Use of the anti-CD70 antibody ARGX-110 for the treatment of acute myeloid leukemia

Abstract

To provide a pharmaceutical composition for treating acute myeloid leukaemia (AML) or myelodysplastic syndrome (MDS).SOLUTION: The present invention provides a pharmaceutical composition for use in a method for treating acute myeloid leukaemia (AML) or myelodysplastic syndrome (MDS) in a subject, where the pharmaceutical composition comprises an anti-CD70 antibody and an antigen-binding fragment thereof. The method comprises administering to the subject one or more doses of the anti-CD70 antibody or antigen-binding fragment thereof, and administering to the subject azacitidine or decitabine.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (Technical field of the invention) The present invention relates to a method for treating acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), and The present invention relates to compositions and combinations suitable for use in the method. [Background technology]

[0002] (background) Acute myeloid leukemia (AML) is characterized by uncontrolled clonal proliferation of hematopoietic progenitor cells. AML is the most common acute leukemia affecting adults and is a heterogeneous disease affecting most people in Europe. The annual incidence rate for adults in the state is 5-8 cases per 100,000, and for those aged 70 years and older The incidence increases dramatically in Japan, reaching 15-25 / 100,000 per year.

[0003] Cancer Research UK (CRUK) data on AML diagnosed in the UK between 2008 and 2010 for all ages. Survival statistics published by Cancer Research UK show that approximately 20% of patients survive for more than five years after diagnosis. The prognostic factors for poor outcome were age of the patient, treatment-induced AML, and a history of myelodysplastic syndrome or another antecedent blood disorder. Age 65 or older The five-year survival rate for patients with this disease is approximately 5%.

[0004] Chemotherapy, either as a single agent or in combination, treats most forms of leukemia. Under favorable conditions, high-dose chemotherapy followed by hematopoietic stem cell transplantation may also be used. Approximately 60% to 70% of adults with AML achieve complete remission (CR) after appropriate induction therapy. Approximately 45% of patients who reach this stage are expected to survive for more than three years and can be cured (US Cancer However, the side effects of chemotherapy can be a significant burden for patients, and many patients AML is not amenable to standard intensive chemotherapy, therefore alternative therapies for AML are desirable. Summary of the Invention

[0005] (Summary of the Invention) CD70 is expressed on a small subset of activated B and T lymphocytes, as well as on mature It is a cell surface antigen normally expressed on mature dendritic cells and is involved in lymphocyte differentiation and its cognate It is involved in survival signaling upon binding to the human cell surface receptor CD27. -induced signaling results in increased production and activation of CD27-expressing regulatory T cells do.

[0006] CD70 expression is low or absent in normal tissues, including all vital organs. CD70 is expressed in some tumor types, sometimes in association with CD27 in hematological malignancies. CD70 is overexpressed in combination with other malignant cells, suggesting its involvement in the proliferation and survival of malignant cells. CD70 also plays a role in evading immune surveillance by inducing Tregs, resulting in It is also clear that steroids may promote tumor growth.

[0007] Inhibition of the CD70-CD27 signaling pathway on regulatory T cells inhibits the recruitment and / or This may prevent activation of the immune system, thereby restoring immune surveillance in the tumor microenvironment. It is believed that

[0008] The present invention is the first to demonstrate the effective treatment of AML and MDS in humans with anti-CD70 antibodies. The treatment according to the present invention is surprisingly effective after a single dose of anti-CD70 antibody. Even low doses are effective. Anti-CD70 antibodies administered as monotherapy are surprisingly effective. In addition to being effective, the combination of anti-CD70 antibodies with nucleoside metabolic inhibitors (NMIs) It is also shown herein that combined therapy results in additional efficacy. As used herein, combination therapy according to the present invention refers to the simultaneous administration of two or more active agents. , or that two or more substances be formulated into a single composition.

[0009] AML patients treated with the therapies provided herein exhibit response rates greater than 90%; Two out of every three patients achieve complete remission. These results are consistent with the results of AML therapy for all patients. represents meaningful progress.

[0010] As demonstrated in the accompanying examples, treatment according to the present invention reduces bone marrow damage in patients. As previously noted, treatment with the present invention resulted in patients achieving complete remission. By the time of admission, the number of blasts had decreased significantly. The decline occurs when patients undergo successful hematopoietic stem cell transplantation (HSCT), a potentially curative treatment. Importantly, treatment according to the present invention allows patients to proceed to transplantation.

[0011] Notably, treatment with the present invention exhibits toxicity comparable to that reported for conventional NMI treatments. It effectively treats AML without increased toxicity.

[0012] This has implications for all patients with AML. Standard intensive chemotherapy for AML is The effective treatment according to the present invention is associated with significant toxicity and many patients experience severe side effects. The associated limited toxicity indicates that the therapy described herein represents an improved AML treatment for all patients. It highlights the possibility of providing

[0013] The surprising efficacy of the treatment according to the present invention is further illustrated by the characteristics of the patients recruited for the clinical trials. It is important that the patients treated are able to tolerate the toxicities associated with conventional intensive chemotherapy. Because they are not a match, they are not eligible for standard chemotherapy.

[0014] Standard intensive chemotherapy is thought to be required to sufficiently reduce bone marrow blasts, HSCT is not currently available for these patients. As mentioned above, the monotherapy and combination therapy according to the present invention can be administered to patients who are not able to undergo standard intensive chemotherapy. Thus, the therapies provided herein significantly reduce AML blasts in patients without AML. This opens up the possibility of predicting successful HSCT in patient populations for whom HSCT was not previously recommended. The D70 antibody has shown promise as monotherapy and in potent combination therapy with nucleoside metabolic inhibitors. A further significant benefit of the fact that both can effectively treat AML is the breadth The use of certain therapeutic models is possible, e.g., nucleoside metabolic inhibitors (NMIs). Combination therapy with EGFR-10 ... This provides a powerful therapeutic approach resulting from the enhanced effects of CD70 antibodies (in vitro and in vivo). This provides an effective therapy with reduced toxicity compared to standard intensive chemotherapy. This is especially important for patients who are not healthy enough to tolerate standard intensive chemotherapy. Monotherapy with CD70 antibodies provides an effective treatment without even the need for NMI. Additionally, by avoiding the effects of NMI on non-blast cells, toxicity is reduced, which may contribute to the prevention of hematopoietic This may lead to a reduced risk in patients who develop cytopenias. This reduces the patient's risk of infection and the need for blood transfusions.

[0015] Further treatment models made available by the present invention are the "induction" and "support" models. That is, the combination of anti-CD70 plus a nucleoside metabolic inhibitor (any loading dose) Induction therapy using IgG1 (with anti-CD70) strongly reduces the proportion of AML blasts in the bone marrow Patients are then treated with anti-CD70 alone or in combination with a lower dose of NMI. This model demonstrates that the reduction in blasts is due to the inhibition of nucleoside metabolism. without the need to expose patients to potential cumulative toxicity from long-term administration of harmful agents. This has the advantage that it can be maintained.

[0016] Furthermore, supportive treatment with such anti-CD70 antibodies alone may be ineffective against other cell types (which may have minimal CD70 expression). It has minimal effect on blast cells (which express CD70) and on blastocytes (which may or may not express CD70). The dosage of nucleoside metabolic inhibitors can be stopped or reduced. In this way, cytotoxic pressure on non-blast cell types is reduced, allowing these cells to proliferate. This is because AML blasts can be targeted by CD70 antibodies while other cell types (e.g. , platelets, red blood cells, and neutrophils) can be recovered, so the blood cell count resulting from long-term NMI treatment is This has the advantage of reducing the risk of attrition.

[0017] Furthermore, administration of anti-CD70 antibodies, alone or in combination with NMI, promoted the differentiation of LSCs into myeloid cells. It is shown herein that such promotion of differentiation is related to self-renewal. This reduces the population of LSCs that can contribute to disease propagation and maintenance. This contributes significantly to the proliferation of malignant cells, providing a self-renewal pool of malignant cells. Administration in combination with MI reduces the LSC population by inducing differentiation of LSCs, thereby promoting tolerance. Increases the chances of resolution and reduces the risk of recurrence.

[0018] Thus, in one aspect, the present invention provides a method for treating acute myeloid leukemia (AML) or myeloid dysplasia in a subject.

[0013] A method for treating myelodysplastic syndrome (MDS) comprising administering to a subject an anti-CD70 antibody or antigen-binding fragment thereof. The present invention provides methods of treating a patient suffering from atopic dermatitis, the methods comprising administering the above dosages.

[0019] In a further embodiment, the method reduces the percentage of blast cells in the bone marrow and / or peripheral blood of a subject with AML or MDS. A method comprising administering to a subject one or more doses of an anti-CD70 antibody or antigen-binding fragment thereof. The present invention provides a method for detecting a stoichiometric amount of a substance comprising:

[0020] In a further embodiment, a method of preparing a subject with AML or MDS for hematopoietic stem cell transplantation (HSCT) administering to the subject a therapeutically effective amount of one or more anti-CD70 antibodies or antigen-binding fragments thereof. The present invention provides a method comprising:

[0021] In a further aspect, the present invention provides a method for administering one or more doses of an anti-CD70 antibody or antigen-binding fragment thereof to a patient. A method for treating acute myeloid leukemia (AML) or myelodysplastic syndrome in a subject, comprising administering to the subject The present invention provides an anti-CD70 antibody or antigen-binding fragment thereof for use in a method for treating myelodysplastic syndromes (MDS). To provide.

[0022] In a further aspect, the present invention provides a method for administering one or more doses of an anti-CD70 antibody or antigen-binding fragment thereof to a patient. for use in a method for reducing AML blast cells in a subject, the method comprising administering to the subject The present invention provides an anti-CD70 antibody or antigen-binding fragment thereof for use in the treatment of cancer.

[0023] In a further aspect, the present invention provides a method for administering one or more doses of an anti-CD70 antibody or antigen-binding fragment thereof to a patient. 2. A method for preparing a subject with AML or MDS for hematopoietic stem cell transplantation (HSCT), comprising administering to the subject The present invention provides anti-CD70 antibodies for use in the methods.

[0024] In a further aspect, the present invention provides an anti-CD70 antibody or antigen-binding fragment thereof, an anti-CD70 antibody or and a pharmaceutical composition comprising the antigen-binding fragment and a pharmaceutically acceptable excipient or carrier. A method for treating acute myeloid leukemia (AML) or myelodysplastic syndromes in a subject, comprising administering the above dose to the subject. The present invention provides pharmaceutical compositions for use in methods for treating myelodysplastic syndromes (MDS).

[0025] In a further embodiment, one or more doses of an anti-CD70 antibody or antigen-binding fragment thereof are administered to a subject. 1. A pharmaceutical composition for use in a method for reducing AML blast cells in a subject, the method comprising administering Pharmaceutical compositions are provided.

[0026] In a further embodiment, one or more doses of an anti-CD70 antibody or antigen-binding fragment thereof are administered to a subject. 1. A method of preparing a subject with AML or MDS for hematopoietic stem cell transplantation (HSCT), comprising administering The present invention provides a pharmaceutical composition for use in

[0027] In a further embodiment, the antibody or antigen-binding fragment is a hypomethylating agent, preferably azacytidine. Anti-CD70 antibody or antigen binding thereof for use in the treatment of AML, administered in combination with a steroid Provide a combined fragment.

[0028] In certain embodiments of the methods of the invention, the anti-CD70 antibody or antigen-binding fragment thereof is administered once. A dose ranging from 0.1 mg / kg to 25 mg / kg per dose, optionally in the range of 1 mg / kg to 20 mg / kg In certain embodiments, the anti-CD70 antibody or antigen-binding fragment thereof is administered in an amount of 1 mg / In certain preferred embodiments, the compound is administered at a dose of 1 mg / kg, 3 mg / kg, 10 mg / kg, or 20 mg / kg. Thus, the anti-CD70 antibody or antigen-binding fragment thereof is administered at a dose of 10 mg / kg.

[0029] In certain embodiments of all aspects of the methods of the invention, the methods include administering a nucleoside inhibitor to a subject. In certain embodiments, the method further comprises administering a non-methylating agent (NMI), such as a hypomethylating agent (HMA). I is azacitidine or decitabine.

[0030] In certain embodiments, the methods of the invention comprise: (i) following a dosing regimen described herein; Phase 1, including administration of an anti-CD70 antibody and a nucleoside metabolic inhibitor as a combination therapy and (ii) administering an anti-CD70 antibody according to the dosing regimen described herein, and a lower dose of a nucleoside metabolic inhibitor than the dose of the nucleoside metabolic inhibitor to be administered; A second step includes administering:

[0031] In certain embodiments, the subject is not eligible for standard intensive chemotherapy prior to treatment according to the present invention. do not have.

[0032] In certain embodiments, the method further comprises performing a hematopoietic stem cell transplant in the subject. Included.

[0033] In certain embodiments, the patient is 60 years of age or older, optionally 75 years of age or older.

[0034] In certain embodiments, the present invention provides a combination of an anti-CD33 antibody, an anti-CD123 antibody, and an anti-CD33 antibody as part of a combination therapy. Antibodies, E-selectin inhibitors, FLT3 inhibitors, cyclin-dependent kinase inhibitors, BCL-2 inhibitors and one or more active substances selected from the group consisting of anti-inflammatory drugs, ... The method further comprises administering

[0035] In a further aspect, the present invention provides a combination comprising an anti-CD70 antibody or antigen-binding fragment thereof and an NMI. In certain embodiments, the NMI is a hypomethylating agent. In an embodiment, the hypomethylating agent is azacitidine or decitabine, preferably azacitidine. It's gin.

[0036] In a further aspect, the present invention provides a method for treating AML or MDS comprising: Combinations are provided that include an anti-CD70 antibody or antigen-binding fragment thereof and an NMI. In certain preferred embodiments, the NMI is a hypomethylating agent. is azacitidine or decitabine, preferably azacitidine.

