated with vehicle, gemtuzumab ozogamicin, venetoclax or decitabine alone or in constant ratios combinations in the presence of CFSE labeled NK cells (ratio 1 : 1). The number of NB4 AML cells per well was counted after 72 hours, the extent of viable cells was determined by annexin V staining and the effect of drug treatment was calculated as the ratio of surviving cells to vehicle treated cells. Combination index (CI) values were calculated and a plot of values between 0 and 10 versus the fraction affected (Fa) values was drawn. The Fa-CI plot [Chou-Talalay plot] is shown to assess synergy and / or antagonism. Fa values of 0, 0.5 and 1 correspond to 0%, 50% and 100% of cells killed. (A) Venetoclax and decitabine; (B) Venetoclax and gemtuzumab ozogamicin; (C) Decitabine and gemtuzumab ozogamicin; (D) Venetoclax, decitabine and gemtuzumab ozogamicin.

[0031] Figure 4 : Combination of gemtuzumab ozogamicin and decitabine synergistically eliminated MOLM-13 AML cells. MOLM-13 AML cells were treated with vehicle, gemtuzumab ozogamicin, venetoclax or decitabine alone or in constant ratios combinations in the presence of CFSE labeled NK cells (ratio 1 : 1). The number of MOLM-13 AML cells per well was counted after 72 hours, the extent of viable cells was determined by annexin V staining and the effect of drug treatment was calculated as the ratio of surviving cells to vehicle treated cells. Combination index (CI) values were calculated and a plot of values versus the fraction affected (Fa) values was drawn. The Fa-CI plot [Chou-Talalay plot] is shown to assess synergy and / or antagonism. Fa values of 0, 0.5 and 1 correspond to 0%, 50% and 100% of cells killed. (A) Venetoclax and decitabine; (B) Venetoclax and gemtuzumab ozogamicin; (C) Decitabine and gemtuzumab ozogamicin; (D) Venetoclax, decitabine and gemtuzumab ozogamicin.

[0032] Figure 5: Combination of venetoclax and inotuzumab ozogamicin treatment synergistically eradicates MV4-11 AML cells in vitro. MV4-11 AML cells were treated with vehicle, inotuzumab ozogamicin, venetoclax or the combination in constant proportions in the presence of CFSE-labeled NK cells (ratio 1 : 1). The number of MV4-11 AML cells per well was counted after 72 hours, the extent of viable cells was determined by annexin V staining and the effect of drug treatment was calculated as the ratio of surviving cells to vehicle-treated cells. Combination index (CI) values were calculated and a plot of values versus fraction affected (Fa) values was drawn. Fa-CI plots [Chou-Talalay plots] assessing synergy and / or antagonism are illustrated. Fa values of 0, 0.5 and 1 correspond to 0%, 50% and 100% of cells killed.

[0033] Figure 6 : Combination of venetoclax and inotuzumab ozogamicin treatment synergistically eradicates leukemia stem cells (LSCs) in vitro. CD34 + CD38 - AML LSCs were cultured overnight in the presence of inotuzumab ozogamicin (Cusa: 0.3 pg / ml) or venetoclax (Ve: 6 nM) alone or in combination with NK cells (ratio 1 : 1), followed by plating in methylcellulose. Colony formation was assessed after 14 days. (A) Absolute number of colonies per well at the first plating after treatment; (B) cells harvested from the first plating were re-plated (second plating) and colonies per well were assessed after 14 days.

[0034] Data are expressed as mean ± S.D. Statistics: one-way ANOVA; Tukey's post-test; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0035] Figure 7 : Combination of venetoclax and inotuzumab ozogamicin treatment synergistically eradicates LSCs in vitro. Data shown correspond to Figure 6 data normalized to the average number of colonies per well after vehicle treatment for each patient. (A) First plating; (B) second plating.

[0036] Figure 8 : Combination of venetoclax and inotuzumab ozogamicin treatment synergistically eradicates LSCs in vitro. CD34 + CD38 -AML LSCs were cultured overnight in the presence of Cusatuzumab (Cusa: 0.3 pg / ml), Venetoclax (Ve: 6 nM) or Decitabine (0.01 pM) alone or in combination thereof in the presence of NK cells (ratio 1 : 1) followed by plating in methylcellulose. Colony formation was assessed after 14 days. Data are presented as mean ± S.D. (A) Results from individual patients; (B) Results from P4 and P5.

[0037] Figure 9 CD70 mRNA expression (percent relative to housekeeping gene) after 24 hours or 48 hours of treatment with vehicle (Veh) or Venetoclax (Ven).

[0038] Figure 10 CD70 protein and mRNA expression of MOLM-13 cells and NOMO-1 cells in the presence (grey bars) and absence (black bars) of Venetoclax. Significance was determined using Student's t-test. MFI = mean fluorescence intensity. ***, P < 0.001. DETAILED DESCRIPTION

[0039] A. Definitions

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0041] “Combination therapy”: The term “combination therapy” as used herein refers to a treatment in which two or more therapeutic agents are administered to a subject, e.g., a human subject. The “combination” described herein is used in combination therapy. Typically, two or more therapeutic agents are administered to treat a single disease, in this case cancer or a malignancy. The combination or combination therapy of the present application comprises an antibody or antigen-binding fragment that binds to CD70 and a BCL-2 inhibitor, preferably the small molecule inhibitor Venetoclax or a pharmaceutically acceptable salt thereof. As described elsewhere herein, the agents included in the combination therapy can be co-formulated for administration, or can be provided separately, e.g., as separate compositions, for administration to a subject or patient in need thereof.

[0042] "Antibody": The term "antibody" as used herein is intended to encompass full-length antibodies and variants thereof, including but not limited to modified antibodies, humanized antibodies, germlined antibodies. The term "antibody" is used herein generally to refer to an immunoglobulin polypeptide having a combination of two heavy chains and two light chains, wherein the polypeptide has significant specific immunoreactive activity to an antigen of interest (in this document, CD70). For antibodies of the IgG class, the antibody comprises two identical light chain polypeptide chains having a molecular weight of about 23,000 daltons and two identical heavy chains having a molecular weight of 53,000-70,000. The four chains are linked by disulfide bonds into a "Y" configuration, with the light chains beginning at the mouth of the "Y" and continuing around the heavy chains through the variable region. The light chains of antibodies are classified as kappa or lambda. Each heavy chain class can be combined with either kappa or lambda light chains. Generally, when an immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, the light and heavy chains are covalently bonded to one another, and the "tail region" portions of the two heavy chains are bonded to one another by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain.

[0043] Those skilled in the art will appreciate that the heavy chains are classified as gamma, mu, alpha, delta, or epsilon, with some subclasses among them (e.g., gamma1-gamma4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well characterized and known to confer functional specialization. The term "antibody" as used herein encompasses antibodies from any class or subclass of antibodies.

[0044] "Antigen binding fragment": The term "antigen binding fragment" as used herein refers to a fragment that is partial or partial to a full-length antibody or antibody chain, which comprises fewer amino acid residues than an intact or complete antibody, while retaining antigen binding activity. An antigen binding fragment of an antibody includes a peptide fragment that exhibits specific immunoreactive activity to the same antigen (e.g., CD70) as the antibody. The term "antigen binding fragment" as used herein is intended to encompass antibody fragments selected from the group consisting of: an antibody light chain variable domain (VL); an antibody heavy chain variable domain (VH); a single chain antibody (scFv); a F(ab')2 fragment; a Fab fragment; a Fd fragment; a Fv fragment; a single arm (monovalent) antibody; a diabody, triabody, tetrabody, or any antigen binding molecule formed by combination, assembly, or conjugation of such antigen binding fragments. The term "antigen binding fragment" as used herein can also encompass an antibody fragment selected from the group consisting of: a unibody; a domain antibody; and a nanobody. Fragments can be obtained, for example, via chemical or enzymatic treatment of an intact or complete antibody or antibody chain or by recombinant means.

[0045] “Specific” and “multispecific antibodies”: Antibodies and antigen-binding fragments used in the combination therapies described herein bind to a particular target antigen (e.g., CD70). Preferably, antibodies and antigen-binding fragments bind “specifically” to their target antigen, where the term “specifically bind” refers to the ability of any antibody or antigen-binding fragment to preferentially bind to a given target (e.g., CD70). Antibodies and antigen-binding fragments of the compositions and methods of the present application can be monospecific and contain one or more binding sites that specifically bind a particular target. Antibodies and antigen-binding fragments of the compositions and methods of the present application can be incorporated into “multispecific antibody” formats, e.g., bispecific antibodies, where the multispecific antibody binds to two or more target antigens. To achieve multiple specificity, “multispecific antibodies” are typically engineered to include different combinations or pairings of heavy and light chain polypeptides with different VH-VL pairs. Multispecific antibodies, particularly bispecific antibodies, can be engineered to adopt the overall conformation of a natural antibody, e.g., a Y-shaped antibody with Fab arms of different specificity conjugated to an Fc region. Alternatively, multispecific antibodies, e.g., bispecific antibodies, can be engineered to adopt a non-native conformation, e.g., where variable domains or pairs of variable domains of different specificity are located at opposite ends of an Fc region.

[0046] “Modified antibodies”: The term “modified antibodies” as used herein includes synthetic forms of antibodies that are altered so that they are not naturally occurring, e.g., antibodies comprising at least two heavy chain portions without containing two complete heavy chains (such as domain-deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) that are altered to bind to two or more different antigens or different epitopes on a single antigen; heavy chain molecules linked to scFv molecules, etc. scFv molecules are known in the art and are described, e.g., in U.S. Patent 5,892,019. In addition, the term “modified antibodies” includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc. antibodies that bind to three or more copies of the same antigen). In another embodiment, the modified antibodies of the present application are fusion proteins comprising at least one heavy chain portion lacking a CH2 domain and comprising a binding domain of a polypeptide comprising a binding portion of one member of a receptor ligand pair.

[0047] "Humanized substitution": The term "humanized substitution" as used herein refers to an amino acid substitution in which an amino acid residue present at a particular position in a VH domain or VL domain of an antibody is replaced with an amino acid residue present at the equivalent position in a reference human VH domain or VL domain. The reference human VH domain or VL domain can be a VH domain or VL domain encoded by a human germline. Humanized substitutions can be made in the framework regions and / or CDRs of the antibodies defined herein.

[0048] "Humanized variant": The term "humanized variant" or "humanized antibody" as used herein refers to a variant antibody that contains one or more "humanized substitutions" as compared to a reference antibody, wherein a portion of the reference antibody (e.g., a VH domain and / or VL domain or a portion thereof containing at least one CDR) has amino acids derived from a non-human species, and the "humanized substitutions" occur within the amino acid sequence derived from the non-human species.

[0049] "Germlined variant": The term "germlined variant" or "germlined antibody" is used herein to specifically refer to a "humanized variant" in which the "humanized substitutions" result in one or more amino acid residues present at one or more particular positions in a VH domain or VL domain of an antibody being replaced with amino acid residues present at the equivalent positions in a reference human VH domain or VL domain encoded by a human germline. It is typical for any given "germlined variant" that the replacement amino acid residues substituted into the germlined variant are taken from only or primarily a single human germline-encoded VH domain or VL domain. The terms "humanized variant" and "germlined variant" are often used interchangeably. The introduction of one or more "humanized substitutions" into a VH domain or VL domain of camelid origin (e.g., a llama origin) results in the creation of a "humanized variant" of the camelid (llama)-origin VH domain or VL domain. If the amino acid residues substituted therein are derived primarily or only from a single human germline-encoded VH domain or VL domain sequence, the result can be a "human-germlined variant" of the camelid (llama)-origin VH domain or VL domain.

[0050] “CD70”: The term “CD70” or “CD70 protein” or “CD70 antigen” as used herein are used interchangeably and refer to a member of the TNF ligand family that is a ligand for TNFRSF7 / CD27. CD70 is also known as CD27L or TNFSF7. The term “human CD70 protein” or “human CD70 antigen” or “human CD70” are used interchangeably to specifically refer to the human homolog, including the native human CD70 protein naturally expressed on the surface of human cells in vivo and / or in cultured human cell lines as well as recombinant forms and fragments thereof. Particular examples of human CD70 include the polypeptide having the amino acid sequence set forth under NCBI Reference Sequence accession number NP_001243 or the extracellular domain thereof.

[0051] “BCL-2 family”: The term “BCL-2 family” or “BCL-2 protein family” as used herein refers to the collection of pro-apoptotic and anti-apoptotic proteins related to BCL-2, see Delbridge et al. (2016) Nat Rev Cancer. 16(2):99-109. At least 16 members of this family are divided into three functional groups: (i) BCL-2-like proteins (e.g. BCL-2, BCL-X L / BCL2L1, BCLW BCL2L2, MCL2, BFL1 / BCL2A1); (ii) BAX and BAK; and (iii) BH3-only proteins (e.g. BIM, PUMA, BAD, BMF, BID, NOXA, HRK, BIK). The BCL-2 protein family plays an indispensable role in regulating the intrinsic apoptotic pathway, with anti-apoptotic members of the family (e.g. BCL-2, BCL-X L ) generally antagonizing pro-apoptotic members (e.g. BAX and BIM). Dysregulation of BCL-2 family members has been observed in many cancers, for example through gene translocation, amplification, overexpression, and mutation. A downstream effect of this dysregulation is often apoptosis resistance, which can promote cancer growth.

[0052] “BCL-2”: As used herein, “BCL-2” or “BCL-2 protein” refers to the first member of the BCL-2 protein family identified in humans, B-cell lymphoma 2. The cDNA encoding human BCL-2 was cloned in 1986, and the key role of the protein in inhibiting apoptosis was elucidated in 1988. BCL-2 has been found to be upregulated in several different types of cancer. For example, BCL-2 is activated by t(14;18) chromosomal translocation in follicular lymphoma. Amplification of the BCL-2 gene has also been reported in different cancers including leukemias (such as CLL), lymphomas (such as B-cell lymphoma), and some solid tumors (e.g., small cell lung cancer). Human BCL-2 is encoded by the BCL2 gene (UniProtKB - P10415) and has the amino acid sequence set forth under NCBI Reference Sequence NP_000624.2 and NP_000648.2.

[0053] “BCL-2 inhibitor”: As used herein, a BCL-2 inhibitor refers to any agent, compound, or molecule capable of specifically inhibiting the activity of BCL-2, particularly an agent, compound, or molecule capable of inhibiting the anti-apoptotic activity of BCL-2. Examples of BCL-2 inhibitors suitable for use in the compositions described herein include B-cell lymphoma homology 3 (BH3) mimetic compounds (Merino et al. (2018) Cancer Cell. 34(6): 879-891). Particular BCL-2 inhibitors include, but are not limited to, venetoclax, ABT-737 (Oltersdorf, T. et al. (2005) Nature 435: 677-681), navitoclax / ABT-263 (Tse, C. et al. (2008) Cancer Res. 68: 3421-3428), BM-1197 (Bai, L. et al. (2014) PLoS ONE 9: e99404), S44563 (Nemati, F. et al. (2014) PLoS ONE 9: e80836), BCL2-32 (Adam, A. et al. (2014) Blood 124: 5304), AZD4320 (Hennessy, E.J. et al. (2015) ACS Medicinal Chemistry annual meeting https: / / www.acsmedchem.org / ama / orig / abstracts / mediabstractf2015.pdf_abstr. 24), and S55746 (International Standard Randomised Controlled Trial Number Registry. ISRCTN http: / / www.isrctn.com / ISRCTN04804337 (2016)). Additional examples of BCL-2 inhibitors are described in Ashkenazi, A et al. (2017) Nature Reviews Drug Discovery 16: 273-284, which is incorporated herein by reference.

[0054] “Venetoclax”: As used herein, the term “venetoclax” refers to a compound having the chemical structure shown below:

[0055]

[0056] The compound is referred to herein as “Compound (I)”. Venetoclax is a potent, selective, orally bioavailable inhibitor of the BCL-2 protein. It has the empirical formula C 45 H50 C1N7O7S and a molecular weight of 868.44. It has very low water solubility. Venetoclax can be chemically described as 4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1 - en-1 -yl]methyl}piperazin-1 -yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4- ylmethyl)amino]phenyl}sulfonyl)-2-(1 H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide). Alternative names for venetoclax include ABT-199; chemical name 1257044-40-8; GDC-0199.

[0057] Venetoclax was approved by the U.S. Food and Drug Administration (FDA) in 2015 for the treatment of adult patients with chronic lymphocytic leukemia (CLL) or small lymphocytic leukemia (SLL) who have received at least one prior therapy. Venetoclax is also approved in the U.S. in combination with azacitidine or decitabine or low-dose cytarabine for the treatment of newly diagnosed acute myeloid leukemia (AML) in adults 75 years or older or in adults with comorbidities that preclude the use of intensive induction chemotherapy.

[0058] “Myeloid malignancy”: As used herein, the term “myeloid malignancy” refers to any clonal disease of hematopoietic stem or progenitor cells. Myeloid malignancies or myeloid malignancies include chronic conditions and acute conditions. Chronic conditions include myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), and chronic myelomonocytic leukemia (CMML), and acute conditions include acute myeloid leukemia (AML).

[0059] “Acute myeloid malignancy”: As used herein, “acute myeloid leukemia” or “AML” refers to a hematopoietic neoplasm that involves cells of the bone marrow. AML is characterized by clonal proliferation of myeloid precursors with reduced differentiation capacity. AML patients exhibit accumulation of blasts in the bone marrow. As used herein, “blast cell” or simply “blast” refers to a clonal myeloid progenitor cell that exhibits disrupted differentiation potential. Blasts also typically accumulate in the peripheral blood of AML patients. Generally, if a patient exhibits 20% or more blasts in the bone marrow or peripheral blood, then AML is diagnosed.

[0060] “Standard intensive chemotherapy”: As used herein, “standard intensive chemotherapy” (also referred to herein as “intensive induction therapy” or “induction therapy”) refers to so-called “7+3” induction chemotherapy, which is characterized by the administration of 7 days of high-dose cytarabine, followed by 3 days of an anthracycline (e.g., daunorubicin or idarubicin). Standard intensive chemotherapy can be given to eligible newly diagnosed AML patients to induce complete remission of AML, often in order to allow the patient to undergo stem cell transplantation after successful chemotherapy. As explained herein, not all newly diagnosed AML patients are eligible to receive such standard intensive chemotherapy.

[0061] “Leukemia stem cell”: As used herein, “leukemia stem cell” or “LSC” is a subset of blast cells associated with AML. LSCs are blast cells that have stem cell properties, and thus, if they are transplanted into an immunodeficient recipient, are able to initiate leukemia. LSCs can self-renew by giving rise to leukemia, and can also partially differentiate into non-LSC conventional blast cells that resemble the original disease, but are not self-renewing. LSCs occur at a frequency that ranges from 1 / 10,000 to 1 / 1,000,000 as a proportion of primary AML blast cells (Pollyea and Jordan (2017) Blood 129: 1627-1635, incorporated herein by reference). LSCs can be characterized as cells that are CD34+, CD38-, optionally also CD45- and / or CD123+. LSCs can also be characterized as CD45dim, SSClo, CD90+ CD34+ cells.

[0062] “Anti-cancer agent”: As used herein, an anti-cancer agent refers to any agent that is capable of directly or indirectly preventing, inhibiting or treating the growth of a cancer. Such agents include chemotherapeutic agents, immunotherapeutic agents, anti-angiogenic agents, radionuclides, and the like, many examples of which are known to those skilled in the art.

[0063] B. Combination therapy using CD70 antibodies and BCL-2 inhibitors

[0064] The present application provides a combination therapy comprising (i) an antibody or antigen-binding fragment thereof that binds to CD70; and (ii) a BCL-2 inhibitor.

[0065] As described elsewhere herein, CD70 has been characterized as an attractive target for anti-cancer therapy. CD70 is constitutively expressed on many types of hematological malignancies and solid cancers and its expression has been associated with poor prognosis for several cancers. Antibodies targeting CD70 have been developed and some have been advanced into clinical development.

[0066] It has been found that antibodies targeting CD70 are particularly effective for treating myeloid malignancies, in particular for treating subjects suffering from acute myeloid leukemia (AML). Results from a phase I / II clinical trial of CD70 antibody ARGX-110 in patients with AML revealed surprising efficacy in this indication, in particular in newly diagnosed patients classified as not suitable for standard intensive chemotherapy (see WO2018 / 229303). It is particularly noteworthy that in the clinical study, the CD70 antibody effectively reduced leukemia stem cells (LSCs) in AML patients when used in combination with azacitidine. Tests performed on LSCs isolated from patients in the trial revealed signs of increased asymmetric division of LSCs, which is indicative of differentiation into myeloid cells. Taken together, these results suggest that the CD70 antibody depletes the LSC pool in AML patients, thereby increasing the likelihood of remission and reducing the risk of relapse.

[0067] The present invention combines a CD70 antibody or antigen binding fragment thereof with a BCL-2 inhibitor.

[0068] BCL-2 proteins are members of the BCL-2 family. This family comprises over 20 proteins. Members of the BCL-2 family are involved in the regulation of the intrinsic apoptosis pathway and play a fundamental role in regulating the balance between cell survival and death.

[0069] BCL-2 proteins are anti-apoptotic members of the BCL-2 family and are upregulated in many different types of cancer. Overexpression of BCL-2 allows tumor cells to escape apoptosis by sequestering pro-apoptotic proteins. BCL-2 is highly expressed in many hematological malignancies and is a major pro-survival protein in diseases such as chronic lymphocytic leukemia (CLL), follicular lymphoma, and mantle cell lymphoma. Inhibition of BCL-2 suppresses the anti-apoptotic or pro-survival activity of this protein.

[0070] It has been reported that anti-apoptotic members of the BCL-2 family, including BCL-2, are overexpressed in primary AML samples (Bogenberger et al. (2014) Leukemia 28(2); 1657-65). It has also been reported that BCL-2 is overexpressed in leukemia stem cells (LSCs) obtained from AML patients (Lagadinou et al. (2013) Cell Stem Cell 12(3); 329-341). Inhibition of BCL-2 in ex vivo LSC populations led to selective eradication of quiescent LSCs (Lagadinou et al. (2013) Cell Stem Cell 12(3); 329-341).

[0071] Without wishing to be bound by theory, it is believed that the compositions of the application are particularly effective for treating AML due to the combined therapeutic effect of the CD70 antibody or antigen binding fragment and the BCL-2 inhibitor, particularly the combined effect at the level of LSCs. The self-renewal capacity of LSCs means that the persistence of these cells is a major factor in disease relapse.

[0072] As demonstrated in the examples, the compositions of the application exhibit synergistic therapeutic efficacy on AML cells, that is to say, the level of inhibition induced by the compositions is greater than the additive effect of the single therapies alone. Methods for determining synergistic interactions are familiar to the person skilled in the art and are described in the examples. The preferred method for determining whether a combination produces a synergistic effect is the Chou-Talalay method (Chou, TC. Cancer Res. (2010) 70(2); 440-6, incorporated herein by reference).

[0073] The synergistic efficacy of the compositions of the application translates into potent inhibition of primary LSC cells from AML patients. The combination therapy of the application thus targets both the blast cell and LSC compartments, thereby increasing the likelihood of disease remission while reducing the risk of relapse.

[0074] In certain preferred embodiments of the compositions of the application, the BCL-2 inhibitor is venetoclax or a pharmaceutically acceptable salt thereof. Venetoclax is a small molecule inhibitor of BCL-2, described in US 2010 / 0305122 (incorporated herein by reference).

[0075] By inhibiting BCL-2, venetoclax inhibits the anti-apoptotic or pro-survival activity of this protein. Venetoclax rapidly induces apoptosis in most CLL cells and lymphoma cell lines overexpressing BCL-2.