[0037] In a further aspect, the present invention provides an anti-CD70 antibody for use in a method according to the present invention. The present invention provides a combination comprising the antibody or antigen-binding fragment thereof and an NMI.

[0038] In all embodiments of the present invention, the anti-CD70 antibody inhibits the interaction of CD70 with its receptor CD27. In certain embodiments, the anti-CD70 antibody or antigen-binding fragment thereof may inhibit CD70-C In certain embodiments, the anti-CD70 antibody or antigen-binding fragment thereof may inhibit CD27 binding. may inhibit CD70-CD27 induced signaling.

[0039] In all embodiments of the invention, the anti-CD70 antibody is capable of inhibiting antibody effector functions, e.g., antibody with dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and / or antibody-dependent phagocytosis (ADCP). In certain embodiments, the anti-CD70 antibody or antigen-binding fragment thereof may be, for example, an anti-CD70 antibody. CD70-expressing cells may be depleted via somatic effector function.

[0040] In certain embodiments, the anti-CD70 antibody is a modified antibody, e.g., an antibody drug conjugate ( As described elsewhere herein, ADCs can be used to bind to active agents such as cytotoxic agents. ADCs also conjugate one or more antibodies in addition to delivering an active agent to a target. It may have an effector function.

[0041] In certain embodiments of all aspects of the invention, the anti-CD70 antibody comprises a heavy chain variable domain (VH ) and a light chain variable domain (VL), wherein the VH and VL domains comprise the following CDRs: SEQ ID NO:3 [ka] HCDR3 comprising or consisting of SEQ ID NO:2 [ka] HCDR2 comprising or consisting of SEQ ID NO:1 [ka] HCDR1 comprising or consisting of SEQ ID NO:7 [ka] LCDR3 comprising or consisting of SEQ ID NO:6 [ka] LCDR2 comprising or consisting of: SEQ ID NO:5 [ka] LCDR1 comprising or consisting of: Includes.

[0042] In certain embodiments, the anti-CD70 antibody or antigen-binding fragment has at least one sequence identical to SEQ ID NO:4. VH that is 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical domain and / or at least 80%, at least 90%, at least 95%, at least 98%, at least 99% identical VL domains. For embodiments in which the domain of a fragment is defined by a specific percentage of sequence identity with a reference sequence, Thus, the VH and / or VL domains may maintain the same CDR sequences as present in the reference sequence. , so that variation exists only within the framework regions.

[0043] In certain embodiments, the anti-CD70 antibody is an IgG1 antibody.

[0044] In all aspects of the present invention, the anti-CD70 antibody is preferably ARGX-110.

[0045] In all aspects of the invention, a preferred embodiment is a combination of ARGX-110 and azacitidine. That is it.

[0046] In all aspects of the present invention, the subject or patient is a human subject or patient. [Brief explanation of the drawings]

[0047] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]Figure 1: αCD70 / decitabine combination therapy eradicates human CD34+CD38- AML stem / progenitor cells in mouse xenografts. (ai) 5 × 106 FACS-purified CD45dimSSClo cells from the BM of newly diagnosed AML patients (patients P10 and P21) were injected intravenously into the tail vein of sublethally irradiated NSG mice. After engraftment (days 32 (P10) and 97 (P25) post-transplant), mice were randomized and treated intraperitoneally with a control mAb and 10 mg / kg αCD70 mAb (41D12-D) (total of three injections), or with decitabine (1.5 mg / kg / day) alone or in combination for five consecutive days. One day after the final treatment, animals were sacrificed, and blood, spleen, and BM were analyzed. (a) Experimental setup. (b) CD70 expression on CD34+CD38- AML stem / progenitor cells. Isotype is shown in gray; CD70 staining is shown in black (P10) and blue (P25). The solid line represents vehicle, and the dashed line represents decitabine treatment on AML stem / progenitor cells. ΔMFI: MFI staining - MFI isotype. (c) Fold change in CD70 expression on non-CD34+CD38- bulk and CD34+CD38- AML stem / progenitor cells (vehicle vs. decitabine). (d) Representative FACS plot of human CD45+ AML cell engraftment in the BM of PDX mice. (e) Frequency of human CD45+ AML cells in the BM of PDX AML mice. (f) Absolute number of CD45dimSSClolin-CD90-CD34+ AML stem / progenitor cells in the BM. (g) Representative FACS plot and (h) quantification showing the frequency of CD38- AML cells within the CD45dimSSClolin-CD90-CD34+ AML stem / progenitor cell population. (i) Frequency of CD45RA+ cells within the CD45dimSSClolin-CD90-CD34+CD38- AML stem / progenitor cell population. Data are presented as mean ± SD. Statistics: (b, c) Student's t-test; (e, f, h, i) One-way ANOVA; Tukey's post-hoc test; P<0.05; **, P<0.01; ***, P<0.001. [Figure 2]Figure 2: HMA treatment induces CD70 expression in primary AML stem / progenitor cells. (a) Representative FACS plot of CD70 expression on lin-CD90+CD34+CD38- AML stem / progenitor cells after culture in the presence or absence of 0.5 mM decitabine (D) or vehicle (Veh). Isotype: gray; CD70: black. (b) Cell viability. (c) Fold change in ΔMFI CD70, and (d) Fold change in mRNA CD70 expression (AML: n=9-15; healthy: n=3). (e) Representative FACS plot and (f) Fold change in ΔMFI CD70 on stem / progenitor cells in peripheral blood of AML patients at diagnosis and after one cycle of decitabine or azacitidine treatment (D(5), 20 mg / kg daily for 5 days; A(7), 75 mg / m2 daily for 7 days). [Figure 3] Figure 3: αCD70 / decitabine co-treatment reduces the replating capacity of human CD34+CD38- AML stem / progenitor cells. (ab) FACS-purified lin-CD90-CD34+CD38- stem / progenitor cells from the BM of newly diagnosed AML patients (P6, P8, P11) were cultured overnight in triplicate in the presence or absence of 10 mg / ml anti-CD70 (αCD70) mAb or 0.5 mM decitabine alone or in combination, and then plated onto methylcellulose containing αCD70 and decitabine or both. Colony and cell numbers were counted after 14 days. (a) Number of colonies per 1 × 103 plated cells. (b) Number of cells per colony. (c) Serial replating experiment. (df) FACS-purified lin-CD90+ CD34+ CD38- stem / progenitor cells from the BM of "healthy" donors were cultured and plated in methylcellulose as described in (ac). (d) Number of colonies per 1 x 103 plated cells. (e) Number of cells per colony. (f) Serial replating experiment. Data are presented as mean ± SD. Statistics: One-way ANOVA. Dunnett's post-hoc test (vs. aCD70 / D); *, P < 0.05; **, P < 0.01; ***, P < 0.001). [Figure 4]Figure 4: Schematic showing the treatment regimen for patients enrolled in an open-label, dose-escalation study with proof of concept cohort of ARGX-110 in combination with AZA. [Figure 5]Figure 5: (a) End of Treatment (EOT): The EOT date and time is the last day ARGX-110 is administered. A clinic visit is scheduled within 7 days after EOT. (b) The time windows are as follows: - Pre-dose time points: up to 4 hours before ARGX-110 infusion (days -14 and 17 of each cycle) or AZA administration (days 1, 3, and 7); - Midnight (end of ARGX-110 infusion) ± 30 minutes and 2 hours (after end of ARGX-110 infusion): ± 30 minutes; - 24 hours: ± 4 hours; - Post-dose time points (day X): within 2 hours after end of ARGX-110 infusion; (c) up to 4 hours before ARGX-110 infusion on day -14, and days 3 and 17 of cycles 1-4 and cycle 8 (if applicable), and at EOT and follow-up visits; (d) Molecular genetic samples can be used for characterization of CD70 and CD11a promoter methylation and genomic DNA analysis of disease and treatment effect on target pathologies. From cycle ≥ 3 onwards, sampling should occur on day 1 of every odd-numbered cycle (i.e., C3D1, C5D1, ...) prior to AZA administration until complete remission (CR, CRi); (e) gene expression samples are used to characterize CD70 mRNA levels, a marker of disease and drug effect. From cycle ≥ 3 onwards, sampling should occur on day 1 of every odd-numbered cycle (i.e., C3D1, C5D1, ...) prior to AZA administration until complete remission (CR, CRi); (f) flow cytometry (FACS) samples may be used for minimal residual disease analysis, CD70 and CD27 expression, and additional characterization of drug effect (e.g., blasts, NK cells, and T cells). From cycle ≥ 3 onwards, sampling should be performed on day 1 of every odd-numbered cycle (i.e., C3D1, C5D1, ...) prior to AZA administration until complete remission (CR, CRi); (g) serum samples may be used for additional characterization of sCD27, markers of disease and drug effect, and inflammatory cytokine analysis. From cycle ≥ 3 onwards, sampling should be performed on day 1 of every odd-numbered cycle (i.e., C3D1, C5D1, ...) prior to AZA administration until complete remission (CR, CRi); (h) stemness determination will be performed on mononuclear cells purified from blood or bone marrow.Depending on the number of cells collected, readings can include Numb staining (to determine the ratio of asymmetric / symmetric divisions), cell and in vivo testing (e.g., CFU methylcellulose colony assay or survival testing of NSG mice injected with patient mononuclear cells to assess stem cell potential); (i) Timing of aspirate biopsy sampling for response assessment of combination therapy: Unless clinically contraindicated, it should be performed on day 1 of every odd-numbered cycle (i.e., C3D1, C5D1, etc.) until complete remission (CR, CRi) and at EOT. For patients who achieve CRMRD, an additional BM aspirate / biopsy should be collected no sooner than 4 weeks after CRMRD to confirm response. Additional aspirate / biopsy samples will be collected as directed by the treating physician and will be treated as study-related samples; (j) additional PK and / or ADA samples may be obtained if evidence emerges regarding decreased serum exposure of ARGX-110 and / or increased ADAs during long-term treatment; (k) bone marrow samples will be collected unless medically contraindicated; (l) bone marrow supernatant samples will be used to assess sCD27 concentrations and treatment effects on disease and target pathologies. [Figure 6] Figure 6: Bone marrow (BM) blast burden was assessed by cell morphology (A.) and flow cytometry using leukemia-associated immunophenotype (LAIP) gating (B.) and blast gating (SSClowCD45dim; C.). The latter method was used to determine the patient's measurable / minimal residual disease (MRD) status. [Figure 7] Figure 7: Peripheral blood (PB) blast load was assessed by cell morphology (A.) and flow cytometry using leukemia-associated immunophenotype (LAIP) gating (B.) and blast gating (SSClowCD45dim; C.). The latter method was used to determine the patient's measurable / minimal residual disease (MRD) status. [Figure 8]Figure 8: Purified AML blasts (FACS gate: CD45dim SSClow AV-CD4-CD8-CD19-) were fixed and permeabilized, and the cells were incubated overnight with α-Numb antibody followed by staining with a fluorescently labeled secondary antibody. DAPI was used to counterstain DNA. Samples were acquired on an ImageStreamX® Mark II imaging flow cytometer (Amnis / EMD Millipore) and analyzed using INSPIRE™ and IDEAS® software (Amnis / EMD Millipore). A.) Total Numb expression (mean fluorescence intensity) was determined across slides for one patient's bone marrow samples collected at baseline ("Screening"), after ARGX-110 monotherapy (C1D1), and after combination therapy (C4D1). B.) Symmetrically dividing (SD) and asymmetrically dividing (AD) cells were scored in bone marrow patient samples at baseline ("pre") and after ARGX-110 monotherapy (post-αCD70). [Figure 9] Figure 9: αCD70 mAb treatment reduced CD34+CD38- AML stem / progenitor cell frequencies in AML patients. (a) Colony formation at P2. 104 BM MNCs from patient P002 at the time of diagnosis (SCR) and 14 days after administration of ARGX110 (1 mg / kg iv, time point 0, or C1D1) were seeded in methylcellulose, and colony formation was assessed 2 weeks later. (b) Fold change in colony formation in different patients treated with the indicated doses of ARGX110. (c) Colony formation at limiting dilution from P2. (d) Stem cell frequencies at SCR and time point 0 determined in an enhanced limiting dilution experiment in different patients treated with P2 and the indicated doses of ARGX110. Data are presented as mean ± SD. Statistics: (a, b, e) Student's t-test; (d): chi-square test; **, P<0.01; ***, P<0.001. (f) Colony formation in P2. At the time of diagnosis (SCR) of patient P002, 14 days after ARGX110 administration (1 mg / kg iv, time 0, or C1D1), and after combination treatment (“combination”), 10, 5 x 10, 10, and 10 BM MNCs were seeded in methylcellulose and colony formation was assessed 2 weeks later. [Figure 10] Figure 10: Serum samples were analyzed using ELISA (Duoset ELISA, human CD27 / TNFRSF7 (catalog number: DY382-05, R&D Systems). The concentration of soluble CD27 in the samples is calculated from a calibration curve. This method allows the measurement of serum concentrations of native human soluble CD27 by a sandwich ELISA. A goat anti-human CD27 capture antibody is coated onto a microplate surface to block nonspecific binding sites. Human serum samples are applied, and bound human soluble CD27 is detected and visualized by the subsequent addition of biotinylated goat anti-human CD27 detection antibody, streptavidin-HRP, and a mixture of the color reagent HO and the chromogenic substrate tetramethylbenzidine (TMB). [Figure 11] Figure 11: Serum concentrations of ARGX-110 were analyzed using a validated enzyme-linked immunosorbent assay (ELISA). Individual patient PK plots are provided for the 10 mg / kg cohort. The PK plot shows data from ARGX-110 Cycle 1 (D-14 pre-dose to Cycle 1 D1 pre-dose). DETAILED DESCRIPTION OF THE INVENTION

[0048] (Detailed Description of the Invention) The so-called “7+3” standard intensive chemotherapy (i.e., 7 days of intensive cytarabine plus Three days of tracycline, typically followed by consolidation chemotherapy or hematopoietic stem cell transplantation (HSCT) Conventional treatment of AML using chemotherapy has been the standard of care for many years. In the current study, the majority of AML patients under the age of 60 fail to survive for more than five years. In older adults who are not well adapted to therapy, the outcome of less intensive treatment is not curative and is associated with HS. CT is typically not feasible, and median overall survival is less than 1 year. AML is a heterogeneous disease. Disease relapse is due to one or more leukemia stem cell (LSC) clones that are resistant to therapy. Although there are many preclinical studies investigating alternative therapies, few are translated into clinical practice. Therefore, there are few effective new AML therapies, especially for patients who are not suitable for standard intensive chemotherapy. Ideally, novel therapies would reduce the blast burden in the bone marrow and blood. Reduction of LSCs not only eliminates the disease but also reduces or eradicates the heterogeneous LSC population. They even do it.

[0049] As disclosed earlier herein, human subjects with AML or MDS may be treated with an anti-CD70 antibody. Such treatment of patients with AML or MDS is often achieved by standard intensive chemotherapy. This is particularly desirable because conventional treatment with the method is associated with significant toxicity and side effects. However, many patients (e.g., elderly patients) have comorbidities that result in them being unable to tolerate standard intensive chemotherapy. They are unable to tolerate the pain, which means that only less effective therapies can be used on them. It is effective even at low doses, with limited side effects and toxicity, which is Treatments for AML are provided herein, meaning they are suitable for administration to all patients. This is particularly relevant for the treatment of patients who are otherwise ineligible for standard intensive chemotherapy. is advantageous.

[0050] Thus, treatment of AML according to the present invention provides surprising and significant benefits over currently available therapies. Aspects and embodiments of the present invention are further described herein. Each embodiment of the present invention may be combined with any other embodiment of the present invention unless technically incompatible. It can be implemented in combination with the above.

[0051] (definition) Acute myeloid leukemia (AML) refers to a hematopoietic neoplasm involving bone marrow cells. AML is a type of hematopoietic neoplasm that lacks differentiation capacity. AML is characterized by the clonal expansion of myeloid precursors, which is reduced in the bone marrow. This shows the accumulation of blast cells, which typically also accumulate in the peripheral blood of AML patients. Typically, AML is diagnosed when a patient exhibits 20% or more blast cells in the bone marrow or peripheral blood. .

[0052] As used herein, "blast cells" or simply "blasts" refer to cells that have perturbed differentiation potential. A subset of blast cells is leukemia stem cells (LSCs). These are blast cells with stem cell properties and therefore may be useful for immunocompromised recipients. When transplanted, LSCs allow leukemia disease to begin. more self-renewing and resembles the underlying disease but is unable to self-renew, a common form of non-LSC They can also partially differentiate into blast cells. LSCs are a fraction of primary AML blast cells in the 10,000 It occurs with a frequency ranging from 1 in 100 to 1 in 1 million (Pollyea and Jordan, Reference Blood 201 7 129:1627-1635, which is incorporated herein by reference). LSCs are CD34+CD38 LSCs may also be characterized as CD45- and / or CD123+ cells. Also characterized as D45dim, SSClo, CD90+CD34+ cells.

[0053] AML is classified and diagnosed according to the WHO 2008 classification, in combination with the 2016 revision to this classification. (Arber et al., Blood, May 19, 2016, Vol. 127, No. 20, which is cited in According to the WHO classification, AML generally encompasses the following subtypes: Acute myeloid leukemia with recurrent genetic abnormalities; AML with myelodysplasia-related changes ;Therapy-related myeloid neoplasms: myeloid sarcoma; Down syndrome-associated myeloproliferative disorder; blastic plasmacytoid dendritic cell tumors; and other classified AML (e.g., acute megakaryoblastic leukemia, acute halophilic leukemia) basal cell leukemia).

[0054] AML can also be classified according to the French-American-British (FAB) classification, which includes the following subtypes: Types include: M0 (acute myeloblastic leukemia, minimally differentiated); M1 (acute myeloblastic leukemia, mature) M2 (acute myeloblastic leukemia, with granulocytic maturation); M3 (proosteoblastic or Acute promyelocytic leukemia (APL); M4 (acute myelomonocytic leukemia); M4eo (with bone marrow eosinophilia) Myelomonocytic; M5 (acute monoblastic leukemia (M5a) or acute monocytic leukemia (M5b)); M6 (erythroleukemia (M acute erythroblastic leukemia, including M6a) and the very rare pure erythroleukemia (M6b); or M7 (acute megaloblastic leukemia) karyoblastic leukemia).

[0055] As used herein, "AML" refers to the AML subtype as defined by the WHO and / or FAB classification, unless otherwise specified. Certain AML subtypes have a relatively good prognosis, Some subtypes are considered to have an intermediate prognosis and some to have a poor or adverse prognosis. know which risk category they fall into.

[0056] Myelodysplastic syndromes (MDS) are characterized by dysplasia, cytopenias, and / or bone marrow cellularity. abnormal changes in myeloid differentiation, such as increased blast cell infiltration, and / or MDS can be classified and diagnosed according to the WHO 2008 classification. According to the WHO classification, MDS generally encompasses the following subtypes: MDS with single-lineage dysplasia (MDS); Until now, this was called "refractory cytopenia with single-lineage dysplasia," which is a condition that can cause refractory anemia, refractory cytopenia, and MDS with ringed sideroblasts, which is a single-lineage variant; This includes a subgroup with refractory anemia and multilineage dysplasia (previously called "ringed sideroblast-positive refractory anemia"). MDS with multilineage dysplasia (previously called "multilineage dysplastic refractory cytopenia") MDS with excess blasts (MDS-EB, previously called "refractory anemia with excess blasts"), which is further Based on the percentage of blast cells, it is subdivided into MDS-EB-1 and MDS-EB-2; MDS with leukemia (l(5q)); and unclassified MDS.

[0057] MDS can also be classified according to the French-American-British (FAB) classification, with the following subtypes: Types include: M9980 / 3 (refractory anemia (RA)); M9982 / 3 (ringed sideroblast-positive refractory anemia (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)).

[0058] As used herein, "MDS" refers to the WHO and / or FAB classification unless otherwise specified. For both AML and MDS, the WHO classification It is preferred in the specification.

[0059] "High-risk" MDS patients - i.e., those at high risk of developing AML and with poor survival prognosis It is particularly desirable to treat MDS patients with associated tumours. The International Prognostic Scoring System for Myelodysplastic Syndrome (IPSS-R) (Greenberg et al., Blood, 2012 Sep 20;120(12):2454-2465, which is incorporated herein by reference. The IPSS-R is used to assign patients to prognostic categories. Consider the patient's bone marrow blast percentage, cytogenetic abnormalities, and number and severity of cytopenias. Patients with a core greater than 4.5 are considered to be "high risk" MDS patients.

[0060] A subject's response to AML therapy can be clinically characterized according to the criteria in Table 1. : Table 1 [Table 1]

[0061] As used herein, "dosage" is used to mean an effective amount of an active substance. The dosage administered to a subject is an effective amount of a particular active substance administered per day. "Dose" refers to a single dose per day if the specified effective amount of the dose is administered to a subject. For example, a dose of 1 mg / kg may be administered as a single dose of 1 mg / kg once daily. kg dose or twice daily for a total of 1 mg / kg It can be given.

[0062] As used herein, the terms "CD70 protein" or "CD70 antigen" or "CD70" or "TNFSF7" or "CD27L" are used interchangeably and are the ligands for TNFSF27 / CD27. refers to a member of the human TNF ligand family. A specific example of human CD70 is found in the NCBI reference sequence. A polypeptide having the amino acid sequence set forth in Accession No. NP_001243 or its extracellular domain Includes.

[0063] As used herein, the term "antibody" refers to an antibody that is directed against an antigen of interest (e.g., human CD70). It is an immunoglobulin having a combination of two heavy chains and two light chains, which has a highly specific immune response activity. The term "CD70 antibody" refers to an antibody that is immunospecific for the human CD70 protein. "Specificity" for human CD70 is used herein to refer to the specificity of the species homologue of CD70. Cross-reaction with the human body is not excluded. Antibodies consist of light and heavy chains with covalent interchain bonds between them. An antigen-binding fragment of an antibody is a fragment that binds to the same antigen as the antibody (e.g., CD70). Examples of antigen-binding fragments include peptide fragments that exhibit specific immune reaction activity against the antigen. :Antibody light chain variable domain (VL); antibody heavy chain variable domain; single chain variable region fragment or single chain antibody (scFv); Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; one-arm (monovalent) antibody; diabody or a combination, assembly or multiple of such antigen-binding fragments. A fragment is, for example, an intact or complete antibody or antigen-binding molecule formed by the fusion of They can be obtained by chemical or enzymatic treatment of the base chain or by recombinant means.

[0064] As used herein, a "nucleoside metabolic inhibitor" (NMI) refers to a compound that inhibits the metabolism of nucleotides (DNA and / or RNA) epigenetic modifications (e.g., methylation, demethylation, acetylation) nucleoside metabolism inhibitors are molecules that interfere with the Antioxidants (HMA), isocitrate dehydrogenase (IDH) inhibitors, histone deacetylase (HDAC) inhibitors Preferred nucleoside metabolic inhibitors include bromodomain and extraterminal (BET) inhibitors. The inhibitors are hypomethylating agents. Hypomethylating agents inhibit the normal methylation of DNA and / or RNA. Examples of hypomethylating agents are azacitidine, decitabine, and guadecitabine.

[0065] As used herein, when two or more active agents are administered as "combination therapy," This requires that the active agents be administered simultaneously or formulated into a single composition. Combination therapy does not include or exclude the administration of two or more active substances. Conventional interpretations are given so that patients can derive benefit from each drug. To avoid confusion, "combination therapy" refers to co-administration, simultaneous administration, or fixed-dose formulations. Not necessary.

[0066] As used herein, "standard intensive chemotherapy" refers to 7 days of high-dose cytarabine, followed by 3 days of anthracycline (e.g., daunorubicin or idarubicin) This refers to the so-called "7+3" induction chemotherapy, which is characterized by the following: In the current study, the induction of complete remission in AML was investigated for the consolidation of patients undergoing stem cell transplantation after successful chemotherapy. It is given for the purpose of guidance.

[0067] Standard intensive chemotherapy is associated with significant toxicity and side effects, which may limit these effects. As used herein, the term "non-steroidal anti-inflammatory drug" means that the drug is not suitable for patients who cannot tolerate non-steroidal anti-inflammatory drugs. For example, patients may be treated with one of the following chemotherapy regimens: or more comorbidities that indicate they cannot tolerate toxicity, or Because prognostic factors characterizing the disease indicate an unfavorable outcome with standard intensive chemotherapy, these Patients may be ineligible for standard intensive chemotherapy. Determination of eligibility is based on individual patient history and clinical guidelines (e.g., National Comprehensive Cancer Network Consider the National Cancer Center (NCCN) guidelines, which are incorporated herein by reference. Patients over 60 years of age who are ineligible for standard intensive chemotherapy are considered to be eligible for this treatment. Other factors that are often evaluated and should be considered are the cytogenetic abnormalities and and / or molecular abnormalities.

[0068] Patients who are ineligible for standard intensive chemotherapy may be offered reduced-intensity chemotherapy, such as low-dose cytarabine (LDAC). Patients who are ineligible for standard intensive chemotherapy and whose LDAC levels are inadequate will receive chemotherapy instead. Patients with ulcerative colitis receive best supportive care (BSC), including hydroxyurea (HU) and transfusion support. It is possible.

[0069] As used herein, "subject" and "patient" are used interchangeably to refer to a human individual. Used.

[0070] (Detailed explanation) As disclosed herein, patients suffering from myeloid neoplasms such as AML and MDS Patients can be treated with anti-CD70 antibodies. After a single administration of anti-CD70 antibodies, the subject's bone The number of leukemic stem cells that can be isolated from the bone marrow is significantly higher than the number of blast cells detected in the bone marrow and peripheral blood. This result was surprising, even at low doses of antibody. It was also recognized.

[0071] Thus, in a first aspect, the present invention provides one or more anti-CD70 antibodies or antigen-binding fragments thereof. 2. A method for treating acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) in a subject, comprising administering a dose of The present invention provides a method for treating myelodysplastic syndrome (MDS). A method for treating acute myeloid leukemia (AML) in a subject, comprising administering to the subject a therapeutically effective amount of one or more of the following: The present invention provides methods for treating myelodysplastic syndromes (MDS) or myelodysplastic syndromes (MDS).

[0072] In a further embodiment, the method reduces the percentage of blast cells in the bone marrow and / or peripheral blood of a subject with AML or MDS. The present invention provides a method for administering to a subject an anti-CD70 antibody or antigen-binding fragment thereof, the method comprising administering to the subject an anti-CD70 antibody or antigen-binding fragment thereof, the ... administering an effective amount to a subject.

[0073] In a further embodiment, an anti-CD70 antibody for use in the methods provided herein or an antigen-binding fragment thereof is provided.

[0074] (Anti-CD70 antibody monotherapy and aCD70+NMI combination therapy reduce bone marrow and peripheral blood blast cells) As described herein, the method of the present invention involves the collection of bone marrow and / or peripheral blood, preferably bone marrow. It reduces the number and / or percentage of blast cells in both the marrow and peripheral blood.

[0075] The percentage of blast cells in the bone marrow or peripheral blood may be determined, for example, from a bone marrow biopsy or peripheral blood smear of the subject. flow cytometry or cytomorphological evaluation of cells obtained from the The percentage of blasts can be assessed by methods known in the art and described herein. For example, flow cytometry can be used to measure CD45 counts relative to total cell counts. dim ,SSC l ow The number of cells can be used to determine the percentage of blast cells. A morphological evaluation was used to determine the number of morphologically identified blastema cells relative to the total number of cells in the field examined. The number of balls can be determined.