[0076] Early studies suggested that venetoclax can be useful as a therapy for AML (Konopleva et al. (2016) Cancer Discov. 6(10); 1106-17). However, it was found to have limited activity as a monotherapy. Subsequent studies explored the efficacy of combinations of venetoclax with hypomethylating agents (i.e. azacitidine and decitabine), and found that these combinations were particularly effective (Bogenberger et al. (2015) Leuk Lymphoma 56(1): 226-229). Clinical trials have been conducted to test combinations of venetoclax with azacitidine, decitabine or low-dose cytarabine (Dinardo et al. (2018) Lancet Oncol. 19(2): 216-228; Dinardo et al. (2019) Blood 133(1); 7-17). The results of these trials have led to FDA approval of venetoclax in combination with azacitidine, decitabine or low-dose cytarabine for the treatment of newly diagnosed acute myeloid leukemia (AML) in adults 75 years or older or with comorbidities that do not allow the use of intensive induction chemotherapy.

[0077] In certain alternative embodiments of the application, the BCL-2 inhibitor is a B-cell lymphoma homology domain 3 (BH3) mimetic compound. In certain embodiments, the BCL-2 inhibitor is selected from ABT-737, navitoclax, BM-1197, S44563, BCL2-32, AZD4320 or S55746.

[0078] CD70 antibodies

[0079] Antibodies or antigen-binding fragments that bind to CD70 and can be incorporated into any of the compositions described herein include, but are not limited to: CD70 antibodies or antigen-binding fragments that inhibit the interaction of CD70 with CD27; CD70 antibodies or antigen-binding fragments that compete with CD27 for binding to CD70; CD70 antibodies or antigen-binding fragments that inhibit CD70-induced CD27 signaling; CD70 antibodies or antigen-binding fragments that inhibit Treg activation and / or proliferation; CD70 antibodies or antigen-binding fragments that deplete cells expressing CD70; CD70 antibodies or antigen-binding fragments that induce lysis of cells expressing CD70; CD70 antibodies or antigen-binding fragments that have ADCC, CDC function and / or induce ADCP.

[0080] Exemplary CD70 antibodies are ARGX-110 described in WO2012 / 123586 (incorporated herein by reference), SGN-70 (WO2006 / 113909 and McEarChern et al. (2008) Clin Cancer Res. 14(23):7763, both of which are incorporated herein by reference), and those described in WO2006 / 044643 and WO2007 / 038637 (each incorporated herein by reference).

[0081] WO2006 / 044643 describes CD70 antibodies containing antibody effector domains that can mediate one or more of ADCC, ADCP or CDC and exert a cytostatic or cytotoxic effect on a CD70-expressing cancer or an immunosuppressive effect on an immune disorder expressing CD70 without being conjugated to a cytostatic or cytotoxic agent. Antibodies exemplified therein are based on the antigen binding regions of two monoclonal antibodies denoted 1F6 and 2F2.

[0082] WO2007 / 038637 describes fully human monoclonal antibodies that bind to CD70. These antibodies are characterized by binding to human CD70 with a K -7 M or less. D and mediating ADCC with an EC50 of 1 x 10

[0083] ARGX-110 is an IgGl anti-CD70 antibody, also known as gantenerimab. ARGX-110 has been shown to inhibit the interaction of CD70 with its receptor CD27 (Silence et al. (2014) MAbs. Mar-Apr; 6(2):523-32, incorporated herein by reference). In particular, ARGX-110 has been shown to inhibit CD70-induced CD27 signaling. The level of CD27 signaling can be determined, for example, by measuring serum soluble CD27 as described in Riether et al. (J. Exp. Med. (2017) 214(2); 359-380), or IL-8 expression as described in Silence et al. (MAbs (2014) 6(2):523-32). Without being bound by theory, it is believed that inhibiting CD27 signaling reduces the activation and / or proliferation of Tregs (regulatory T cells), thereby reducing the suppression of anti-tumor effector T cells. ARGX-110 has also been shown to deplete tumor cells expressing CD70. In particular, ARGX-110 has been shown to lyse tumor cells expressing CD70 via antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and also to increase antibody-dependent cellular phagocytosis (ADCP) of cells expressing CD70 (Silence et al., supra).

[0084] The CDR, VH and VL amino acid sequences of ARGX-110 or gantenerimab are shown in the table below.

[0085] Table 1

[0086]

[0087]

[0088] In certain embodiments, the antibody or antigen-binding fragment thereof that binds to CD70 comprises a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH domain and VL domain comprise CDR sequences:

[0089] HCDR3 comprising or consisting of SEQ ID NO: 3;

[0090] HCDR2 comprising or consisting of SEQ ID NO: 2;

[0091] HCDR1 comprising or consisting of SEQ ID NO: 1;

[0092] LCDR3 comprising or consisting of SEQ ID NO: 7;

[0093] LCDR2 comprising or consisting of SEQ ID NO: 6; and

[0094] LCDR1 comprising or consisting of SEQ ID NO: 5.

[0095] In certain embodiments, the antibody or antigen-binding fragment thereof that binds to CD70, optionally having the CDR sequences shown above, is an IgG, preferably an IgG1. In certain embodiments, the antibody or antigen-binding fragment thereof that binds to CD70 comprises a variable heavy chain domain (VH domain) comprising or consisting of a sequence that is at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 4, and a variable light chain domain (VL domain) comprising or consisting of a sequence that is at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 8. In certain embodiments, the antibody molecule that binds to CD70 comprises a variable heavy chain domain (VH domain) comprising or consisting of SEQ ID NO: 4 and a variable light chain domain (VL domain) comprising or consisting of SEQ ID NO: 8. For embodiments in which the VH domain and / or VL domain is defined as having a particular percentage of identity to a reference sequence, the VH domain and / or VL domain can retain the CDR sequences of the reference sequence. In particular, the CD70 antibodies or antigen-binding fragments defined herein with reference to SEQ ID NO: 4 and 8 can retain the CDR sequences as shown by SEQ ID NO: 1-3 and 5-7.

[0096] CD70 antibodies or antigen-binding fragments thereof that can be incorporated into the compositions described herein include antibody drug conjugates (ADCs). ADCs are antibodies linked to active agents, such as auristatins and maytansines or other cytotoxic agents. Certain ADCs maintain antibody blocking and / or effector functions (e.g., ADCC, CDC, ADCP), while also delivering the conjugated active agent to cells expressing the target (e.g., CD70). Examples of anti-CD70 ADCs include vorsetuzumab mafodotin (also known as SGN-75, Seattle Genetics), SGN-70A (Seattle Genetics), and MDX-1203 / BMS936561 (Bristol-Myers Squibb), each of which can be used according to the present application. Suitable anti-CD70 ADCs are also described in WO2008074004 and WO2004073656 (each incorporated herein by reference).

[0097] Venetoclax

[0098] In certain preferred embodiments of the application, the CD70 antibodies or antigen-binding fragments described herein are combined with venetoclax or a pharmaceutically acceptable salt thereof. Venetoclax is a small molecule inhibitor of BCL-2 as described elsewhere herein.

[0099] Venetoclax for use in the combination therapies described herein can be provided in any suitable form such that it is effective to inhibit BCL-2 protein. These forms include, but are not limited to, any suitable polymorphic, amorphous or crystalline form or any isomeric or tautomeric form. In certain embodiments, the combination therapies described herein comprise venetoclax synthesized according to the methods described in US2010 / 0305122 (incorporated herein by reference). In alternative embodiments, the combination therapies described herein comprise venetoclax according to the form described in or synthesized according to the methods described in any one of EP3333167, WO2017 / 156398, WO2018 / 029711, CN107089981(A), WO2018 / 069941, WO2017 / 212431, WO2018 / 009444, CN107648185(A), WO2018 / 167652, WO2018 / 157803, CZ201769 (each incorporated herein by reference). In certain embodiments, the combination therapies described herein comprise venetoclax in any of the crystalline forms or salt forms described in WO2012 / 071336 (incorporated herein by reference).

[0100] Pharmaceutically acceptable salts for use according to the application include salts of acidic or basic groups. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l,l'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Pharmaceutically acceptable salts can be formed with a variety of amino acids. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts.

[0101] Venetoclax for use in the combination therapies described herein can also be provided in the form of a hydrate, an anhydrate, or a solvate.

[0102] Venetoclax is marketed and sold under the trade name VENCLYXTO® by AbbVie Inc and Genentech. In certain embodiments, the combination therapies described herein comprise an antibody or antigen-binding fragment thereof that binds CD70 and VENCLYXTO®.

[0103] Additional agents

[0104] The compositions of the application can include one or more additional agents, for example one or more additional anti-cancer agents.

[0105] In certain embodiments, the compositions comprise one or more “nucleoside metabolism inhibitors” (NMIs). NMIs are molecules that interfere with epigenetic modifications (e.g. methylation, demethylation, acetylation or deacetylation) of nucleic acids (DNA and / or RNA). Examples of nucleoside metabolism inhibitors include hypomethylating agents (HMAs), isocitrate dehydrogenase (IDH) inhibitors, histone deacetylase (HDAC) inhibitors, and bromodomain and extraterminal domain (BET) inhibitors. Preferred nucleoside metabolism inhibitors are hypomethylating agents. Hypomethylating agents inhibit the normal methylation of DNA and / or RNA. Examples of hypomethylating agents are azacitidine, decitabine and guadecitabine.

[0106] In preferred embodiments, the compositions of the application additionally comprise azacitidine (also referred to herein as azacytidine, AZA or aza). Thus, in preferred embodiments, the application provides a composition comprising (i) an antibody or antigen binding fragment thereof that binds to CD70; (ii) venetoclax or a pharmaceutically acceptable salt thereof; and (iii) azacitidine.

[0107] In further preferred embodiments, the compositions of the application additionally comprise decitabine. Thus, in preferred embodiments, the application provides a composition comprising (i) an antibody or antigen binding fragment thereof that binds to CD70; (ii) venetoclax or a pharmaceutically acceptable salt thereof; and (iii) decitabine.

[0108] Azacitidine is an analogue of cytidine and decitabine is its deoxy derivative. Azacitidine and decitabine are DNA methyltransferase (DNMT) inhibitors known to upregulate gene expression by promoter hypomethylation. Such hypomethylation disrupts cellular function, resulting in a cytotoxic effect.

[0109] In certain embodiments, the compositions of the application additionally comprise cytarabine. Cytarabine (also known as “cytosine arabinose” or “ara-C”) is a chemotherapy drug commonly used to treat AML. High-dose cytarabine forms part of the “7+3” standard induction chemotherapy commonly used for newly diagnosed AML patients. Low-dose cytarabine can be used for AML patients who are not suitable for standard induction chemotherapy. For example, low-dose cytarabine is prescribed in combination with venetoclax for newly diagnosed AML patients who are not suitable for standard induction chemotherapy. The compositions of the application can additionally comprise low-dose cytarabine.

[0110] In certain embodiments, the compositions of the application comprise an additional anticancer agent. The one or more additional anticancer agents can be selected from any agent useful in the treatment of myeloid malignancies, preferably AML. Preferred agents can be selected from: Selectin inhibitors (e.g. GMI-1271); FMS-like tyrosine kinase receptor 3 (FLT3) inhibitors (e.g. midostaurin or gilteritinib); cyclin-dependent kinase inhibitors; aminopeptidase inhibitors; JAK / STAT inhibitors; cytarabine; fludarabine; anthracyclines (e.g. daunorubicin, idarubicin); doxorubicin; hydroxyurea; Vyxeos; IDH1 or IDH2 inhibitors, such as Idhifa (or Enasidenib) or Tibsovo (or ivosidenib); Smoothened inhibitors, such as glasdegib; BET bromodomain inhibitors; CD123 or CD33 targeting agents; HDAC inhibitors; LSC targeting agents; AML bone marrow niche targeting agents; NEDD8 activating enzyme inhibitors, such as pevonedistat; G-CSF and topoisomerase inhibitors, such as mitoxantrone, selinexor and etoposide.

[0111] Formulation of compositions

[0112] The agents in the combinations described herein can be combined or formulated in any manner suitable for administering the combination therapy to a subject or patient in need thereof, preferably a human subject or patient in need thereof. The combinations can be formulated for single dose administration or multiple dose administration.