[0076] In certain embodiments, the bone marrow Reduce the percentage of blast cells - for example, reduce the percentage of blast cells in the bone marrow from 30% to 25% In certain embodiments, the treatment reduces, in absolute terms, At least 10%, preferably at least 15% in absolute terms, preferably at least 20 %, preferably at least 25%, preferably at least 30%, preferably at least 40% , preferably at least 50%, preferably at least 55%, preferably at least 60% This can be characterized as a decrease in the percentage of blast cells in the bone marrow. In this treatment, the percentage of blast cells in the bone marrow is reduced by at least 50% compared to before treatment.

[0077] In certain embodiments, the percentage of myeloblasts is measured by morphological evaluation of the cells. In certain embodiments, the percentage of myeloblasts is measured by flow cytometry assessment. In certain embodiments, the percentage of bone marrow blasts is measured according to minimal residual disease (MRD) assessment. can be.

[0078] In certain embodiments, the percentage of blast cells in the peripheral blood is increased by at least 5% in absolute terms. Reduce the proportion of blast cells in peripheral blood from 30% to 25% or less In certain embodiments, the percentage of blast cells in peripheral blood is measured in absolute terms. by at least 5% in absolute terms, optionally by at least 10% in absolute terms, Preferably, it is reduced by at least 15%, preferably by at least 20%, preferably by at least 25%. In certain embodiments, the percentage of peripheral blood (PB) blasts is determined by cell morphology. In certain embodiments, the percentage of PB blasts is measured by flow cytometry. In certain embodiments, the percentage of PB blasts is measured by minimal residual disease (MD). Measured according to the RD) assessment.

[0079] In certain embodiments, the percentage of blast cells in the bone marrow is reduced to less than 40%, optionally less than 20%, e.g., In certain embodiments, methods are provided for reducing the number of blast cells in the bone marrow to, for example, less than 10%. Methods are provided to reduce the rate to less than 5%.

[0080] In certain embodiments, the percentage of blast cells in the peripheral blood is reduced to less than 40%, optionally less than 20%, e.g., In certain embodiments, methods are provided for reducing the number of blast cells in peripheral blood to, for example, less than 10%. A method is provided for reducing the percentage of cells to less than 5%.

[0081] In certain embodiments, the subject has a reduction in blood cholesterol level by at least 20%, optionally at least 40%, of the pre-treatment level. Optionally, the myeloblast percentage is at least 60%, optionally at least 70%.

[0082] In certain embodiments, the subject has a schizophrenia of at least 5% of pre-treatment, optionally at least 5% of pre-treatment. at least 8%, optionally at least 10%, optionally at least 15%, optionally at least 20%, optionally at least 30%, optionally at least 40%, optionally at least 50%, optionally at least 60% have a peripheral blood blast rate of

[0083] Clinical determination of the blast percentage is typically based on cytomorphology (also known as cytomorphology). Knowledge) evaluation is preferred.

[0084] (Anti-CD70 antibody monotherapy and aCD70+NMI combination therapy reduced the levels of leukemia stem cells and differentiated (promoting An important subset of blast cells in AML are leukemia stem cells (LSCs). LSCs are the cells that initiate leukemia. LSCs are cancer stem cells that can self-renew by giving rise to leukemia. and normal non-LSCs that resemble the original disease but are unable to self-renew. LSCs are CD34+CD38-, optionally also CD45- and LSCs may also be characterized as cells that are CD45dim, SSClo, lin-C These may also be characterized as D90+CD34+ cells. A decrease in LSC numbers in AML patients is associated with poor remission outcomes. This significantly improves congestion and reduces the chances of recurrence.

[0085] As disclosed herein, the monotherapy and combination treatments according to the present invention provide patients with Reduce the proportion of LSCs in the Thus, treatment according to the present invention not only reduces the overall blast percentage in a patient, but also It is also expected that the likelihood of disease recurrence after surgery will be reduced.

[0086] Thus, in certain embodiments, the methods of the present invention reduce the number of LSCs as a percentage of mononuclear cells. In certain embodiments, the LSC frequency is reduced by at least A method is provided to reduce the

[0087] In certain embodiments, assessment of the proportion of LSCs in bone marrow is performed by, for example, measuring the percentage of LSCs on methylcellulose. , can be determined by serial dilution plating.

[0088] The monotherapy and combination treatments according to the present invention also promote the differentiation of LSCs into myeloid cells. Such promotion of differentiation reduces the proportion of LSCs capable of self-renewal, thereby reducing the likelihood of remission. Increased odds and reduced risk of recurrence.

[0089] Myeloid differentiation of LSCs occurs through asymmetric cell division, as opposed to symmetric cell division, which gives rise to two daughter stem cells. Asymmetric division can be characterized by microscopic techniques or by the use of proteins such as Numb. This can be assessed by measuring cell fate determination markers. As reported in

[2014] , LSCs derived from patients receiving anti-CD70 antibody monotherapy expanded compared to before treatment. Numb expression (and therefore increased differentiation) indicates that patients with NMI are more likely to develop cytotoxic T cell death than those with NMI. These data are further enhanced when treated with anti-CD70 antibodies. This is the first clinical demonstration that the body can increase LSC differentiation in vivo, thereby enabling R In vitro studies reported in J. Exp. Med. 2017 Feb;214(2):359-380 Moving data forward.

[0090] In a particular embodiment, the method according to the invention promotes differentiation of leukemic stem cells. In one embodiment, the method according to the invention promotes asymmetric division of leukemic stem cells. In one embodiment, the method according to the invention promotes the expression of Numb on leukemic stem cells.

[0091] (AML can be effectively treated even with low doses of anti-CD70 antibodies) Data from the clinical trials presented herein demonstrate that patients treated with CD70 antibodies had at least Even at this dose, the proportion of blast cells was reduced. The intensity of the antibody administered to patients is easily determined to ensure the safety of this antibody treatment and to determine dose-limiting toxicity. Therefore, even after a single dose of the lowest dose of antibody, blast cell division was significantly reduced. It was quite surprising that the number of cases was significantly reduced.

[0092] Thus, the data presented herein demonstrate that AML or MDS can be effectively treated with anticoagulants, including at unexpectedly low doses. - demonstrating that all tested doses of CD70 antibody are therapeutic.

[0093] Thus, in certain embodiments of all of the methods of the present invention, the anti-CD70 antibody or its antigen-binding The fragments are administered in a range of 0.1 mg / kg to 25 mg / kg per dose, for example in a range of 0.1 mg / kg to 20 mg / kg. In certain embodiments, the anti-CD70 antibody or antigen-binding fragment thereof is administered at a dose ranging from 0.1 to 1.0 mg / kg. The compound is administered at a dose ranging from 1 mg / kg to 20 mg / kg per dose. Ranges include endpoints within the range unless otherwise specified (e.g., 0.1 to 25 mg / kg administration at a dose of 0.1 mg / kg and administration at a dose of 25 mg / kg; and all doses between these two endpoints).

[0094] In certain embodiments of the methods of the invention, the anti-CD70 antibody or antigen-binding fragment thereof is administered at a concentration of 0.1 In certain embodiments, the anti-CD70 antibody or its derivatives are administered at a dose ranging from 1 mg / kg to 15 mg / kg. The antigen-binding fragment is administered at a dosage ranging from 0.5 to 2 mg / kg. The anti-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 anti-CD70 antibody or antigen-binding fragment thereof is administered In certain preferred embodiments, the anti-CD70 antibody or its The antigen-binding fragment is administered at a dose of 10 mg / kg.

[0095] In certain embodiments, multiple doses of the CD70 antibody or antigen-binding fragment are administered. In certain such embodiments, each dose of the anti-CD70 antibody or antigen-binding fragment thereof is administered in a dose of 10 In certain embodiments, each administration of the anti-CD70 antibody is spaced apart by 12 to 20 days, and optionally 12 to 18 days. Doses are spaced 14 to 17 days apart.

[0096] In certain embodiments, treatment according to the present invention results in at least a partial response. In certain embodiments, treatment results in the patient exhibiting a morphologically leukemia-free state. In some cases, treatment results in at least a complete remission with incomplete recovery (CRi). In embodiments, the treatment results in at least a complete remission.

[0097] Therefore, treatment with the present invention reduces the number of blast cells (including leukemia stem cells) in the bone marrow and peripheral blood. By reducing LSC levels, AML or MDS can be treated. It is particularly effective in that it specifically reduces the level.

[0098] (Patient characteristics) Treatment according to the present invention is achieved without the significant toxicity and morbidity associated with standard intensive chemotherapy. This is particularly advantageous as it provides an effective means of inducing remission (partial or complete) of AML. Providing a therapy without these side effects is a significant advantage of the present invention.

[0099] Reducing the side effects of AML therapy is clearly desirable for the treatment of AML patients overall. This is particularly advantageous for patients who cannot receive more intensive AML treatments than usual. For example, patients aged 60 years or older and / or ineligible for standard intensive chemotherapy due to comorbidities. There is a need for effective therapies for these patients. Although chemotherapy and low-dose cytarabine (LDAC) are available, these have limited potential for achieving remission. In contrast, treatment with the present invention is more effective than standard intensive therapy and is still associated with significant side effects. An effective treatment for AML that can be tolerated by patients who cannot tolerate the toxicity of chemotherapy Provide the law.

[0100] Thus, in certain embodiments of the invention, the subject is eligible for standard intensive chemotherapy prior to treatment. In certain such embodiments, the subject is at least 60 years old, optionally at least He is also 70 years old.

[0101] Further conventional therapy for AML involves engraftment using either allogeneic or autologous stem cells. Patients who are ineligible for standard intensive chemotherapy usually undergo HSCT. Patients are excluded from receiving CT because their chances of successful transplant are lower than those receiving low-dose chemotherapy. This is because the risk of death is reduced in patients receiving only can receive standard intensive chemotherapy intended to eliminate bone marrow blasts in preparation for HSCT can be performed using less intensive chemotherapy, but residual blasts may be The chances of success are lower because of the greater likelihood of the remaining in the bone marrow.

[0102] An individual patient's suitability for HSCT depends on the likelihood of transplant success and remaining treatment options. The clinical evaluation of the case will depend on the patient and other factors such as quality of life. An illustrative but important factor to consider is the percentage of bone marrow blasts exhibited by the patient. be.

[0103] As demonstrated herein, treatment according to the present invention significantly reduces the percentage of bone marrow blasts. The bone marrow blast rate in patients ineligible for standard intensive chemotherapy By providing a means to reduce the risk of HSCT, the methods of the present invention may be useful in cases where HSCT has not previously been recommended. This opens up new prospects for successful HSCT in this patient population.

[0104] Thus, in certain embodiments of all aspects of the invention, the subject is not receiving standard reinforcement therapy prior to treatment. In patients who are ineligible for chemotherapy, this treatment reduces the bone marrow blast percentage, thereby making the subject eligible for HSCT. In certain embodiments, the treatment further comprises administering a hematopoietic stem cell transplant to the subject. Includes.

[0105] administering to a subject one or more doses of an anti-CD70 antibody or antigen-binding fragment thereof. Provided herein are methods for preparing a subject with AML or MDS for hematopoietic stem cell transplantation (HSCT). .

[0106] In certain embodiments, and as described elsewhere herein, the methods include administering an NMI (e.g., azathioprine). The method may further include administering to the subject an HMA such as cytidine.

[0107] In certain embodiments, the method further comprises performing HSCT in the subject.

[0108] Treatment according to the present invention is particularly effective in treating patients with abnormally high levels of soluble CD27 in their serum. It is predicted that there will be.

[0109] Without wishing to be bound by theory, this suggests that CD27-CD70 binding is a signaling pathway that This is thought to be because cloning leads to the release of soluble CD27 (sCD27). Therefore, soluble CD27 serves as a biomarker for the extent of CD70 / CD27 interaction (Riet et al., 2004). Her et al., J. Exp. Med. 2017 Feb;214(2):359-380, which is incorporated herein by reference. In particular, sCD27 correlates with the percentage of blast cells in the bone marrow, as well as the percentage of blast cells in patients. sCD27 acts as a marker of stemness in the thymus, and increased sCD27 indicates increased levels of stemness. This is thought to be the case (Riether et al., J. Exp. Med. 2017 Feb;214(2):359-380).

[0110] CD70 / CD27-mediated signaling promotes abnormal cell division and leads to high levels of serum sC D27 is thought to be correlated with poor prognosis in AML patients. It reduces the percentage of blast cells in the bone marrow (see Examples) and blocks the CD27 / CD70 interaction. This reduces the level of stemness of the blasts and promotes differentiation. AML patients who express sCD27 may derive particular benefit from treatment with the anti-CD70 antibodies of the present invention. It is expected that this will be possible.

[0111] Samples from selected healthy individuals showed serum sCD27 levels of 10-200 U / ml, whereas AML patients , showing elevated serum sCD27 (Riether et al., J. Exp. Med. 2017 Feb;214(2):359-380) Thus, in certain embodiments of the invention, the patient being treated has a serum sCD greater than 200 U / ml. 27 concentrations. All AML patients across ages and AML subtype risk categories (i.e., A threshold of 577 U / Patients with serum sCD27 levels above 1 ml were predicted to be at a higher risk of developing sCD27 than patients with sCD27 levels below this threshold. It has also been determined that the prognosis is poorer after the first trimester than after the second trimester. , the treated patient has a serum sCD27 concentration greater than 577 U / ml.

[0112] Serum sCD27 levels above the threshold of 470 U / ml when analyzed within AML subtype risk categories Patients with AML of favorable subtypes who have serum sCD27 levels above this threshold were those with AML of favorable subtypes and It was determined that patients with sCD27 levels below 100 mg / mL had a worse prognosis than patients with sCD27 levels below 100 mg / mL. In certain embodiments, the patient being treated has a favorable or low-risk AML subtype, and and have a serum sCD27 concentration greater than 470 U / ml.