[0113] In certain embodiments, the agents in the combinations can be co-formulated, i.e. formulated into a single pharmaceutical composition. For embodiments in which the agents are co-formulated, the combination or composition is suitable for simultaneous administration of the agents.

[0114] In preferred embodiments, the agents in the combinations described herein are formulated as separate compositions or pharmaceutical compositions. For embodiments in which the agents are formulated separately, there is the possibility of simultaneous or separate administration of the different agents or compositions. If the different compositions are administered separately, the agents can be administered sequentially in any preferred order. The time interval between administration of the agents can be any suitable time interval. Administration of the different compositions can be performed once (for single dose administration) or repeatedly (for multiple dose administration).

[0115] The CD70 antibody or antigen-binding fragment in the compositions described herein can be formulated using any suitable pharmaceutical carrier, adjuvant and / or excipient. Techniques for formulating antibodies for human therapeutic use are well known in the art and are reviewed, for example, in Wang et al. (2007) Journal of Pharmaceutical Sciences, 96: 1-26, the contents of which are incorporated herein in their entirety. Pharmaceutically acceptable excipients that can be used in formulating antibody compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (such as sodium carboxymethyl cellulose), polyethylene glycols, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin.

[0116] The BCL-2 inhibitor, preferably venetoclax or a pharmaceutically acceptable salt thereof, can be formulated using any suitable pharmaceutical carrier, adjuvant and / or excipient. Suitable agents include, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffering agents, coating agents, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, release agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof.

[0117] In certain embodiments, the compositions are formulated for administration to a subject via any suitable route of administration, including but not limited to intramuscular, intravenous, intradermal, intraperitoneal injection, subcutaneous, epidural, nasal, oral, rectal, topical, inhalation, buccal (e.g., sublingual) and transdermal administration. In certain embodiments, the compositions are formulated as an aqueous solution, tablet, capsule, powder or any other suitable dosage form.

[0118] Excipients used to prepare compositions comprising a BCL-2 inhibitor, preferably venetoclax, to be administered orally in solid dosage form include, for example, agar, alginic acid, aluminum hydroxide, benzyl alcohol, benzyl benzoate, 1,3-butanediol, carbomer, castor oil, cellulose, cellulose acetate, cocoa butter, com starch, com oil, cottonseed oil, cross-linked polyplione, diglycerides, ethanol, ethyl cellulose, ethyl laurate, ethyl oleate, fatty acid esters, gelatin, germ oil, glucose, glycerol, groundnut oil, hydroxypropyl methyl cellulose, isopropyl alcohol, isotonic saline, lactose, magnesium hydroxide, magnesium stearate, malt, mannitol, monoglycerides, olive oil, peanut oil, potassium phosphate, potato starch, povidone, propylene glycol, Ringer's solution, safflower oil, sesame oil, sodium carboxymethyl cellulose, sodium phosphate, sodium lauryl sulfate, sodium sorbitol, soybean oil, stearic acid, stearol, sucrose, surfactants, talc, tragacanth, tetrahydrofurfuryl alcohol, triglycerides, water, and mixtures thereof. Excipients used to prepare compositions comprising a BCL-2 inhibitor, preferably venetoclax, to be administered orally in liquid dosage form include, for example, 1,3-butanediol, castor oil, com oil, cottonseed oil, ethanol, fatty acid esters of sorbitan, germ oil, groundnut oil, glycerol, isopropyl alcohol, olive oil, polyethylene glycol, propylene glycol, sesame oil, water, and mixtures thereof. Excipients used to prepare compositions comprising a BCL-2 inhibitor, preferably venetoclax, to be administered by penetration include, for example, chlorofluorocarbons, ethanol, water, and mixtures thereof. Excipients used to prepare compositions comprising a BCL-2 inhibitor, preferably venetoclax, to be administered parenterally include, for example, 1,3-butanediol, castor oil, com oil, cottonseed oil, dextrose, germ oil, groundnut oil, liposomes, oleic acid, olive oil, peanut oil, Ringer's solution, safflower oil, sesame oil, soybean oil, U.S.P. or isotonic sodium chloride solution, water, and mixtures thereof.

[0119] For embodiments in which the agents in the composition are formulated separately, i.e., as separate compositions, the separate compositions can be formulated for the same route of administration. For embodiments in which the agents in the composition are formulated separately, i.e., as separate compositions, the separate compositions can be formulated for different routes of administration. For example, the CD70 antibody or antigen binding fragment can be formulated for intravenous administration, and the BCL-2 inhibitor, preferably venetoclax, can be formulated for oral administration.

[0120] As described above, the combination therapy of the application can comprise Venetoclax is a product sold and distributed by AbbVie Inc. and Genentech, Inc. Venetoclax for oral administration is marketed under the trade name VENCLYXTO®. Tablets are supplied as light yellow or beige tablets containing 10 mg, 50 mg or 100 mg of venetoclax as the active ingredient. Each tablet also contains the following inactive ingredients: copovidone, colloidal silicon dioxide, polysorbate 80, sodium stearyl fumarate, and tribasic calcium phosphate. In addition, the 10 mg and 100 mg coated tablets include the following: iron oxide yellow, polyvinyl alcohol, polyethylene glycol, talc, and titanium dioxide. The 50 mg coated tablets also include the following: iron oxide yellow, iron oxide red, iron oxide black, polyvinyl alcohol, talc, polyethylene glycol, and titanium dioxide. For embodiments in which the CD70 antibody or antigen-binding fragment thereof is combined with a BCL-2 inhibitor, the CD70 antibody or antigen-binding fragment can be formulated for intravenous administration, while the BCL-2 inhibitor can be formulated for oral administration.

[0121] For compositions of the application that comprise or consist of an additional agent in addition to a CD70 antibody or antigen-binding fragment and a BCL-2 inhibitor (preferably venetoclax), the one or more additional agents can be formulated for administration by the same route or a different route as the other agents. For example, in preferred embodiments in which the composition comprises (i) an antibody or antigen-binding fragment thereof that binds to CD70; (ii) venetoclax or a pharmaceutically acceptable salt thereof; and (iii) azacitidine, the antibody or antigen-binding fragment can be administered intravenously, venetoclax or a pharmaceutically acceptable salt thereof can be administered orally, and azacitidine can be administered subcutaneously by injection. In preferred embodiments in which the composition comprises (i) an antibody or antigen-binding fragment thereof that binds to CD70; (ii) venetoclax or a pharmaceutically acceptable salt thereof; and (iii) decitabine, the antibody or antigen-binding fragment can be administered intravenously, venetoclax or a pharmaceutically acceptable salt thereof can be administered orally, and decitabine can be administered subcutaneously by injection.

[0122] C. Methods of treatment

[0123] The combination therapy described according to the first aspect of the application can be used in a method of treating a malignant tumor, in particular a myeloid malignant tumor, in a human subject.

[0124] The present application provides an antibody or antigen-binding fragment thereof that binds to CD70 for use in treating a malignant tumor, in particular a myeloid malignant tumor, in a human subject, wherein the antibody or antigen-binding fragment thereof is administered in combination with a BCL-2 inhibitor. The present application also provides a BCL-2 inhibitor for use in treating a malignant tumor, in particular a myeloid malignant tumor, in a human subject, wherein the BCL-2 inhibitor is administered in combination with an antibody or antigen-binding fragment that binds to CD70.

[0125] In preferred embodiments, the BCL-2 inhibitor is venetoclax or a pharmaceutically acceptable salt thereof.

[0126] The present application also provides a composition according to the first aspect of the application for use in the treatment of a malignant tumor, in particular a myeloid malignancy, in a human subject.

[0127] In yet another aspect, the present application provides a method for the treatment of a malignant tumor, in particular a myeloid malignancy, in a human subject, the method comprising administering to the subject a composition according to the first aspect of the application. The present application also provides a method for the treatment of a malignant tumor, in particular a myeloid malignancy, in a human subject, the method comprising the steps of: (i) administering to the subject an antibody or antigen-binding fragment thereof that binds to CD70; and (ii) administering to the subject a BCL-2 inhibitor, preferably venetoclax or a pharmaceutically acceptable salt thereof. Steps (i) and (ii) of the method can be performed in any order.

[0128] All embodiments described above in relation to the composition of the first aspect of the application equally apply to the method described herein.

[0129] The term "malignant tumor" encompasses a disease in which abnormal cells proliferate in an uncontrolled way and invade surrounding tissues. Malignant cells that have entered the body's blood and lymphatic systems are able to travel to distant sites in the body and seed at secondary locations. In certain embodiments, the methods described herein are used to treat a malignant tumor that includes the production of cancer progenitor or stem cells that express CD70, CD27, or both. As described elsewhere herein, upregulation of CD70 expression has been detected in different types of cancer including renal cell carcinoma, metastatic breast cancer, brain tumors, leukemia, lymphoma, and nasopharyngeal carcinoma. Co-expression of CD70 and CD27 has also been detected in hematopoietic lineage malignancies including acute lymphoblastic lymphoma and T-cell lymphoma. In certain embodiments, the methods described herein are used to treat any of the above-mentioned malignant tumors that are associated with CD70 expression, CD27 expression, or both.

[0130] In particular embodiments, the methods described herein are used to treat a myeloid malignancy, wherein a myeloid malignancy refers to any clonal disease of hematopoietic stem or progenitor cells. The myeloid malignancy treated according to the methods of the present application can be a newly diagnosed myeloid malignancy or a relapsed / refractory myeloid malignancy.

[0131] In certain embodiments, the myeloid malignancy is selected from the group consisting of: acute myeloid leukemia (AML); myelodysplastic syndrome (MDS); myeloproliferative neoplasm (MPN); chronic myeloid leukemia (CML); and chronic myelomonocytic leukemia (CMML). In preferred embodiments, the myeloid malignancy is acute myeloid leukemia (AML).

[0132] Myeloid malignancies can be classified and diagnosed according to the WHO 2008 classification, with updates to the classification in 2016, see in particular Arber et al. (2016) Blood 127(20): 2391-2405, incorporated herein by reference.

[0133] Acute myeloid leukemia (AML) refers to a hematopoietic neoplasm that involves cells of the bone marrow. AML is characterized by clonal proliferation of myeloid precursors with reduced differentiation capacity. AML patients exhibit accumulation of blasts in the bone marrow. Blasts also accumulate in the peripheral blood of AML patients. Generally, if a patient exhibits 20% or more blasts in the bone marrow or peripheral blood, then AML is diagnosed.

[0134] According to the WHO classification, AML generally encompasses the following subtypes: AML with recurrent genetic abnormalities; AML with myelodysplasia-related changes; therapy-related myeloid neoplasm; myeloid sarcoma; myelodysplasia associated with Down syndrome; blastic plasmacytoid dendritic cell neoplasm; and AML not otherwise classified (e.g. acute megakaryoblastic leukemia, acute basophilic leukemia).

[0135] AML can also be classified according to the French-American-British (FAB) classification, encompassing the following subtypes: M0 (acute myeloblastic leukemia, minimally differentiated); Ml (acute myeloblastic leukemia, immaturity); M2 (acute myeloblastic leukemia, with maturation); M3 (promyelocytic leukemia or acute promyelocytic leukemia (APL)); M4 (acute myelomonocytic leukemia); M4eo (myelomonocytic leukemia with myeloid metamyelocyte increase); M5 (acute monocytoid leukemia (M5a) or acute monocytic leukemia (M5b)); M6 (acute erythroid leukemia, including erythroleukemia (M6a) and very rare pure erythroid leukemia (M6b)); or M7 (acute megakaryoblastic leukemia).

[0136] Unless otherwise specified, "AML" as used herein refers to any of the disorders encompassed by the WHO classification and / or the FAB classification. Certain AML subtypes are considered to have a more favorable prognosis, some have an intermediate prognosis and some have a poor prognosis. The skilled person is aware of which subtypes will fall into which risk category.

[0137] Myelodysplastic syndromes (MDS) are characterized by dysplasia, cytopenias, and / or abnormal changes in bone marrow cellularity and / or bone marrow differentiation, e.g., increased blasts infiltration. According to the WHO classification, MDS generally encompasses the following subtypes: MDS with unilineage dysplasia (previously known as "refractory cytopenia with unilineage dysplasia"), including refractory anemia, refractory neutropenia, and refractory thrombocytopenia; MDS with ring sideroblasts, including subgroups with unilineage dysplasia and multilineage dysplasia (previously known as "refractory anemia with ring sideroblasts"); MDS with multilineage dysplasia (previously known as "refractory cytopenia with multilineage dysplasia"); MDS with excess blasts (MDS-EB, previously known as "refractory anemia with excess blasts"), which can be further subdivided into MDS-EB-1 and MDS-EB-2 based on the percentage of blasts; MDS with isolated del(5q); and unclassifiable MDS.

[0138] MDS can also be classified according to the French-American-British (FAB) classification, encompassing the following subtypes: M9980 / 3 (refractory anemia (RA)); M9982 / 3 (refractory anemia with ring sideroblasts (RARS)); M9983 / 3 (refractory anemia with excess blasts (RAEB)); M9984 / 3 (refractory anemia with excess blasts in transformation (RAEB-T)); and M9945 / 3 (chronic myelomonocytic leukemia (CMML)).

[0139] Unless otherwise specified, "MDS" as used herein refers to any of the disorders encompassed by the WHO classification and / or the FAB classification. For both AML and MDS, the WHO classification is preferred herein.

[0140] Myeloproliferative neoplasms (MPN) are similar to MDS, but according to the WHO classification, MPN generally encompasses the following subtypes: chronic myelogenous leukemia (CML); chronic neutrophilic leukemia (CNL); polycythemia vera (PV); primary myelofibrosis (PMF); essential thrombocythemia (ET); chronic eosinophilic leukemia, not otherwise specified; and unclassifiable MPN.

[0141] According to the WHO classification, chronic myelomonocytic leukemia (CMML) and atypical chronic myeloid leukemia (aCML) fall within the category of MDS / MPN disorders because they represent myeloid neoplasms with overlapping clinical, laboratory, and morphologic features between MDS and MPN.

[0142] Patient characteristics

[0143] Patients or subjects treated according to the methods described herein, particularly those with AML, can have newly diagnosed disease, relapsed disease, or primary refractory disease.

[0144] The standard treatment method for newly diagnosed AML patients is the "standard 7+3 intensive chemotherapy" method, which features the administration of 7 days of high-dose cytarabine followed by 3 days of an anthracycline (e.g., daunorubicin or idarubicin). The purpose of giving intensive chemotherapy is to induce complete remission of AML, usually in order to allow the patient to receive a stem cell transplant after successful chemotherapy.

[0145] Standard intensive chemotherapy is associated with significant toxicities and side effects, meaning that it is not suitable for patients who cannot tolerate these effects. These patients are said to be "unfit for standard intensive chemotherapy." A patient can be unfit for standard intensive chemotherapy because, for example, they exhibit one or more comorbidities, indicating that they cannot tolerate the toxicities, or prognostic factors characterizing their disease indicate an adverse outcome for standard intensive chemotherapy. The fitness of an individual patient for standard intensive chemotherapy will be determined by a clinician taking into account the individual patient's medical history and clinical guidelines (e.g., the National Comprehensive Cancer Network (NCCN) guidelines, incorporated herein by reference). AML patients over the age of 60 are often assessed as unfit for standard intensive chemotherapy, with other factors to be considered including the cytogenetic and / or molecular abnormalities of the AML being treated.

[0146] Patients who are unfit for standard intensive chemotherapy can instead receive reduced-intensity chemotherapy, such as low-dose cytarabine (LDAC). Patients who are unfit for standard intensive chemotherapy and unfit for LDAC can receive best supportive care (BSC), including hydroxyurea (HU) and transfusion support.

[0147] Patients or subjects treated according to the methods described herein can be those classified as "unfit for standard intensive chemotherapy." The compositions of the present invention comprise targeted therapies that can be predicted to have fewer side effects. Thus, patients deemed unfit for standard intensive chemotherapy for any of the reasons identified above can be treated with the combination according to the present invention.

[0148] As described above, venetoclax is approved in the United States in combination with azacitidine, decitabine, or low-dose cytarabine for the treatment of newly diagnosed AML in adults 75 years of age or older or adults with comorbidities that do not allow for the use of intensive induction chemotherapy. Thus, in certain embodiments, particularly those in which the BCL-2 inhibitor is venetoclax or a pharmaceutically acceptable salt thereof, the patient or subject treated according to the methods described herein is a newly diagnosed AML patient 75 years of age or older. In further embodiments, the patient or subject treated according to the methods described herein is a newly diagnosed AML patient with comorbidities that do not allow for the use of intensive induction therapy. A patient with comorbidities that do not allow for the use of intensive induction chemotherapy can be so classified based on at least one of the following criteria: baseline Eastern Cooperative Oncology Group (ECOG) performance status is 2-3 points; severe cardiac or pulmonary comorbidities; moderate liver impairment; or CLcr < 45 mL / min. These embodiments are particularly preferred when the BCL-2 inhibitor in the compositions according to the application is venetoclax or a pharmaceutically acceptable salt thereof.

[0149] The patient or subject treated according to the methods described herein can be eligible for other treatments, such as standard intensive chemotherapy, but can receive the combination therapies described herein as an alternative treatment option. For example, the patient or subject treated according to the methods described herein can be a newly diagnosed AML patient who is otherwise eligible for standard intensive chemotherapy.

[0150] Additional therapeutic agents

[0151] The methods described herein can include administration of one or more additional therapeutic agents, such as additional anti-cancer agents. In certain embodiments, the methods include administration of one or more agents useful for treating myeloid malignancies, such as agents useful for treating AML. Such agents include, but are not limited to: selectin inhibitors (e.g., GMI-1271); FMS-like tyrosine kinase receptor 3 (FLT3) inhibitors (e.g., midostaurin or gilteritinib); cyclin-dependent kinase inhibitors; aminopeptidase inhibitors; JAK / STAT inhibitors; cytarabine; fludarabine; anthracyclines (e.g., daunorubicin, idarubicin); doxorubicin; hydroxyurea; Vyxeos; IDH1 or IDH2 inhibitors, such as Idhifa (or enasidenib) or Tibsovo (or ivosidenib); Smoothened inhibitors, such as glasdegib; BET bromodomain inhibitors; CD123 or CD33 targeting agents; HDAC inhibitors; LSC targeting agents; AML marrow niche targeting agents; NEDD8 activating enzyme inhibitors, such as pevonedistat; G-CSF; and topoisomerase inhibitors, such as mitoxantrone, selinexor, and etoposide.

[0152] In preferred embodiments, the methods described herein comprise administering another agent that is a nucleoside metabolism inhibitor, preferably a hypomethylating agent. Particularly preferred hypomethylating agents are azacitidine and decitabine. As described above, in certain embodiments, the compositions of the application comprising (i) a CD70 antibody or antigen-binding fragment thereof; and (ii) a BCL-2 inhibitor, preferably venetoclax or a pharmaceutically acceptable salt, can be formulated to include an additional agent, such as azacitidine or decitabine.

[0153] Alternatively, for embodiments wherein the composition consists of (i) an antibody or antigen-binding fragment that binds to CD70; and (ii) a BCL-2 inhibitor, preferably venetoclax or a pharmaceutically acceptable salt thereof, the method of administering the composition to a subject can comprise an additional step of administering an additional agent, such as azacitidine or decitabine. Thus, in one preferred embodiment, the present application provides a method for treating a myeloid malignancy, preferably AML, in a human subject, the method comprising administering to the subject: (i) an antibody or antigen-binding fragment thereof that binds to CD70; (ii) a BCL-2 inhibitor, preferably venetoclax or a pharmaceutically acceptable salt thereof; and (iii) azacitidine or decitabine. Also provided herein is a composition for treating a myeloid malignancy, preferably AML, in a human subject, the composition comprising: (i) an antibody or antigen-binding fragment thereof that binds to CD70; (ii) a BCL-2 inhibitor, preferably venetoclax or a pharmaceutically acceptable salt thereof; and (iii) azacitidine or decitabine.

[0154] Dosing

[0155] As demonstrated in the Examples, the compositions of the application exhibit synergistic therapeutic efficacy on AML cells, that is, the level of inhibition induced by the compositions is greater than the additive effect of the single therapies alone.

[0156] Methods for determining synergistic interactions are familiar to the person skilled in the art and are described in the Examples. A preferred method for determining whether a composition produces a synergistic effect is the Chou-Talalay method (Chou, TC. Cancer Res. 2010 Jan 15;70(2):440-6, incorporated herein by reference).

[0157] According to the Chou-Talalay method, CI < 1 indicates synergy, CI = 1 indicates additivity, and CI > 1 indicates antagonism. As Figure 1As shown in the Examples, the presence and extent of synergy produced by the compositions of the application varies depending on the strength of the inhibitory effect of the composition. The strength of the inhibitory effect of the composition itself depends on the total concentration of the composition.

[0158] Thus, preferably, in embodiments of all aspects of the application, the dose of CD70 antibody or antigen-binding fragment thereof administered and / or provided in the composition and the dose of BCL-2 inhibitor administered and / or provided in the composition are each selected so that the composition provides synergistic treatment, i.e., wherein the composition exhibits a CI of less than 1 as determined by the Chou-Talalay method. Preferably, the doses are such that the composition exhibits a CI of less than 0.5.

[0159] Preferably, in certain embodiments, the dose of CD70 antibody or antigen-binding fragment thereof administered and / or provided in the composition and the dose of BCL-2 inhibitor administered and / or provided in the composition are each selected so that the composition exhibits a CI of less than 1 and a Fa > 0.5 as determined by the Chou-Talalay method.

[0160] As shown in the Examples, synergy was also observed for compositions of anti-CD70 antibody (ARGX-110), BCL-2 inhibitor (venetoclax), and HMA (decitabine). Thus, in certain preferred embodiments of aspects of the application wherein the composition comprises an HMA, the dose of CD70 antibody or antigen-binding fragment thereof administered and / or provided in the composition, the dose of BCL-2 inhibitor administered and / or provided in the composition, and the dose of HMA administered and / or provided in the composition are each selected so that the composition provides synergistic efficacy in the treatment.

[0161] It has been found that CD70 antibodies, particularly ARGX-110, are effective in treating myeloid malignancies, particularly AML, at relatively low doses. Thus, in certain embodiments of all methods of the application, the CD70 antibody or antigen-binding fragment thereof is administered at a dose ranging from 0.1 mg / kg to 25 mg / kg per dose, for example, ranging from 0.1 mg / kg to 20 mg / kg per dose. In certain embodiments, the CD70 antibody or antigen-binding fragment thereof is administered at a dose ranging from 1 mg / kg to 20 mg / kg per dose. Unless otherwise specified, ranges recited herein include the endpoints of the ranges, e.g., administration at a dose ranging from 0.1 mg / kg to 25 mg / kg per dose includes administration at a dose of 0.1 mg / kg per dose and administration at a dose of 25 mg / kg per dose, as well as all doses in between these two endpoints.

[0162] In certain embodiments of the methods of the application, the CD70 antibody or antigen-binding fragment thereof is administered at a dose ranging from 0.1 mg / kg to 15 mg / kg. In certain embodiments, the CD70 antibody or antigen-binding fragment thereof is administered at a dose ranging from 0.5 mg / kg to 2 mg / kg. In certain embodiments, the CD70 antibody or antigen-binding fragment thereof is administered at a dose of 1 mg / kg, 3 mg / kg, 10 mg / kg, or 20 mg / kg. In certain preferred embodiments, the CD70 antibody or antigen-binding fragment thereof is administered at a dose of 1 mg / kg. In certain preferred embodiments, the CD70 antibody or antigen-binding fragment thereof is administered at a dose of 10 mg / kg.