[0113] For patients with intermediate-risk AML subtype, patients with serum sCD27 levels above the threshold of 586 U / ml were Patients with intermediate-subtype AML and sCD27 levels below this threshold have a poorer prognosis than patients with AML. Thus, in certain embodiments, the patient being treated has an intermediate-risk AML subtype. and have a serum sCD27 concentration greater than 586 U / ml.

[0114] For patients with high-risk subtypes of AML, patients with serum sCD27 levels above the threshold of 714 U / ml have a better prognosis than patients with high-risk subtypes of AML and with sCD27 levels below this threshold. Thus, in certain embodiments, the patient being treated has a high-risk AML subtype. and have a serum sCD27 concentration greater than 714 U / ml.

[0115] Patients to be treated according to the present invention should be evaluated prior to testing above or below any of the described thresholds. It will be clear that the serum sCD27 concentration of those determined may be In particular embodiments, the treatments and methods of the present invention include measuring the serum CD27 concentration in a patient. It further includes a process.

[0116] As already described herein, administration of anti-CD70 antibodies according to the present invention reduces the number of myeloblasts. It is possible to block the interaction between CD27 and CD70. The decrease in blast cells paralleled by the injury was due to the sCD27 shed light, as demonstrated in the Examples. This significantly reduces the level of noise.

[0117] In certain embodiments, the methods according to the invention (e.g., monotherapy with an anti-CD70 antibody or The combination therapy of anti-CD70 antibody and NMI reduces serum sCD27 compared to pre-treatment. In the method, serum sCD27 is reduced by at least 200 pg / ml, optionally by at least 500 pg / ml. In certain embodiments, serum CD27 is at least 1000 pg / ml lower than before treatment. The concentration is reduced by at least 1500 pg / ml or at least 10,000 pg / ml.

[0118] In the Examples herein, the level of sCD27 was determined to be a function of the binding of an anti-CD70 antibody to its target antigen. It has further been shown that soluble CD27 levels can be used as a correlate of: The response to CD70 antibody treatment decreased and continued to increase when therapy was subsequently stopped. Thus, in some embodiments, the method further comprises detecting serum sCD27. This includes monitoring therapeutic efficacy.

[0119] The administration of the anti-CD70 antibody according to the present invention not only reduces the percentage of blast cells but also inhibits the expression of CD70. Preferentially reduces the number of CD70-expressing blast cells (CD70+ blasts). Abnormal AML blast cells are expressed by normal precursors. These cells are understood to overexpress CD70 compared to normal cells, thus targeting CD70-expressing blast cells. reduces the number of pathogenic blasts with negligible effect on normal progenitor cells, which reduces the patient's risk of developing cytopenias.

[0120] In certain embodiments of the invention, the percentage of CD70+ blasts is reduced. In this study, the percentage of CD70+ blasts was at least 5% in absolute terms, optionally at least 10%. In certain embodiments, the percentage of CD70+ cells is reduced by less than 20%, optionally by 10%. In certain embodiments, the percentage of CD70+ blast cells is reduced to less than 5%. The percentage of CD70+ blasts may be reduced by a specified amount in the bone marrow and / or peripheral blood. .

[0121] Regarding the treatment of MDS, patients with "high-risk" MDS - i.e., those with poorer survival prognosis and faster disease progression - are at increased risk. It is particularly desirable to treat MDS patients with a higher incidence of MDS and a higher probability of progression to AML. Thus, in certain embodiments, the patient is a "high-risk" MDS patient. In this case, the patient has an IPSS-R score of greater than 4.5.

[0122] (Anti-CD70 antibody therapy in combination with nucleoside metabolic inhibitors (NMI)) As previously described herein, monotherapy with anti-CD70 antibodies is effective in the treatment of AML or MDS. The data herein provide an effective treatment, resulting in a significant reduction in the blast cell percentage. In addition to providing monotherapy, nucleoside metabolic inhibitors (NMIs), such as azacitidine ( Hypomethylated steroids such as azacytidine (also referred to herein as AZA or aza) or decitabine Further therapeutic efficacy of anti-CD70 antibodies when administered as part of a combination therapy with a human methicillin-resistant antigen (HMA) It reveals:

[0123] Thus, in a further embodiment, a combination comprising an anti-CD70 antibody or antigen-binding fragment thereof and an NMI is provided. In certain embodiments, the NMI is a hypomethylating agent, preferably azacitidine. It's gin.

[0124] In a further embodiment, an anti-CD70 antibody is provided for use in a method of treating AML or MDS. Combinations are provided that contain the antibody or antigen-binding fragment thereof and an NMI. The NMI is a hypomethylating agent, preferably azacitidine.

[0125] In a further embodiment, an anti-CD70 antibody or its antibody is provided for use in the methods of the present invention. In certain embodiments, the NMI is a low A methylating agent, preferably azacitidine.

[0126] The following embodiments are illustrative of all aspects and aspects of the present invention provided herein unless otherwise specified. It is applicable to the above method.

[0127] Azacitidine is an analog of cytidine, and decitabine is its deoxy derivative. AZA and decitabine upregulate gene expression through promoter hypomethylation. It is an inhibitor of DNA methyltransferases (DNMTs) known to inhibit such low methyltransferases. Thiolation disrupts cellular function, thereby producing cytotoxic effects.

[0128] Without wishing to be bound by theory, CD70 antibodies and nucleoside such as AZA The therapeutic effect resulting from treatment with both cyclohexyl benzoate metabolic inhibitors is due to the combination of these two active substances. This is thought to be due to the enhanced effect of the anti-CD70 antibody alone. depletes bone marrow and circulating blasts (i.e., blasts in the bone marrow, peripheral blood, or both); and Nucleoside metabolism inhibitors alone (such as AZA) disrupt cellular activity.

[0129] In addition, upregulation of CD70 on the surface of AML blasts and LSCs is associated with nucleoside metabolism. This is induced by treatment with metabolic inhibitors (azacytidine or decitabine) (see Examples and Figure 1). As demonstrated in the accompanying examples, this antigen-upregulating The efficacy of anti-CD7 antibodies when used in combination with nucleoside metabolic inhibitors such as azacitidine This correlates with increased efficacy of the 0 antibody, compared with that after administration of either active agent alone. This results in a further reduced number of blast cells, and the anti-CD70 antibody is administered at a low dose. This may explain the surprising effectiveness of the combined treatment.

[0130] Thus, in certain preferred embodiments, the treatments and methods of the present invention involve the treatment of nucleoside metabolism In certain embodiments, the method further comprises administering an inhibitor of nucleoside metabolism to the subject. In certain embodiments, the inhibitor of nucleoside metabolism is a hypomethylating agent. In certain preferred embodiments, the nucleoside metabolite is zacitidine or decitabine. The inhibitor is azacitidine.

[0131] In certain embodiments, the nucleoside metabolic inhibitor (e.g., azacitidine) is administered at a dose of 100 mg / kg / day or 100 mg / kg / day. 50-100mg / m 2 As noted above, the ranges described herein are used in the administration of is inclusive of the range endpoints unless otherwise specified - e.g., 50 to 100 mg / m per day 2 of Dosage ranges from 50 mg / m per day 2 and administered at a dose of 100 mg / m per day 2 of This includes administration of any dose in any range, as well as all doses between these two endpoints. In this case, the nucleoside metabolic inhibitor is 70-80 mg / m per day. 2 It is administered in doses ranging from In certain preferred embodiments, the nucleoside metabolic inhibitor is administered at a dose of 75 mg / m per day. 2 Throw It is administered in doses.

[0132] In certain embodiments, the nucleoside metabolism inhibitor is administered over a 5 to 9 day daily dose period. That is, doses of nucleoside inhibitors are administered over a period of 5, 6, 7, 8, or 9 days in length. In certain preferred embodiments, the nucleoside metabolic inhibitor is , administered over a 7-day daily dosing period.

[0133] In certain embodiments, the nucleoside metabolic inhibitor is administered in a dosing regimen with repeated dosing periods. The end of one dosing period and the beginning of the next dosing period are separated by 18 to 25 days. That is, this dosing regimen involves administering at least one dose of the nucleoside inhibitor daily. at least two dosing periods (e.g., periods of 5, 6, 7, 8, or 9 days in length), where one dose The end of a medication period and the beginning of the next medication period must be separated by 18, 19, 20, 21, 22, 23, 24, or 25 days. In certain embodiments, the end of one dosing period and the beginning of the next dosing period are separated by 21 days. They are separated.

[0134] In certain embodiments, each dosing period is the same length (e.g., 7 days). In embodiments, the end of each dosing period and the beginning of the next dosing period are separated by the same number of days (e.g., 21 days). can be done.

[0135] In certain embodiments, the initial dose of the nucleoside metabolic inhibitor is administered with an anti-CD70 antibody or The antigen-binding fragment is administered 7 to 21 days after the first dose. The initial dose of the nucleotide metabolic inhibitor is the same as the initial dose of the anti-CD70 antibody or its antigen-binding fragment. In certain embodiments, the initial dose of the nucleoside metabolic inhibitor is administered for 10 to 17 days. The doses are administered 14 days after the first dose of anti-CD70 antibody or antigen-binding fragment thereof.

[0136] In certain embodiments, one of the daily doses of the nucleoside metabolism inhibitor is administered in combination with an anti-CD70 antibody or i.e., the dose of the anti-CD70 antibody (or its antigen-binding fragment) is administered on the same day.

[0023] The practice of the method of the present invention in which both a nucleoside binding fragment and a nucleoside metabolic inhibitor are administered to a subject In embodiments, the dosing regimen for both the anti-CD70 antibody and the nucleoside metabolic inhibitor comprises At least one of the scheduled doses of the antibody is administered after the scheduled administration of the nucleoside metabolic inhibitor. The day is set to be the same day as one of the administered daily doses. This may be on the first, second, third, fourth, fifth, sixth or seventh day of the dosing period of the inhibitor.

[0137] In certain embodiments, the dose of the anti-CD70 antibody or antigen-binding fragment thereof is administered for 14 to 17 days. It is administered daily, and the nucleoside metabolic inhibitor is administered in a dosing regimen with a 7-day daily dose repeat dosing period. The end of one dosing period and the beginning of the next dosing period are separated by 21 days. and wherein the first daily dose of the first dosing period is an anti-CD70 antibody or antigen-binding fragment thereof. The vaccine will be administered for 14 days after the first dose.

[0138] Following the initiation period of combination therapy, administration of the NMI (e.g., aza) may be tapered or discontinued. It is a further advantage of the present invention that, for example, as disclosed herein, The initial period of combination therapy can significantly reduce the blast percentage in patients. The potential for cytopenias resulting from prolonged NMI treatment, such as those resulting from the effects of NMI on non-blast cell types, may be a contributing factor. There is a possibility of cumulative toxicity due to NMI. After the initiation period, tapering or stopping the NMI dose is recommended. This reduces the risk of such toxicity and allows recovery of non-blast cell types. However, the blast percentage can still be controlled by maintaining the dose of anti-CD70 antibody. In other words, patients will be treated with the induction regimen of the combination therapy already described. This can be followed by anti-CD70 therapy with tapering doses of NMI, or simply with the CD70 antibody alone. Patients can then move on to maintenance therapy, including drug therapy.

[0139] Therefore, in a particular embodiment, the treatment according to the invention is carried out in the first stage (induction therapy). and administering to a patient an anti-CD70 antibody and an NMI as a combination therapy according to any of the embodiments described above. In a subsequent second step, an anti-CD70 antibody is administered to the patient and the first step This includes administering a dose of NMI below the dose of NMI prescribed (maintenance therapy). The dose of NMI may be zero - i.e., the second phase involves administration of anti-CD70 antibody only. It is possible.

[0140] In such embodiments, the dose of the CD70 antibody administered in the second phase (i.e., maintenance therapy) The dosage is any dosage according to the embodiments previously described. In this case, the dosage is 0.1 mg / kg to 25 mg / kg, for example, 0.1 mg / kg to 20 mg / kg, for example, 1 mg / kg to 20 In certain embodiments, the dosage ranges from 0.1 mg / kg per dose. In certain embodiments, the dosage ranges from 0.5 mg / kg to 2 mg / kg. In certain embodiments, the dosage ranges from 1 mg / kg, 3 mg / kg, 10 mg / kg, or In certain embodiments, the dosage is 1 mg / kg. In an embodiment, the dosage is 10 mg / kg.

[0141] The length of the first phase (i.e., induction therapy), the timing of transition to the second phase (i.e., maintenance therapy), The rate at which the dose of NMI is tapered or completely stopped will vary for each individual patient. The dose is adjusted and administered by the patient's physician according to the individual patient's response to therapy and their medical history. The following embodiments are therefore provided as non-limiting examples.

[0142] In certain embodiments, induction therapy is administered to patients with bone marrow and / or peripheral blood blast percentages of 10 In certain embodiments, the patient is administered an amount of 500 mg / kg of erythrocyte sedimentation product (E1) or 200 mg / kg of erythrocyte sedimentation product (E2) until the amount of erythrocyte sedimentation product (E1) is less than 5%, optionally less than 5%. The therapy comprises at least 5 NMI dosing periods, optionally at least 6, 7, 8, 9, or at least 10 It is administered during the NMI dosing period.

[0143] In certain embodiments, the dosage of NMI during the maintenance period is 50 mg / m per day 2 Not exceeding 40 mg / m per day 2 Not to exceed 30 mg / m² per day, optionally 2 Not exceeding 20m per day at will g / m 2 does not exceed.

[0144] (Pharmaceutical composition) Also provided herein are pharmaceutical compositions for use in the methods described herein. Therefore, in a further aspect of the invention, there is provided a method for use in a method according to the invention. and a pharmaceutical composition comprising the anti-CD70 antibody of the present invention and a pharmaceutically acceptable excipient or carrier. Suitable pharmaceutically acceptable carriers and excipients will be familiar to those skilled in the art. Examples of pharmaceutically acceptable carriers and excipients suitable for inclusion in pharmaceutical compositions of the present invention include: Examples include sodium citrate, glycine, polysorbates (e.g., polysorbate 80), and Examples include saline and water.