[0163] In certain embodiments, multiple doses of the CD70 antibody or antigen-binding fragment are administered. In certain such embodiments, each dose of the CD70 antibody or antigen-binding fragment thereof is separated by 10-20 days, optionally 12-18 days. In certain embodiments, each dose of the anti-CD70 antibody is separated by 14-17 days.

[0164] The BCL-2 inhibitor, preferably venetoclax or a pharmaceutically acceptable salt thereof, in the composition can be administered according to any regimen determined to be effective for the compound. The FDA has established a label for the use of Information for the treatment of AML proposes a dosing regimen in which there is a first ramp-up period followed by a maintenance period. In the case of In the case of a prescription in combination with azacitidine or decitabine, a dosing regimen consisting of 100 mg of 200 mg of 400 mg of and 400 mg of every day thereafter is recommended. 2 In the case of a prescription in combination with 75 mg / m 2 of azacitidine or 20 mg / m 2 of decitabine until disease progression or unacceptable toxicity is observed. In the case of a prescription in combination with low-dose cytarabine, a dosing regimen consisting of 100 mg of 200 mg of 400 mg of and 600 mg of every day thereafter is recommended. 2 In the case of a prescription in combination with 20 mg / m 2 of cytarabine until disease progression or unacceptable toxicity is observed.

[0165] In certain embodiments, each dose, e.g., oral dose, of venetoclax or a pharmaceutically acceptable salt thereof is in the range of 100 mg - 600 mg. In certain embodiments, venetoclax or a pharmaceutically acceptable salt thereof is administered daily at 400 mg. In certain embodiments, venetoclax or a pharmaceutically acceptable salt thereof is administered daily at 600 mg. As described above, a run-in period, e.g., 3 days, can precede the daily fixed dosing of venetoclax, in which an escalating dose of venetoclax is administered to the patient until the maintenance daily dose is reached.

[0166] For embodiments of the application in which the composition comprises a nucleoside metabolic inhibitor or the method involves administration of a nucleoside metabolic inhibitor, the nucleoside metabolic inhibitor can be administered at a dose in the range of 20 mg / m 2 - 100 mg / m 2 . As described above, unless otherwise specified, ranges recited herein include the endpoints of the ranges, e.g., in the range of 20 mg / m 2 - 100 mg / m 2 , includes administration at a dose of 20 mg / m 2 per day and administration at a dose of 100 mg / m 2 per day, and all doses in between these two endpoints.

[0167] In certain embodiments, the nucleoside metabolic inhibitor is azacitidine and is administered at a dose in the range of 70 mg / m 2 - 80 mg / m 2 . In certain preferred embodiments, the nucleoside metabolic inhibitor is azacitidine and is administered at a dose of 75 mg / m 2 per day.

[0168] In certain embodiments, the nucleoside metabolic inhibitor is decitabine and is administered at a dose in the range of 15 mg / m 2 - 25 mg / m 2 . In certain preferred embodiments, the nucleoside metabolic inhibitor is decitabine and is administered at a dose of 20 mg / m 2 per day.

[0169] For embodiments of the application in which the composition comprises a nucleoside metabolic inhibitor or the method involves administration of a nucleoside metabolic inhibitor, the nucleoside metabolic inhibitor can be administered at a daily dose for a dosing period of 5 days - 10 days. That is, one dose of the nucleoside inhibitor is administered per day for a period of 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. In certain preferred embodiments, the nucleoside metabolic inhibitor is administered at a daily dose for a dosing period of 7 days. A preferred nucleoside metabolic inhibitor is azacitidine.

[0170] In certain embodiments, the nucleoside metabolic inhibitor is administered according to a dosing regimen of repeated dosing periods, wherein the end of one dosing period and the start of the next dosing period are separated by 18-25 days. That is, the dosing regimen comprises at least 2 dosing periods in which a dose of the nucleoside inhibitor is administered each day (e.g., for a period of 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days), wherein the end of one dosing period and the start of the next dosing period are separated by 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, or 25 days. In certain embodiments, the end of one dosing period and the start of the next dosing period are separated by 21 days.

[0171] In certain embodiments, each dosing period has the same length (e.g., 7 days). In certain embodiments, the end of each dosing period and the start of the next dosing period are separated by the same number of days (e.g., 21 days).

[0172] In certain embodiments, the first dose of the nucleoside metabolic inhibitor is administered 7-21 days after the first dose of the CD70 antibody or antigen-binding fragment thereof. In certain embodiments, the first dose of the nucleoside metabolic inhibitor is administered 10-17 days after the first dose of the CD70 antibody or antigen-binding fragment thereof. In certain embodiments, the first dose of the nucleoside metabolic inhibitor is administered 14 days after the first dose of the CD70 antibody or antigen-binding fragment thereof.

[0173] In certain embodiments, one of the daily doses of the nucleoside metabolic inhibitor is administered on the same day as a dose of the CD70 antibody or antigen-binding fragment thereof. That is, in embodiments of the methods of the application in which both a CD70 antibody (or antigen-binding fragment thereof) and a nucleoside metabolic inhibitor are administered to a subject, the dosing regimen for both the CD70 antibody and the nucleoside metabolic inhibitor is such that at least one of the scheduled doses of the CD70 antibody is administered on the same day as one of the scheduled daily doses of the nucleoside metabolic inhibitor. This day can be the first, second, third, fourth, fifth, sixth, or seventh day of a dosing period of the nucleoside metabolic inhibitor.

[0174] In certain embodiments, a dose of the CD70 antibody or antigen-binding fragment thereof is administered every 14-17 days and the nucleoside metabolic inhibitor is administered for 7 days according to a dosing regimen of repeated daily doses of dosing periods, wherein the end of one dosing period and the start of the next dosing period are separated by 21 days, and wherein the first daily dose of the first dosing period is administered 14 days after the first dose of the anti-CD70 antibody or antigen-binding fragment thereof.

[0175] In certain embodiments, a patient treatment cycle consists of 28 days, and the nucleoside metabolism inhibitor, preferably azacitidine or decitabine, is administered daily for a period of 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, starting on day 1 of the cycle. The treatment methods described herein can comprise multiple treatment cycles. Each treatment cycle can repeat the previous treatment cycle. In certain embodiments, a patient treated with a CD70 antibody, a BCL-2 inhibitor, preferably venetoclax or a pharmaceutically acceptable salt thereof, and azacitidine is treated according to a cycle consisting of 28 days, wherein azacitidine is administered daily for the first 7 days of the 28-day cycle. In certain embodiments, a patient treated with a CD70 antibody, a BCL-2 inhibitor, preferably venetoclax or a pharmaceutically acceptable salt thereof, and decitabine is treated according to a cycle consisting of 28 days, wherein decitabine is administered daily for the first 5 days of the 28-day cycle. For embodiments wherein a patient is treated with a CD70 antibody, a BCL-2 inhibitor, preferably venetoclax or a pharmaceutically acceptable salt thereof, and a nucleoside metabolism inhibitor, preferably azacitidine or decitabine, according to a 28-day cycle, the CD70 antibody can be administered on day 3 and / or day 17 of the 28-day cycle. In preferred embodiments, the CD70 antibody is ARGX-110. In further preferred embodiments, the CD70 antibody (e.g. ARGX-110) is administered at a dose of 10 mg / kg.

[0176] Another advantage of the present application is that after the initial phase of the combination therapy, the administration of the NMI (e.g. azacitidine) can be gradually reduced or stopped. Long-term NMI treatment can result in cumulative toxicity, e.g. cytopenia due to the effect of the NMI on non- blast cell types. By gradually reducing or stopping the dose of the NMI after the initial phase, the risk of such toxicity is reduced and the non-blast cell types can recover. In certain embodiments, the treatment according to the present application comprises administering to the patient a CD70 antibody, a BCL-2 inhibitor (e.g. venetoclax) and an NMI as a combination therapy (induction therapy) in a first phase according to any of the above embodiments; and administering to the patient a CD70 antibody, a BCL-2 inhibitor (e.g. venetoclax) and an NMI as a combination therapy in a subsequent second phase, but wherein the dose of the NMI in the second phase (maintenance therapy) is lower than the dose of the NMI administered in the first phase. The dose of the NMI in the second phase can be zero.

[0177] In such embodiments, the dose of CD70 antibody administered in the second phase (i.e., maintenance therapy) is any dose according to the embodiments already described. That is, in certain embodiments, the dose is in the range of 0.1 mg / kg to 25 mg / kg, for example, in the range of 0.1 mg / kg to 20 mg / kg, for example, in the range of 1 mg / kg to 20 mg / kg. In certain embodiments, the dose is in the range of 0.1 mg / kg to 15 mg / kg per dose. In certain embodiments, the dose is in the range of 0.5 mg / kg to 2 mg / kg. In certain embodiments, the dose is 1 mg / kg, 3 mg / kg, 10 mg / kg, or 20 mg / kg. In certain embodiments, the dose is 1 mg / kg. In certain embodiments, the dose is 10 mg / kg.

[0178] The duration of the first phase (i.e., induction therapy), the time to transition to the second phase (i.e., maintenance therapy), and the extent of the gradual reduction or complete cessation of the dose of NMI are factors that will be adjusted for individual patients and determined by their clinicians based on the individual patient’s response to therapy and their medical history. Accordingly, the following embodiments are provided by way of non-limiting example. In certain embodiments, the patient is administered induction therapy until their bone marrow and / or peripheral blood blast percentage is less than 10%, optionally less than 5%. In certain embodiments, the induction therapy is administered for at least 5 NMI dosing periods, optionally at least 6, 7, 8, 9, or at least 10 NMI dosing periods.

[0179] In certain embodiments, the dose of NMI in the maintenance period does not exceed 50 mg / m 2 , optionally does not exceed 40 mg / m 2 , optionally does not exceed 30 mg / m 2 , optionally does not exceed 20 mg / m 2 , per day. In certain embodiments, the dose of NMI in the maintenance period is zero.

[0180] As explained elsewhere herein, the agents in the composition can be formulated for administration by any suitable route of administration. Accordingly, administration of the agents according to the methods of the application can be by any suitable route, and need not be by the same route for individual agents. For example, the CD70 antibody or antigen-binding fragment thereof can be administered intravenously, while the BCL-2 inhibitor (e.g., venetoclax) is administered orally. For embodiments in which the patient or subject receives a hypomethylating agent such as azacitidine or decitabine, this agent can be administered intravenously or subcutaneously by injection.

[0181] Treatment results

[0182] In certain embodiments, the methods described herein include monitoring the patient for blast cell count, i.e., the number of blast cells. As used herein, "blast cell" or "blast" refers to a myeloblast or myeloblastic cell, which is a myeloid progenitor cell within the bone marrow. In a healthy individual, there are no blast cells present in the peripheral blood circulation, and the blast cells in the bone marrow should be less than 5%. In subjects with myeloid malignancies, particularly AML and MDS, there is an increased production of abnormal blast cells with disrupted differentiation potential, and the excess production of these abnormal blast cells can be detected by monitoring the patient for blast cell count in the peripheral blood circulation or the bone marrow, or both.

[0183] The proportion of blast cells in the bone marrow or peripheral blood can be assessed by methods known in the art, such as flow cytometric or cell morphological assessment of cells obtained from a bone marrow biopsy or peripheral blood smear of the subject. The proportion of blast cells is determined relative to the total cells in the sample. For example, flow cytometry can be used to determine the proportion of blast cells using CD45 暗 , SSC 低 cells relative to the total number of cells. As a further example, cell morphological assessment can be used to determine the number of morphologically identified blast cells relative to the total number of cells in the field examined.

[0184] In certain embodiments, methods are provided for reducing the proportion of blast cells in the bone marrow to less than 25%, less than 20%, such as less than 10%. In certain embodiments, methods are provided for reducing the proportion of blast cells in the bone marrow to less than 5%. In certain embodiments, methods are provided for reducing the proportion of blast cells in the bone marrow to about 5% to about 25%, wherein the percentage of bone marrow blast cells is also reduced by more than 50% compared to the percentage of bone marrow blast cells prior to (or prior to treatment with) the method.

[0185] In certain embodiments, methods are provided for reducing the proportion of blast cells in the peripheral blood to less than 25%, less than 20%, such as less than 10%. In certain embodiments, methods are provided for reducing the proportion of blast cells in the peripheral blood to less than 5%. In certain embodiments, methods are provided for reducing the proportion of blast cells in the peripheral blood to about 5% to about 25%, wherein the percentage of peripheral blood blast cells is also reduced by more than 50% compared to the percentage of peripheral blood blast cells prior to (or prior to treatment with) the method.

[0186] For clinical determination of blast cell percentage, cell morphological (also known as cytomorphological) assessment is generally preferred.

[0187] In particular embodiments, the methods described herein induce a complete response. In the context of AML treatment, a complete response or “complete remission” is defined as: bone marrow blasts <5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC >1.0 x 109 / L (1000 / µL); platelet count >100 x 109 / L (100,000 / µL), see Schuurhuis et al. (2017) Blood 129(4):424-447. 9 In particular embodiments, the methods described herein induce a complete response. In the context of AML treatment, a complete response or “complete remission” is defined as: bone marrow blasts <5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC >1.0 x 109 / L (1000 / µL); platelet count >100 x 109 / L (100,000 / µL), see Schuurhuis et al. (2017) Blood 129(4):424-447. 9 In particular embodiments, the methods described herein induce a complete response. In the context of AML treatment, a complete response or “complete remission” is defined as: bone marrow blasts <5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC >1.0 x 109 / L (1000 / µL); platelet count >100 x 109 / L (100,000 / µL), see Schuurhuis et al. (2017) Blood 129(4):424-447. In particular embodiments, the methods described herein induce a complete response. In the context of AML treatment, a complete response or “complete remission” is defined as: bone marrow blasts <5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC >1.0 x 109 / L (1000 / µL); platelet count >100 x 109 / L (100,000 / µL), see Schuurhuis et al. (2017) Blood 129(4):424-447. In particular embodiments, the methods described herein induce a complete response. In the context of AML treatment, a complete response or “complete remission” is defined as: bone marrow blasts <5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC >1.0 x 109 / L (1000 / µL); platelet count >100 x 109 / L (100,000 / µL), see Schuurhuis et al. (2017) Blood 129(4):424-447.

[0188] The methods can achieve a complete response with platelet recovery, i.e., a response in which the platelet count is >100 x 109 / L (100,000 / µL). The methods can achieve a complete response with neutrophil recovery, i.e., a response in which the neutrophil count is >1.0 x 109 / L (1000 / µL). 9 The methods can achieve a complete response with platelet recovery, i.e., a response in which the platelet count is >100 x 109 / L (100,000 / µL). The methods can achieve a complete response with neutrophil recovery, i.e., a response in which the neutrophil count is >1.0 x 109 / L (1000 / µL). 9 Alternatively or additionally, the methods can induce non-transfusion dependence of red blood cells or platelets or both for 8 weeks or more, 10 weeks or more, 12 weeks or more.

[0189] In particular embodiments, the methods described herein induce a negative minimal or measurable residual disease (or MRD) state, see Schuurhuis et al. (2018) Blood. 131(12):1275-1291.

[0190] In certain embodiments, the methods described herein induce a complete response (CR MRD- ) without minimal residual disease, see Schuurhuis et al. (supra). In certain embodiments, the methods described herein induce a complete response (CR

[0191] The methods can achieve a partial response or induce partial remission. In the context of AML treatment, a partial response or partial remission includes a reduction in the percentage of bone marrow blasts to 5-25% and a reduction of at least 50% in the percentage of bone marrow blasts relative to pre-treatment, see Schuurhuis et al. (supra). In certain embodiments, the methods described herein induce a complete response (CR

[0192] The methods described herein can increase survival. The term "survival" as used herein can refer to overall survival, 1-year survival, 2-year survival, 5-year survival, event-free survival, progression-free survival. The methods described herein can increase survival as compared to the gold standard treatment for the particular disease or condition to be treated. The gold standard treatment can also be identified as the best practice, standard of care, standard medical care, or standard therapy. For any given disease, there can be one or more gold standard treatments depending on different clinical practices, e.g., in different countries. Treatments that have been available for myeloid malignancies are diverse and include chemotherapy, radiation, stem cell transplant, and certain targeted therapies. In addition, clinical guidelines in the United States and Europe have led to standard treatments for myeloid malignancies, such as AML, see O'Donnell et al. (2017) Journal of the National Comprehensive Cancer Network 15(7):926-957; and et al. (2017) Blood 129(4):424-447, both of which are incorporated herein by reference.

[0193] The methods of the present application can increase or improve survival relative to patients receiving any standard treatment for myeloid malignancies.

[0194] The methods described herein can include the additional step of performing a bone marrow transplant on the patient or subject. The methods described herein can also be used to prepare a patient or subject having a myeloid malignancy to receive a bone marrow transplant. As described above, the methods of the present application can be performed to reduce the absolute or relative number of blast cells in the bone marrow or peripheral blood. In certain embodiments, the methods are performed to reduce the blast cell count in the bone marrow and / or peripheral blood prior to transplant. The methods can be used to reduce the blast cell count to less than 5% to prepare the patient or subject to receive a bone marrow transplant.

[0195] D. Kits

[0196] The compositions of the present application described herein can be packaged for provision in the form of a kit including instructions for use.

[0197] Incorporated by reference

[0198] In the foregoing description and throughout the entire embodiments, various publications are referenced. Each of the references cited herein is incorporated by reference in its entirety.

[0199] Examples

[0200] In a recent phase 1 clinical trial, the combination of the ADCC-enhanced humanized monoclonal anti-CD70 antibody (mAb) inotuzumab ozogamicin (also referred to herein as ARGX-110) with HMAs showed promising clinical activity and favorable tolerability profile in older and unfit AML patients.

[0201] The BCL-2 antagonist venetoclax targets and eliminates leukemic stem cells (LSCs) by inhibiting oxidative phosphorylation and showed very promising activity in older AML patients in combination with standard-of-care in phase I and II clinical studies (Pollyea et al., Nature Medicine (2018) 24; 1859-1866). However, even with novel agents such as venetoclax, there are patients who become refractory or relapse. It was hypothesized that the combination of venetoclax and inotuzumab ozogamicin, which would have different but complementary mechanisms of action, could successfully eliminate LSCs.

[0202] Methods

[0203] To test this hypothesis, drug combination studies were performed in CD70-expressing AML cell lines such as MOLM-13, NB-4 and NOMO-1 cells in vitro according to the Chou-Talalay method (Chou TC, Cancer Research (2010) 70(2); 440-6). MOLM-13 AML cells express FLT3-ITD and NOMO-1 cells express t(9; 11) (p22; q23). Each of these genetic aberrations express a poor prognosis of patient outcome. NB-4 is an acute promyelocytic leukemia (APL) cell line, where APL is a subset of AML patients. Notably, NOMO-1 cells and MOLM-13 cells express high levels of CD70 as measured on mRNA and protein levels. MOLM-13 AML cells also express BCL-2 and are sensitive to BCL-2 inhibition (Lin et al., Scientific Reports (2016) 6; 27696).

[0204] MOLM-13 cells (Matsuo et al., Leukemia (1997) 11(9): 1469-77), NOMO-1 cells (Kato et al., Acta Haematol Jpn, 1986), MV4-11 cells, and NB4 cells (Lanotte et al., Blood (1991) 77(5): 1080-86) were purchased from ATCC. The cell lines were tested to be mycoplasma-free and cultured at 37°C in a humidified atmosphere of 95% air and 5% CO2 in an ATCC-recommended FCS-containing medium containing GlutaMAX supplement, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0205] Initially, cells from each AML cell line were treated with decitabine, gustuzumab, or veneclade alone to determine the IC50 for each treatment. 50 In the presence of CFSE-labeled NK cells derived from healthy individuals (1:1 ratio), patients were treated for 10 days with a range of concentrations of gaustuzumab (0.1 μg / ml, 1.0 μg / ml, and 10 μg / ml), veneclax (0.5 nM and 200 nM), decitabine (0.01 μM–1 μM), or a mediator. 5 AML cells. Perform the assay in triplicate.

[0206] IC 50 The following working concentrations were determined for subsequent synergistic effect experiments:

[0207] 1) IC 50 s, MOLM-13: (decitabine: 0.01 nM; veneclax: 0.42 nM; goutuzumab: 0.68 μg / ml)

[0208] 2) IC 50 s, NOMO-1: (decitabine: 0.001 nM; veneclax: 3.4 nM; goutuzumab: 0.14 μg / ml)

[0209] 3) IC 50 s, NB4: (Decitabine: 4.8 nM; Venecla: 17.3 nM; Gustuzumab: 0.3 μg / ml)

[0210] 4) IC 50 s, MV4-11: (decitabine: 2.36 nM; veneclax: 5 nM; goutuzumab: 1.2 μg / ml)

[0211] To determine synergism, constant-combination ratio experiments were performed at equivalent dose ratios (IC501 / IC502or IC501 / IC502 / IC503) so that the contribution of each drug to cell killing was equal. Combination drug dose responses were assessed in two technical replicates for each dose / dose combination, as previously described (Riether et al., Sci Transl Med (2015) 7(298); 298ral 19).

[0212] NOMO-1, MOLM-13, NB-4 or MV4-11 AML cell lines were treated with single agents, decitabine, gemtuzumab ozogamicin or venetoclax; double combinations of venetoclax and decitabine, decitabine and gemtuzumab ozogamicin or venetoclax and gemtuzumab ozogamicin; or triple combinations of decitabine, gemtuzumab ozogamicin and venetoclax. All combinations were tested at three high and three low concentrations (above and below the determined IC 50 s) and at constant ratios. Cells were cultured in the presence of CFSE-labelled NK cells (ratio 1 : 1). The number of viable AML cells was assessed by annexin V staining after 72 hours and the effect of drug treatment was calculated as the ratio of surviving cells to vehicle-treated cells.

[0213] The combination index (CI) was calculated and the combination index versus fraction affected (Fa) plot was drawn using the CompuSyn software. The resulting combination index (CI) versus fraction affected (Fa) plot for all combinations is shown in Figure 1 Figure 2, with individual combinations shown in Figure 2 Figure 3.

[0214] Fa values of 0, 0.5 and 1 correspond to 0%, 50% and 100% of cells killed. CI values of <1, 1, >1 indicate synergism, additivity and antagonism, respectively. IC 50 s indicate the Fa values reached by combinations at the corresponding IC 50 concentrations. The principle and advantages of the Fa-CI plot method to assess synergism are provided in, for example, Chou TC, Cancer Research (2010) 70(2); 440-6; and Zhao et al., Front Biosci (Elite Ed). (2010) 2; 241-249 (each incorporated by reference in its entirety).

[0215] Furthermore, the effect of gemtuzumab ozogamicin / venetoclax and gemtuzumab ozogamicin / venetoclax / HMA combinations was tested on primary LSCs from AML patients. Primary CD34 + CD38 -Leukemia stem cells (LSC) were isolated from newly diagnosed AML patients and treated with ulocuplumab, decitabine or venetoclax monotherapy or combinations thereof. The impact on the colony forming and replating capacity of LSC was then assessed.

[0216] Specifically, CD34+CD38- cells from three AML patients (P1, P2 and P3) were treated in duplicate with ulocuplumab (Cusa: 0.3 pg / ml), decitabine (0.01 pM) or venetoclax (Ve: 6 nM) or combinations thereof as single agents or in combination with NK cells (ratio 1 :1 ) for 24 hours. Colony formation was assessed after 14 days. + CD38 - AML LSC were cultured overnight in the presence of NK cells (ratio 1 :1 ) with ulocuplumab (Cusa: 0.3 pg / ml) or venetoclax (Ve: 6 nM) as single agents or combinations thereof. CD34 + CD38 - AML LSC were cultured overnight in the presence of NK cells (ratio 1 :1 ) with ulocuplumab (Cusa: 0.3 pg / ml), decitabine (0.01 pM) or venetoclax (Ve: 6 nM) as single agents or combinations thereof. Colony formation was assessed after 14 days.