[0145] In the combination of the present invention, the anti-CD70 antibody or antigen-binding fragment may be administered with or without a nucleoside metabolic inhibitor. may be formulated for administration via the same route or via a different route compared to

[0146] In certain embodiments, the anti-CD70 antibody is administered parenterally, preferably intravenously (iv): In certain embodiments, the anti-CD70 antibody is administered to a subject at a dose that reaches a desired level. It is administered as a continuous iv infusion until

[0147] In certain embodiments, the nucleoside metabolism inhibitor is administered parenterally, preferably subcutaneously (sc .) is administered.

[0148] (combination therapy) Treatment according to the present invention may involve the administration of therapeutic or palliative substances (e.g., radiation therapy, analgesics, or antibiotics). The compound may be incorporated into a combination therapy with one or more additional active agents, such as a biologic. The active agent may be a therapeutic agent for improving a patient's response to therapy and / or the patient's quality of life. Thus, in certain embodiments, a CD70 antibody (optionally in combination with an NMI) may be administered. In the method, the compound (or compounds) is administered in the present invention in combination therapy with one or more additional active agents.

[0149] AML or MD treated with anti-CD70 antibodies (as monotherapy or in combination with NMIs such as azathioprine) Surprisingly, treatment with S is particularly effective in reducing blasts in the bone marrow and peripheral blood. A particular advantage is that CD70 antibodies are selective for blasts over other cell types (C D70 is minimally expressed in normal tissues) and induces stem cell differentiation / these stem cells It is possible to reduce the activity of CD27 / CD70-mediated signaling (e.g., by interfering with CD27 / CD70-mediated signaling). It is possible to do this.

[0150] These effects on blasts and leukemia stem cells suggest that treatment with the present invention may also have an effect on blasts and leukemia stem cells. CD33 is primarily expressed on myeloblasts and LSCs, suggesting that it can be advantageously combined with agents targeting LSCs. and LSCs, but is absent or present at low levels on normal hematopoietic stem cells CD33 is a receptor that binds to AML cells, but is expressed on a variety of other cell types that are less relevant to AML. CD33 is expressed on the majority of AML cells, and the level of CD33 appears to correlate with the disease prognosis. The bispecific antibody AMG330 and the antibody-drug conjugate (ADC) vadatuximab-butarilin (va Antibodies against CD33, such as dastuximab talirine, have been shown to effectively treat AML. and also known as vadatuximab butarilin (SGN-CD33A from Seattle Genetics). Anti-CD33 antibodies (known in the art) are being tested in phase III clinical trials. in combination with other treatments (anti-CD70 antibody monotherapy or a combination of anti-CD70 antibody and NMI (e.g., aza) It is expected that this will provide a particularly effective therapy for AML. In this study, patients were randomly assigned to receive an anti-CD70 antibody of the present invention (and optionally an NMI) as part of a combination therapy. -CD33 agents, such as anti-CD33 antibodies, are administered.

[0151] CD123 is associated with myeloblasts and LSCs, but also with other cells such as normal hematopoietic stem cells and lymphocytes. It is another receptor that is expressed at low levels on certain cell types. AML cells with high CD123 expression are CD Higher expression of CD123 among AML blasts indicates a lower complete remission rate. It is associated with poorer remission rates and overall survival. Therefore, agents targeting CD123 are of interest. This represents another preferred combination for use in the treatment of AML. The anti-CD123 agents used are toxin-linked natural ligands (e.g., DT 388 IL3), and CSL360 This includes antibodies (DART modified antibodies) such as CSL362 and MGD006. In an embodiment, the patient receives IFN-γ as part of a combination therapy with an anti-CD70 antibody of the invention (and optionally an NMI). An anti-CD123 agent, such as an anti-CD123 antibody, is then administered.

[0152] Treatment of AML with anti-CD70 antibodies (either as monotherapy or together with NMIs (e.g., aza)) is more effective than other AMs. L therapeutic agents, such as E-selectin inhibitors, FMS-like tyrosine kinase receptor 3 (FLT3) inhibitors, Cyclin-dependent kinase inhibitors, BCL-2 inhibitors, aminopeptidase inhibitors and JAK / STA inhibitors It can be combined more advantageously in combination therapy with T inhibitors and the like.

[0153] As previously mentioned, other AML treatments include cytarabine, anthracycline compounds (e.g., These include daunorubicin, idarubicin, and hydroxyurea.

[0154] (Anti-CD70 antibody or antigen-binding fragment thereof) In all aspects of the invention described herein, the subject being treated is a patient receiving an anti-CD70 antibody or its As used herein, an "antibody" refers to an antibody that binds to an antigen of interest. A combination of two heavy chains and two light chains with specific immunoreactive activity against (e.g., human CD70) The term "CD70 antibody" or "anti-CD70 antibody" refers to an immunoglobulin having the human CD70 antigen. The terms "antibody" and "antibody-specific" are used interchangeably herein to refer to antibodies that exhibit immunospecificity for a protein. In this context, "specificity" for human CD70 does not include cross-reactivity with species homologues of CD70. Do not exclude.

[0155] As used herein, "antibody" refers to any class of human antibody (e.g., IgG, Ig IgM, IgA, IgD, IgE), and their subclasses / isotypes (e.g., IgG1, IgG2, Ig Antibodies as used herein also include modified Modified antibodies also refer to antibodies. Modified antibodies include synthetic forms of antibodies, which do not occur in nature. Antibodies that are modified, e.g., contain at least two heavy chain portions but do not contain two complete heavy chains (e.g., for example, domain deleted antibodies or minibodies; Multispecific forms of antibodies (e.g., bispecific, trispecific, etc.); heavy chain molecules linked to scFv molecules and the like. In addition, the term " "Modified antibodies" includes multivalent forms of antibodies (e.g., those that bind three or more copies of the same antigen). (e.g., trivalent, tetravalent antibodies).

[0156] The antibodies described herein may be used to inhibit, for example, one or more of antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or other cellular cytotoxicity. They can have antibody effector functions, such as antibody-mediated phagocytosis (CDC) and antibody-dependent phagocytosis (ADCP).

[0157] CD70 antibodies suitable for use in accordance with all aspects of the present invention are described in WO2012123586 (incorporated herein by reference). ARGX-110, described in WO2006113909, and McGraw-Hill University Press, both of which are incorporated herein by reference, and SGN-70 (described in WO2006113909, and McGraw-Hill University Press, both of which are incorporated herein by reference). EarChern et al., Clin Cancer Res 2008;14(23) p7763, both incorporated herein by reference. (including those incorporated in

[0158] CD70 antibodies suitable for use in accordance with all aspects of the present invention may also be antibody-drug conjugates (ADCs). ADCs may be used to treat cancers such as auristatins and maytansines or other cytotoxic agents. An antibody attached to an active agent. Certain ADCs utilize antibody blocking and / or effector Targeting (e.g., cytotoxicity) of conjugated active substances while maintaining target functions (e.g., ADCC, CDC, ADCP) An example of an anti-CD70 ADC is borsetuzumab mafodoxin. vorsetuzumab mafodotin (also known as SGN-75, Seattle Genetics), SGN-70A (Seattle Genetics), These include MDX-1203 / BMS936561 (Britsol-Myers Squibb), and MDX-1203 / BMS936561 (Britsol-Myers Squibb). Each may be used in accordance with the present invention. Suitable anti-CD70 ADCs are also included herein, each of which is incorporated by reference. It is also described in WO2008074004 and WO2004073656, which are incorporated herein.

[0159] "Antigen-binding fragment" refers to a polypeptide fragment of an antibody that retains binding specificity to CD70. and includes: antibody light chain variable domains (VL); antibody heavy chain variable domains; single chain variable Single-chain antibody (scFv); Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment: one arm ( monovalent) antibodies; diabodies; triabodies; tetrabodies; or such antigen-binding Any antigen-binding molecule, including fragments thereof (e.g., chimeric antigen receptors), or such antigen-binding fragments. Any antigen-binding molecule formed by the combination, assembly, or conjugation of by chemical or enzymatic treatment of intact or intact antibodies or antibody chains, or by recombinant means. and can be obtained.

[0160] As shown in the Examples, effective treatment of AML patients with the CD70 antibody ARGX-110 has been demonstrated. ARGX-110 has been shown to inhibit the interaction of CD70 with its receptor CD27. It is an IgG1 anti-CD70 antibody (Silence et al., MAbs. 2014 Mar-Apr;6(2):523-32, which (The text is incorporated herein by reference.) In particular, ARGX-110 inhibits CD70-induced CD27 signaling. It has been shown that CD27 signaling inhibits IL-1 signaling. Measurement of serum soluble CD27 as described in the literature by [J. Exp. Med. 2017 Feb;214(2):359-380]. or for measuring IL-8 expression as described in Silence et al. (MAbs. 2014 Mar-Apr;6(2):523-32). Without being bound by theory, inhibition of CD27 signaling may enhance Treg It is believed to reduce cell activation and / or proliferation, thereby promoting the proliferation of anti-tumor effector T cells. Reduces cell inhibition.

[0161] Thus, in all embodiments of the present invention, anti-CD70 antibodies inhibit the interaction of CD70 with its receptor CD27. In certain such embodiments, the anti-CD70 antibody is In certain embodiments, the anti-CD70 antibody may compete with CD27 for CD70 binding. In certain embodiments, the anti-CD70 antibody may inhibit CD27 signaling induced by CD70. , may inhibit the activation and / or proliferation of Tregs.

[0162] AGRX-110 has also been shown to deplete CD70-expressing tumor cells. ARGX-110 inhibits CD70-expressing cells by antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Lyses expressing tumor cells and enhances antibody-dependent phagocytosis (ADCP) of CD70-expressing cells It has been shown to be effective (Silence et al., MAbs. 2014 Mar-Apr;6(2):523-32).

[0163] Thus, in certain embodiments, the anti-CD70 antibody is an antibody that depletes CD70-expressing cells. In certain embodiments, the anti-CD70 antibody induces lysis of CD70-expressing cells. In certain embodiments, the anti-CD70 antibody has ADCC and / or CDC functionality. In certain embodiments, the anti-CD70 antibody may induce ADCP.

[0164] The Fc region of ARGX-110 is defucosylated using the Potelligent™ system. and ARGX-110 exhibits increased ADCC functionality compared to its fucosylated counterpart. (Silence et al., MAbs. 2014 Mar-Apr;6(2):523-32). In an embodiment, the anti-CD70 antibody is fully or partially defucosylated.

[0165] In certain embodiments, the CD70 antibody comprises a nucleotide sequence at Lys433, Phe434, and Tyr436 (EU numbering). The Fc domain, CH3 domain, or Fc-hinge derived from human IgG1 includes the Fc domain, CH3 domain, or Fc-hinge derived from human IgG1. In embodiments, the anti-CD70 antibody further comprises a nucleotide sequence at Tyr252, Thr254, and Glu256 (according to EU numbering). These residues at these positions have been shown to extend the circulation time of antibodies. It has been revealed.

[0166] In certain embodiments, the anti-CD70 antibody is an IgG antibody, preferably an IgG1 antibody.

[0167] In light of the data previously described and initially provided herein, anti-CD7 0 antibodies, e.g., that inhibit the interaction of CD70 with CD27, compete with CD27 for CD70 binding, and inhibit CD7 Inhibits CD27 signaling induced by CD70, inhibits Treg activation and / or proliferation, and inhibits CD70-expression Depletion of CD70-expressing cells, induction of lysis of CD70-expressing cells, preservation of ADCC, CDC functionality, and CD70 antibodies that induce ADCP are expected to be effective treatments according to the present invention. Alternative CD70 antibodies, such as SGN-70 and other CD70 antibodies, are expected to be useful in immunotherapy. These are described in WO2006044643 and WO2007038637, which are incorporated herein by reference. ADCs SGN-75, SGN-70A and MDX-12 are known in the art. Modified CD70 antibodies, such as 03 / BMS936561, are believed to be effective in the treatment of the present invention. Further examples of potential anti-CD70 antibodies.

[0168] In certain non-limiting embodiments, the anti-CD70 antibody comprises the CDR sequences of ARGX-110. That is, in certain embodiments, the anti-CD70 antibody may comprise a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and VL domains comprise the following CDRs (as defined by Kabat): (Depending on): SEQ ID NO:3 [ka] HCDR3 comprising or consisting of SEQ ID NO:2 [ka] HCDR2 comprising or consisting of SEQ ID NO:1 [ka] HCDR1 comprising or consisting of SEQ ID NO:7 [ka] LCDR3 comprising or consisting of SEQ ID NO:6 [ka] LCDR2 comprising or consisting of: SEQ ID NO:5 [ka] LCDR1 comprising or consisting of:

[0169] In certain embodiments, the VH and / or VL domains of the anti-CD70 antibody are identical to the VH and VL domains of ARGX-110. and / or VL domains (VH: SEQ ID NO: 4; VL: SEQ ID NO: 8), respectively, at least 70%, at least At least 80%, at least 90%, at least 95%, at least 98%, at least 99% of the sequence Identification: A domain of an antibody or antigen-binding fragment has a specific percentage of sequence identity with a reference sequence. For the embodiment defined by, the VH and / or VL domains are present in the reference sequence. The CDR sequences may be kept identical to those of the corresponding CDRs in the framework regions, so that variations are not limited to those within the framework regions. In a specific embodiment, the anti-CD70 antibody is ARGX-110.

[0170] Table 2 [Table 2] [Example]

[0171] (Example) Example 1: Monotherapy or Decantation of Anti-CD70 Antibody Against Human AML LSCs Transplanted into Mice (Effect of using with bottle) NSG mice, 5 × 10 6 CD45 dim SSC lo Human AML cells were transplanted. 32 days after engraftment (in PB), NSG mice were treated with either vehicle (Veh), aCD70 mAb (aCD70, ARGX-110, 10 mg / mL), or 14.45 ± 0.95% CI. They were randomized to 5 days of treatment with 1.5 mg / kg / day of aCD70, decitabine (D, 1.5 mg / kg / day), or the combination (aCD70 / D). , and bone marrow, spleen and blood were analyzed.

[0172] Both anti-CD70 and decitabine alone resulted in a decrease in total engraftment in the bone marrow, spleen, and blood. The combination of anti-CD70 and decitabine significantly improved engraftment compared with either therapy alone (Figure 1). This resulted in an enhanced reduction in the percentage of cells (Figure 1).

[0173] The combination therapy also demonstrated better bone marrow activity than either decitabine or anti-CD70 alone. In addition, anti-CD70 treatment reduced the number of CD34+ AML cells (a marker of progenitor cells) that were associated with AML (Figure 1). The numbers of both CD38- and CD34+CD45- cells were reduced, and both cell populations were leukemia stem cells (LSCs). Decitabine alone had minimal effect on reducing LSC numbers, The combination of anti-CD70 and decitabine resulted in greatly enhanced LSC depletion (Figure 1).

[0174] Example 2: Enhancement of AML colony formation by anti-CD70 antibodies in combination with hypomethylating agents (HMA) Increased CD70 expression on primary AML stem cells ex vivo and in vivo correlated with a significant decrease in Pre-regulating HMA) Anti-CD70 antibodies in combination with nucleoside metabolic inhibitors (NMIs), such as the hypomethylating agent decitabine The finding that treatment resulted in an enhanced reduction in AML blast engraftment in mice was compared with primary human AML L Further investigation was carried out in SC.

[0175] CD70 expression by AML LSCs following NMI treatment (e.g., HMAs such as azacitidine or decitabine) CD34+CD38- cells were isolated from AML patients and treated with 0.5 mM decitabine or bisphosphonates. Cultures were carried out in the presence of vehicle.

[0176] The data in Figure 2A show that CD70 expression by AML LSC cells increases when the cells are cultured with decitabine. The results showed that CD70 expression in response to decitabine was increased in The increase in AML was observed across all disease risk categories (favorable, intermediate, and adverse). This occurs when measuring protein or transcript levels in the treated cells (Figures 2C and 2D).

[0177] Importantly, increased CD70 expression in response to hypomethylating agents (HMA) was observed in vivo. Patients with newly diagnosed AML who were treated with decitabine or azacitidine LSCs harvested from patients showed increased CD70 expression in response to HMA treatment compared to expression levels at diagnosis. (Figures 2E and 2F).

[0178] Considering the increased CD70 expression in response to HMA, anti-CD70 antibody / decitabine for AML LSCs The effect of the combination was investigated ex vivo.

[0179] Figure 3A shows that both anti-CD70 and decitabine monotherapy significantly inhibited the proliferation of leukocytes in colony-seeding assays. , which have been shown to reduce the number of colonies formed by AML LSCs. In conclusion, the combination of anti-CD70 and decitabine resulted in greater colony expansion than either monotherapy alone. This effect was observed in all disease risk categories: good (P6), intermediate (P8) and good (P9). The reseeding ability of AML LSCs was also reduced (Figure 3C).

[0180] Importantly, anti-CD70 antibodies had no adverse effects on normal stem cells. (Figures 3D-F) Anti-CD70 as monotherapy has no effect on normal cells compared with vehicle alone. , and when used in combination, they have been shown to be no more effective than decitabine used alone.

[0181] These data support the use of anti-CD70 as monotherapy for AML, as well as anti-CD70 antibodies, e.g., Azacitabine. In combination with a nucleoside metabolic inhibitor (NMI), such as an HMA, such as thiazolidine or decitabine, I'm wearing it.

[0182] Example 3: Comparison of Standard-Dose AZA in Combination with Benzaliphatic Agents in Subjects with Previously Untreated AML and High-Risk MDS Phase I / II clinical trial of ARGX-110, an anti-CD70 antibody A Phase I / II clinical trial was conducted in patients with previously untreated AML and high-risk MDS who were eligible for AZA treatment. Efficacy / clinical benefit and safety of ARGX-110 in combination with standard doses of AZA in subjects and to investigate tolerability.

[0183] (Trial Regimen Protocol and Sample Assay) This clinical trial consisted of a screening phase (days -35 to -14), a loading dose of ARGX-110 (day -14), and ), and the open-label treatment phase ( -14 days until disease progression, and end of treatment (EOT) performed within 7 days of the last ARGX-110 treatment Additional follow-up assessments were planned at 30 and 60 days (± 7 days) after the EOT date. The eye follow-up visit was also the end of study (EOS) visit.

[0184] Failure to comply with standard intensive chemotherapy, blast count >20%, histologically confirmed (bone Newly diagnosed with AML or high-risk myelodysplastic syndrome (MDS) and at least 1 year of age Male and female subjects aged 8 years and older were eligible for enrollment in this study. Subjects had a life expectancy of 3 months or more. and have an Eastern Cooperative Oncology Group (ECOG) activity level of 0-2 at the time of screening. Subjects received a loading dose of ARGX-110 on day -14, followed by AZA standard To determine dose-limiting toxicity, patients received ARGX-110 in combination with a standard dose of A dose of ARGX-110 was administered intravenously every 2 weeks (loading dose on day -14) in combination with 100 mg of AZA. and additional doses on days 3 and 17) (see Figure 4). Patients were monitored until day 7 before inclusion. As no further data were obtained, additional patients in each cohort were enrolled. The 0 dose level was 1 mg / kg body weight.

[0185] All subjects in this trial will receive a single loading dose of ARGX-110 on Day -14 and will receive a second dose of the same Phase I subjects in this trial received one of the following treatments: Receiving: Cohort 1: 1 mg / kg body weight IV on days 3 and 17 of a 28-day cycle Cohort 2: 3 mg / kg body weight IV on days 3 and 17 of a 28-day cycle Cohort 3: 10 mg / kg body weight IV, days 3 and 17 of a 28-day cycle Cohort 4: 20 mg / kg body weight IV on days 3 and 17 of a 28-day cycle.

[0186] The infusion was initiated at a rate of 10 mL / h, which was then increased depending on the patient's tolerance of the drug. The number of cases increased.

[0187] Clinical response to treatment was classified according to Table 3: Table 3 [Table 3] *All criteria must be met; bone marrow evaluation by spicule The count should be based on 200 nucleated cells in the aspirate; if equivocal, 5-7 Consider repeating the test after 2 days; flow cytometry evaluation may reveal persistent leukemia and positive bone marrow biopsy can help distinguish between normal and regenerating bone marrow; bone marrow biopsy is recommended in non-aspirateable cases or Used if spicules are not obtained; no minimum reaction period is required.

[0188] Serum concentrations of ARGX-110 were measured using a validated enzyme-linked immunosorbent assay (ELISA) method. The following pharmacokinetic parameters were assessed: C max :Maximum measurable concentration;C trough : Trough concentration; AUC∞: Serum concentration from zero to infinity - time Area under the curve; AUC tau : Area under the serum concentration-time curve during the dosing interval; V d : Apparent volume of distribution ;CL: systemic clearance of the drug after IV administration; t1 / 2: half-life.

[0189] For all subjects in this study, venous blood samples were collected to assess anti-drug antibodies (ADA). The immunogenicity of ARGX-110 was confirmed by ELI, which can detect any class of ADA. The SA method was used to evaluate the antibody in serum samples. Reactive samples were then subjected to a confirmatory assay to verify specificity. The analysis was carried out in the following manner.

[0190] Biomarker assessment was performed using bone marrow aspirates and / or smears collected at time points as specified in Figure 5. It can be performed in whole blood. Pharmacodynamics measures a range of biomarkers including: Tested by: Molecular Genetics: To identify recurrent AML genomic abnormalities and to address disease and target pathology Characterization of CD70 and CD11a promoter methylation for genomic DNA analysis of therapeutic efficacy in patients with rhesus macular degeneration . Gene expression: Characterization of mRNA levels of CD70, disease and drug effect markers. Flow cytometry (FACS): CD70 (e.g., before ARGX-110 treatment, after relapse) and CD27 expression and further characterization of drug effects (e.g., blasts, NK cells, and T cells), and minimal residual disease. Mutation (MRD) analysis. Serum protein quantification: sCD27, additional characterization of disease and drug effect markers (e.g., IL-8) If the infusion-related reactions are more severe than those seen in earlier ARGX-110 trials, In this case, inflammatory cytokine analysis can be evaluated.

[0191] Stem cell determination was performed on mononuclear cells purified from blood or bone marrow. Depending on the individual, measurements may be made using Numb staining (to determine the ratio of asymmetric / symmetric divisions), cell testing, and in vitro cell division. Bovine serum albumin (CFU) assay (e.g., CFU methylcellulose colony assay or NSG assay using injected mononuclear cells from the patient) These included mouse survival studies (to assess stem cell potential) or gene expression analysis.

[0192] Minimal residual disease (MRD) assessment was performed on bone marrow aspirates and / or whole blood collected at the time points specified in Figure 5. Flow cytometry was used as the primary method for MRD analysis. Suitable flow cytometry markers for AML subtyping include CD16, CD13, CD34, and CD117. , CD11b, CD10, HLA-DR, CD45, CD35, CD64, IREM-2, CD36, CD105, CD14, CD33, CD71, CD36, CD105, CD33, CD71, cTdT, CD56, CD7, CD19, cMPO, c-lactoferrin, c-lysozyme Additional panels include CD70, CD27 expression, and indicators of stem cell potential or myeloid differentiation. Markers as: CD27, CD70, CD34, CD117, CD11b, HLA-DR, CD45, CD38, and CD123 and were used as part of this study. The tree results were compared with other molecular approaches, such as polymerase chain reaction (PCR) testing.

[0193] Bone marrow aspirate or whole blood is cultured for NK cells, T cells, and other potential immune cell subpopulations. Immunophenotyping (performed by flow cytometry or mass cytometry), including analysis of It can be used for the following purposes:

[0194] (result) (Overall clinical results) Importantly, more than 90% of patients recruited to this trial responded to anti-CD70 therapy (response Ten of 11 patients were treated long enough to assess efficacy.

[0195] Table 4 shows the number of patients who had sufficient time in the study to be evaluated for response. The best response was observed across the 1 mg / kg, 3 mg / kg and 10 mg / kg cohorts. Two of the three patients achieved complete remission. In cohort 1, the third patient had incomplete hematological remission. Complete remission with progressive recovery was achieved.

[0196] Response levels of over 90% compared to the approximately 25% seen with aza alone This is clearly evident (Dombret et al., Blood 2015;126(3):291-299, is incorporated herein by reference).

[0197] Table 4 [Table 4]

[0198] Of note, morphologically leukemia-free state (MLFS) was not achieved after monotherapy with ARGX-110 alone. A 71-year-old patient who showed 20% myeloblasts at the time of recruitment in Cohort 2 was reached and A 74-year-old patient who showed >50% bone marrow blasts at recruitment in Phase 3, by time point C1D1 - i.e., ARGX- After the loading dose of 110 and before the first aza dose - MLFS characterized by the absence of blasts reached.

[0199] This clearly demonstrates that anti-CD70 antibody therapy alone can be an effective treatment for AML. It's like a crab.

[0200] Furthermore, complete remission was also induced by the combination of ARGX-110 / aza therapy. Six patients achieved complete remission after combination therapy, and an additional two patients achieved CRi after combination therapy. Reached.

[0201] More importantly, this combination treatment enabled one patient to proceed to bone marrow transplant. This means that many AML patients, especially elderly patients, do not meet transplant criteria. are typically unable to undergo transplantation due to the invasive nature of conventional therapies that require This combination treatment is significant because of the fact that a 75-year-old AML patient cannot proceed to transplant. The fact that this combination was effective enough to allow It shows advantages over

[0202] (Monotherapy and combination therapy reduce blasts in bone marrow and peripheral blood) For each patient, the number of blasts in the bone marrow and peripheral blood was measured at various time points, as outlined in Figure 5. was evaluated.

[0203] Bone marrow aspirates taken at various time points were analyzed to assess myeloblasts. Monocytes (mononuclear cells (CD45 dim ) was performed using Annexin V and antibodies to CD19 and CD4 / CD8. Selectively selected by eliminating red blood cells, dead cells, and lymphocytes The number of LSCs in the bone marrow was assessed using a chilled cellulose colony assay. The percentage was also assessed by cell morphology and flow cytometry analysis.

[0204] The bone marrow blast results for each patient are collated and shown in Figure 6. These data are consistent with the majority of patients. Regarding the half, the bone marrow blast percentage remained at least It indicates a partial or complete reduction.

[0205] Furthermore, once aza therapy is initiated (from C1D1), the bone marrow blast percentage remains constant for the remainder of the treatment. This is due to the fact that the blasts are further reduced and maintained at a low level. This is the case regardless of whether an assay is used.

[0206] Bone marrow blasts, measured according to minimal residual disease (MRD) criteria, were significantly higher in patients treated with ARGX-110 monotherapy and ARGX-110 alone. Both X-110 and aza combination therapy demonstrated a significant reduction in MRD. This is particularly noteworthy given the clinical significance of the findings. Two patients (one in cohort 1 and one in cohort 2) One of the 2 patients achieved MRD status.

[0207] Similar results are observed when peripheral blood blast percentage is assessed (Figure 7). The changes in the data are more susceptible to external factors and therefore more Nevertheless, peripheral blood (PB) results after single-agent and combination therapy This corresponds to the changes observed in the bone marrow (Fig. 7).

[0208] Figure 7 shows that, similar to the bone marrow derived data, PB blast percentages generally increased after ARGX-110 monotherapy (i.e., decreased at C1D1 compared to earlier data points, and then further decreased under combination therapy (C1D1 and beyond) Again, this effect was confirmed by cell morphology or flow-based blast assays. is accepted regardless of whether or not the MRD evaluation criteria are used, and That's certain.