[0217] Results

[0218] 1. Synergy of gemtuzumab ozogamicin used in combination with venetoclax and / or decitabine in the elimination of AML cell lines in vitro Figure 1

[0219] The combination of venetoclax and / or decitabine with ulocuplumab synergistically eliminated CD70 expressing NOMO-1 AML cells over a wide dose range (see Figure 1 1 ). Figure 2 and Figure 3 B-D).

[0220] At higher levels of effect (Fa > 0.7) which are more relevant for tumor killing (Chou, 2010), the combinations exhibited strong synergism. Importantly, the CI of venetoclax and ulocuplumab (Ven / Cusa) as well as venetoclax, ulocuplumab and decitabine (Ven / Cusa / Dec) was close to 0.1, indicating very strong synergism. Venetoclax and decitabine (Ven / Dec) as well as ulocuplumab and decitabine (Cusa / Dec) also achieved synergism at higher levels of effect with a maximum CI of about 0.5. The synergism of the Ven / Cusa combination was similar to that observed for the Ven / Cusa / Dec combination.

[0221] Similar results were observed in the NB4 and MV4-1 1 AML cell lines, where all ulocuplumab combinations exhibited strong synergism at high levels of effect (Fa > 0.5) (see Figure 12). Figure 5 andFigure 4 ).

[0222] All double combinations showed synergism at lower levels of effect in the MOLM-13 cell line. Venetoclax / decitabine and futuxumab / decitabine showed synergism only at drug concentrations lower than 0.4 and 0.6 Fa, respectively. At higher levels of effect (Fa of about 0.7 to 0.8) more relevant to tumor killing, the combination of futuxumab / venetoclax showed strong synergism (see 2. Synergy of gemtuzumab ozogamicin used in combination with venetoclax and / or decitabine in the elimination of primary human AML leukemic stem cells (LSC) in vitro ), although some antagonism was observed at the highest level of effect. The triple combination of venetoclax, decitabine and futuxumab showed low levels of antagonism at all levels of effect.

[0223] Figure 6 Figure 7

[0224] To assess the effect of futuxumab / venetoclax combination on primary human AML LSC, CD34 + CD38 - LSC from 5 AML patients (P1-P5) were treated. Patient characteristics are shown below.

[0225] Table 2: Patient characteristics P1-P5

[0226]

[0227]

[0228] Results for patients P1, P2 and P3 are shown in Figure 6 and Figure 7 . Figure 6 The absolute number of colonies formed per well after treatment is shown, indicating the number of LSC. Figure 7 The same data is shown, expressed as the ratio to the mean number of colonies per well of the vehicle-treated group for each patient.

[0229] Figure 6 and Figure 7 show the synergism between futuxumab and venetoclax observed in AML cell lines, translated into a strong and significant reduction of the number of LSC compared to either treatment alone Figure 6 A and Figure 7 A).

[0230] Figure 8 B and Figure 1 B show that the effect on reducing the number of LSC is maintained when the first colonies are replated in the absence of the treatment molecules.

[0231] Results for patients P4 and P5 are shown in 3. Venetoclax increases CD70 expression In these patients, the triple combination of inotuzumab ozogamicin, decitabine and venetoclax showed equivalent efficacy to the double combination of inotuzumab ozogamicin and venetoclax. This is consistent with the fact that no increase in synergy was observed for the triple combination compared to the Ven / Cusa combination Figure 9 ).

[0232] Figure 10

[0233] The effect of venetoclax on CD70 expression was assessed at the mRNA and protein level. Results are shown in Conclusions and Figure 9 and clearly demonstrate the upregulation of CD70 expression in AML cells in the presence of venetoclax.

[0234] Figure 10

[0235] Experiments of the present application were performed to determine whether combination therapy using inotuzumab ozogamicin and venetoclax and / or a hypomethylating agent (e.g. azacitidine or decitabine) can provide effective AML treatment to a greater extent than each single therapy alone. It was further explored whether the combination therapy can exhibit a synergistic therapeutic effect.

[0236] The combination index (CI) provided by the Chou-Talalay method is an established means for determining whether a drug combination interacts in a synergistic, additive or antagonistic manner. It was hypothesized that venetoclax and inotuzumab ozogamicin can act in a synergistic manner, as it can be demonstrated that treatment with venetoclax causes upregulation of CD70 on AML cells Figures 1-5 and Figures 6-8 This can mean that venetoclax makes LSCs more susceptible to the cytolytic killing effect of inotuzumab ozogamicin. However, as described in Chou 2010 (Chou Cancer Res. (2010) January 15; 70(2):440-6, incorporated herein by reference), it is difficult to predict synergy between drugs even with some understanding of the mechanism of action of each individual drug.

[0237] ​ The data shown in

[0238] This synergistic effect is particularly advantageous as it indicates that when using a combination of drugs, significantly lower concentrations of each drug can be used compared to the concentrations required to achieve the same effect as a single therapy.

[0239] Since cell line experiments showed strong synergy for the venetoclax / epratuzumab combination, the combination was tested on primary AML LSCs. Primary LSCs provide a stringent and more clinically relevant assessment of potential therapeutic benefit since these cells drive the aberrant proliferation characteristic of AML.

[0240] The synergy observed for Ven / Cusa combination therapy in cell lines translated into potent reduction of primary AML leukemia stem cells (LSCs) from human patients ​ ). These data demonstrate that for all patient samples the combination therapy inhibited LSC colony formation to a significantly greater extent than either therapy alone. The data appear to confirm synergy between the components of the Ven / Cusa combination. (Testing for synergy using the Chou-Talalay method was not feasible due to the low number of primary cells available).

[0241] When the impact of epratuzumab / venetoclax treatment on LSC function was analyzed in a more stringent manner by in vitro serial replating experiments, the impaired colony formation observed after combination treatment was maintained during subsequent replating. This was true even in the absence of epratuzumab and venetoclax at the time of replating, indicating an effective reduction in LSC and their proliferative potential.

[0242] The triple combination epratuzumab / venetoclax / decitabine reduced the colony and replating capacity of primary human LSCs to the same extent as epratuzumab / venetoclax combination treatment. Thus, effective treatment of AML can be achieved using the combination of epratuzumab and venetoclax without the need to include an HMA such as decitabine, thereby reducing patient exposure to toxic therapies.

Claims

1. A composition comprising: (i) an antibody or antigen-binding fragment thereof that binds to CD70; and (ii) a BCL-2 inhibitor, wherein the antibody or antigen-binding fragment thereof that binds to CD70 and the BCL-2 inhibitor are each present in the composition in an amount sufficient to provide synergistic elimination of acute myeloid leukemia (AML) stem cells, wherein the antibody or antigen-binding fragment thereof that binds to CD70 comprises a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH domain and the VL domain comprise the following CDR sequence: HCDR3 consisting of SEQ ID NO: 3; HCDR2 consisting of SEQ ID NO: 2; HCDR1 consisting of SEQ ID NO: 1; LCDR3 consisting of SEQ ID NO: 7; LCDR2 consisting of SEQ ID NO: 6; and LCDR1, consisting of SEQ ID NO: 5, wherein the BCL-2 inhibitor is compound (I) as shown below or a pharmaceutically acceptable salt thereof. Compound (I).

2. The composition according to claim 1, wherein the VH domain comprises at least 70% of the same amino acid sequence as SEQ ID NO: 4, and the VL domain comprises at least 70% of the same amino acid sequence as SEQ ID NO:

8.

3. The composition according to claim 2, wherein the VH domain comprises the amino acid sequence represented by SEQ ID NO: 4, and the VL domain comprises the amino acid sequence represented by SEQ ID NO:

8.

4. The composition according to claim 1, wherein the antibody is IgG.

5. The composition according to claim 1, wherein the antibody has ADCC activity, CDC activity or ADCP activity.

6. The composition of claim 1, wherein the antibody comprises a defucosylated antibody domain.

7. The composition according to claim 1, wherein the antibody is ARGX-110 (Gustuzumab).

8. The composition of claim 1, wherein the antigen-binding fragment is selected from the group consisting of: antibody light chain variable domain (VL); antibody heavy chain variable domain (VH); single-chain antibody (scFv); F(ab')2 fragment; Fab fragment; Fd fragment; Fv fragment; single-arm (monovalent) antibody; bisomatic antibody, trisomatic antibody, tetrasomatic antibody and any antigen-binding fragment formed by combination, assembly or conjugation of the antigen-binding fragment.

9. The composition of claim 1, wherein the antibody or its antigen-binding fragment and the BCL-2 inhibitor are formulated as separate compositions.

10. The composition according to claim 1, wherein the composition comprises at least one additional anticancer agent.

11. The composition according to claim 10, wherein the anticancer agent is a medicament for treating myeloid malignant tumors.

12. The composition according to claim 11, wherein the anticancer agent is an agent for treating acute myeloid leukemia (AML).

13. The composition of claim 1, wherein the composition further comprises a low-methylating agent.

14. The composition according to claim 13, wherein the hypomethylating agent is azacitidine.

15. The composition according to claim 13, wherein the hypomethylating agent is decitabine.

16. Use of the composition according to any one of claims 1-15 in the preparation of a medicament for treating AML in human subjects.

17. The use according to claim 16, wherein the subject is additionally administered azacitidine or decitabine.

18. The use according to claim 16, wherein the drug is formulated to be administered separately of the CD70-binding antibody or its antigen-binding fragment and the BCL-2 inhibitor.

19. The use according to claim 16, wherein the AML is selected from newly diagnosed AML or relapsed / refractory AML.

20. The use according to claim 19, wherein the subject is a newly diagnosed AML patient who is not suitable for standard intensive chemotherapy.

21. The use according to claim 20, wherein the newly diagnosed AML patient is 75 years of age or older or has comorbidities that preclude the use of standard intensive chemotherapy.

22. The use according to claim 16, wherein the drug is formulated to be administered at a dose ranging from 0.1 mg / kg to 25 mg / kg to the CD70 antibody or its antigen-binding fragment.

23. The use according to claim 22, wherein the dose of the CD70 antibody or its antigen-binding fragment is 10 mg / kg.

24. The use according to claim 16, wherein the drug is formulated for administration of the BCL-2 inhibitor at a dose in the range of 100 mg to 600 mg.

25. The use according to claim 16, wherein the composition further comprises azacitidine, or the treatment comprises additional administration of azacitidine to the subject.

26. The use according to claim 25, wherein the concentration is 70 mg / m³. 2 -80 mg / m 2 The azacitidine was administered at the prescribed dosage.

27. The use according to claim 26, wherein the dosage of said azacitidine is 75 mg / m². 2 .

28. The use according to claim 16, wherein the composition further comprises decitabine, or the treatment comprises additional administration of decitabine to the subject.

29. The use according to claim 16, wherein the concentration is 15 mg / m³. 2 -25 mg / m 2 The dosage of decitabine was administered.

30. The use according to claim 29, wherein the dose of decitabine is 20 mg / m². 2 .

31. The use according to claim 16, wherein the treatment further includes monitoring the patient's blastocyst count.

32. The use according to claim 31, wherein the patient's myeloid blast count is reduced to less than 5%.

33. The use according to claim 31, wherein the patient’s myeloblast count is reduced to 5% to 25%, and the percentage of myeloblasts is reduced by more than 50% compared to before treatment.

34. The use according to claim 16, wherein the drug induces a partial or complete response.

35. The use according to claim 34, wherein the complete response includes platelet recovery.

36. The use according to claim 34, wherein the complete response includes neutrophil recovery.

37. The use according to claim 16, wherein the drug induces transfusion-independent red blood cells or platelets, or both, for a duration of 8 weeks or longer.

38. The use according to claim 16, wherein the drug increases survival rate.

39. The use according to claim 16, wherein the drug induces a negative minimal residual disease state.

40. The use according to claim 16, further comprising administering a bone marrow transplant to the subject.

41. The use according to claim 16, wherein the medicament further comprises one or more additional anticancer agents suitable for treating AML.

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