[0209] Therefore, anti-CD70 antibody monotherapy and the combination of anti-CD70 with HMAs such as aza, each have a negative effect on bone marrow and It can reduce the blast percentage in the peripheral blood and, in the case of combination therapy, this reduced percentage , maintained over a number of time points.

[0210] (Single-agent and combination therapy increases myeloid differentiation of LSCs) Persistence of leukemic stem cells (LSCs) in AML is a significant obstacle to successful treatment of AML patients. LSCs are responsible for disease recurrence in AML, and their numbers are significantly increased by symmetric cell division. Through this process, each LSC divides symmetrically to produce two daughter LSCs. Such symmetric division is a driver of AML progression and patient relapse.

[0211] An alternative cell fate for LSCs is to undergo myeloid differentiation. LSCs undergo asymmetric division to generate myeloid differentiated daughter cells along with daughter stem cells.

[0212] Such asymmetric divisions greatly reduce the pool of LSCs in patients and increase non- Symmetric division is an indication of an improved response by the patient.

[0213] The level of asymmetric division in a population of LSCs is related to the expression of cell fate determinants such as Numb proteins. This can be determined by measuring the levels (Riether et al., J Exp Med. 201 7 Feb; 214(2): 359-380, which is incorporated herein by reference). umb expression is an indicator of increased asymmetric division and therefore increased myeloid differentiation and decreased LSC population is an indicator of.

[0214] Figure 8 shows that Numb expression increased after ARGX-110 monotherapy (C1D1) and then under combination ARGX-110 / AZA therapy. showed that both therapies increased the asymmetric division and myeloid differentiation of LSCs. This indicates that

[0215] These data suggest that the dose of anti-CD70 monotherapy and aza combination therapy modulates the CD70-CD27 pathway. (thereby promoting T cell reactivity) to reduce the patient's blast count Furthermore, anti-CD70 antibodies have also been shown to promote LSC differentiation. This may deplete the stem cell population and thus further contribute to the reduction in blasts observed in the patients in this study.

[0216] Both monotherapy and combination therapy reduce colony formation ex vivo. To further explore the impact of single-agent and combination therapies on LSC populations in patients, Methylcellulose colony assays using FACS-sorted monocytes were performed in vivo. Mononuclear cells (CD45 dim ) used annexin V and antibodies to CD19 and CD4 / CD8, and doublets, Selectively sorted by excluding dead cells and lymphocytes and then deposited on methylcellulose. were seeded for 14 consecutive days.

[0217] Figures 9A-E show the 1-week mean HR of 1000 cells before treatment (SCR) and after monotherapy with ARGX-110 (time point 0, corresponding to C1D1). Representative data on colony formation (and therefore LSC numbers) per well are provided.

[0218] Figure 9F shows the results before treatment and after monotherapy with ARGX-110 (i.e., with C1D1) and combination therapy (C4D 1) provides representative data on colony formation per well (and therefore LSC number). These data correspond to an approximately 24-fold decrease in LSC frequency compared to baseline levels. is doing.

[0219] In addition, the treatment not only reduced the number of LSCs, but also the number of cells per colony. However, this also indicates that the proliferative capacity of LSCs is reduced even after therapy (data not shown). .

[0220] Again, these data suggest that circulating LSC levels are also reduced following monotherapy with ARGX-110, It has been revealed that the reduction is even greater after the combined use of ARGX-110 and aza. The proliferative capacity of these LSCs is also reduced by the therapy.

[0221] (Both monotherapy and combination therapy reduce soluble CD27 levels) Soluble CD27 (sCD27) has been shown to serve as a biomarker for the extent of CD70 / CD27 interaction. CD70 / CD27-mediated signaling promotes abnormal cell division and high levels of Serum sCD27 is thought to correlate with poor prognosis in AML patients. This correlates with the proportion of blast cells in the patient's blood and further serves as a marker of blast stemness, increasing sCD27 is thought to indicate increased levels of stemness (Riether et al., J. Exp. Med. ed. 2017 Feb;214(2):359-380).

[0222] To evaluate the effect of anti-CD70 monotherapy and combination therapy on CD27 / CD70 interactions and to determine the effect of blast To further explore the effect on the percentage of CD27 and stemness, we also measured soluble CD27 levels in patients. Ta.

[0223] Figure 10 shows representative sCD27 levels for patients during treatment cycles. Drug therapy (C1D1) reduced sCD27 levels compared to pre-treatment ("loading"), and this reduction was accompanied by It continues to decrease during therapy until it reaches levels typical of healthy patients.

[0224] These data are consistent with results from other assays, and both single-agent and combination therapy treatments significantly improved CD 27 / CD70 interaction and reduced the percentage of blasts and stemness. There are.

[0225] In addition, Figure 10 shows that sCD27 levels increase after the end of treatment (EOT). This is probably due to an increase in CD27 / CD70 interactions once the anti-CD70 antibody is removed. This study explores the role of sCD27 in the treatment of rhesus mast cell carcinoma (HMD) and the role of sCD27 as an effective marker of anti-CD70 drug engagement in patients. This indicates that it can be used.

[0226] (ARGX-110 therapy does not increase toxicity) An important factor in the suitability of a therapeutic regimen is the associated toxicity to the patient. Advantageously, the anti-CD70 antibody ARGX-110, when used as monotherapy or in combination with azathioprine, Neither resulted in any observed increase in toxicity.

[0227] Adverse events and hematologic toxicities were observed for each of the 1 mg / kg, 3 mg / kg, and 10 mg / kg cohorts. This does not differ from the general safety profile of azacitidine. This is particularly surprising and advantageous since addition often results in increased toxicity. However, the combination of ARGX-110 and aza did not increase toxicity and showed a significant increase in efficacy. was induced.

[0228] Table 5 [Table 5]

[0229] (Pharmacokinetic (PK) data) Serum concentrations of ARGX-110 were measured using a validated enzyme-linked immunosorbent assay (ELISA) method. Figure 11 provides the individual patient PK plots for the 10 mg / kg cohort. The lot was administered during Cycle 1 of ARGX-110 (from the D-14 pre-dose to the Cycle 1 D1 pre-dose). Represents data related to

[0230] Across the cohort, the pharmacokinetics of ARGX-110 demonstrated Cmax and AUC across the dose range tested. It was dose proportional and showed a half-life of approximately 9.2 days at 10 mg / kg.

[0231] The concentration of ARGX-110 in bone marrow aspirates was measured in limited samples and in matched blood samples. The ARGX-110 concentration in bone marrow aspirates was compared with that in plasma samples in the 3 mg / kg and 10 mg / kg dose cohorts. were comparable to the plasma levels for patients 1001005 and 1001007, respectively.

[0232] Table 6 [Table 6]

[0233] (Patient characterization and case studies) Table 7 provides a summary of newly diagnosed AML patients recruited into each cohort: Table 7 [Table 7]

[0234] (The following are case studies of individual patients recruited in this clinical trial) Patient 1001001 was an 80-year-old woman with a therapy-related complication 5 years after adjuvant chemotherapy for breast cancer. He had AML, with a blast count of 90% in the bone marrow (BM) and 80% in the peripheral blood (PB) (39.5 G / L). FAB subtype: M4 myelomonocytic and WHO 2016 classification: Provisional subentity: Mutated The patient had AML with RUNX1. Patients were treated according to the protocol in the 1 mg / kg arm.

[0235] The bone marrow aspirate collected on day 1 was compared with the bone marrow aspirate collected before the ARGX-110 loading dose. FACS-sorted monocytes (mononuclear cells (CD45 dim ) are annexin V and CD19 and CD4 / CD8 Selective selection using antibodies to eliminate doublets, dead cells, and lymphocytes The number of LSCs in the bone marrow was assessed using a methylcellulose colony assay using .

[0236] These results show that a single dose of ARGX-110 reduced the number of LSCs in the bone marrow by 140,000-fold. (ELDA: Extreme Limiting Dilution Analysis (http: / / bioinf.wehi.e du.au; Hu and Smyth, (2009) ELDA: Journal of Immunological Methods 347, 70- 78).

[0237] In addition, cell morphology and flow cytometry analysis of the blast cell population demonstrated that a single dose of ARGX-110 significantly improved the survival of the blast cell population. The results show that the levels of α-glutamyltransferase in the bone marrow and peripheral blood decreased after the loading dose. After continued treatment with AZA according to the protocol, BM blasts were C3D1 (i.e., after 2 cycles of treatment). By the time of the study, the incidence had decreased to 2.8% (by flow cytometry). Clinical response assessment for patients was complete remission with incomplete recovery (CRi). D) was determined to be 0.2%. CRi status by bone marrow aspirate and MRD assessment was low. In peripheral blood, blasts were maintained at least until C5D1. By C3D1, peripheral blood MRD was 0.3% and C4D1 By the time of diagnosis, the peripheral blood blast percentage was 0.8% blasts by flow cytometry and 0% by cell morphology. On C4D17, the number of peripheral blood blast cells was sufficient to classify the patient as MRD-negative. It was rather low.

[0238] Patient 1001002 was diagnosed with AML with myelodysplasia-related changes (WHO classification: M1 / M2 according to FAB classification). The patient was a 75-year-old man with AML. The patient had 40% bone marrow blasts before treatment. Treatment was performed according to the protocol for the mg / kg therapy arm.

[0239] The bone marrow aspirate collected on day 1 was compared with the bone marrow aspirate collected before the ARGX-110 loading dose. The results were compared and evaluated using a methylcellulose colony assay on monocytes. Similar to patient 1001001, a single dose of ARGX-110 monotherapy reduced the number of LSCs in the bone marrow. In subject 1001002, LSCs were reduced by 2-fold after a loading dose of ARGX-110. , reduced by more than 50% at all significant serial dilutions, and cells per colony The numbers also decreased.

[0240] After a single dose of ARGX-110, the percentage of total blasts in the bone marrow was assessed by flow cytometry When the cells were incubated, the cells appeared to decrease, but when assessed by cell morphology, an increase was observed. .

[0241] Similar to patient 1001001, the overall blast percentage in peripheral blood decreased after the ARGX-110 loading dose (C1D1 ,vs. ,Screening), the duration of Cycle 1 continued to decrease, until the end of Cycle 1 (C2D1) By C2D17, the peripheral blood blast percentage was 0.8% by flow cytometry. After 3 cycles (C3D17), peripheral blood counts were recovered (Hb 1 0.2 g / dL; platelets 128 G / L; absolute neutrophil count (ANC) 0.97 G / L), and bone marrow analysis at C4D1 , CRi was shown.

[0242] Patient 1001003 was diagnosed with AML with myelodysplasia-related changes according to the WHO classification (A according to the FAC classification). The patient was a 77-year-old man with ML-M2 disease who showed 24% myeloblasts. The patient showed no peripheral blasts and short-term thrombocytopenia and lymphopenia (platelets 98 g / L; ANC 0 The patient recovered after a 2-week follow-up (Hb 9.2 g / dL; 0.38 G / L). The blast percentage remained stable at C1D1.

[0243] Data from these patients suggest that treatment with anti-CD70 antibody (ARGX-110) as monotherapy is effective in treating AML Reduce the number of leukemia stem cells in the patient's bone marrow and lower the percentage of total blasts in the bone marrow and peripheral blood. It has been revealed that the anti-CD70 antibody (ARGX-110) and nucleoside Combination therapy with azacitidine, a cytotoxic inhibitor, increased the percentage of total blasts in the bone marrow and peripheral blood compared with azacitidine. patients with minimal residual disease, an outcome not typically achieved with rheumatoid arthritis therapy alone. It further decreases to the extent that it can be classified as The present application provides the following aspects of the invention. (Aspect 1) A method for treating acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) in a subject. and administering to the subject one or more doses of an anti-CD70 antibody or antigen-binding fragment thereof. , the method. (Aspect 2) A method for reducing the percentage of blast cells in the bone marrow and / or peripheral blood of a subject with AML or MDS, comprising administering to said subject a therapeutically effective amount of erythropoietin (ERA) to said subject. administering to the animal a therapeutically effective amount of one or more anti-CD70 antibodies or antigen-binding fragments thereof; The method. (Aspect 3) A method of preparing a subject with AML or MDS for hematopoietic stem cell transplantation (HSCT), comprising administering to the subject an antibody against - administering one or more doses of a CD70 antibody or antigen-binding fragment thereof. (Aspect 4) The method of any one of embodiments 1 to 3, wherein the method reduces the percentage of blasts in the bone marrow of the subject. Law. (Aspect 5) 5. The method of any one of embodiments 1 to 4, wherein the method reduces the percentage of blasts in the peripheral blood of the subject. method. (Aspect 6) The anti-CD70 antibody or antigen-binding fragment thereof is administered in a single dose ranging from 0.1 mg / kg to 25 mg / kg. The method according to any one of embodiments 1 to 5, wherein the compound is administered at a dose ranging from 0.1 to 1.0. (Aspect 7) The anti-CD70 antibody or antigen-binding fragment thereof is administered in a dose range of 1 mg / kg to 20 mg / kg. The method of any one of embodiments 1 to 6, wherein the compound is administered at a dosage of (Aspect 8) Aspects 1-7, wherein the anti-CD70 antibody or antigen-binding fragment thereof is administered at a dose of 10 mg / kg. The method according to any one of the preceding claims. (Aspect 9) Each dose of the anti-CD70 antibody or antigen-binding fragment thereof is administered for 10 to 20 days, optionally 12 to 18 days, or The method of any one of embodiments 1 to 8, wherein the administrations are optionally separated by 14 to 17 days. (Aspect 10) Any one of aspects 1 to 9, further comprising administering to the subject a nucleoside metabolism inhibitor. The method described in section. (Aspect 11) The nucleoside metabolic inhibitor is a hypomethylating agent, optionally azacitidine or decitabine. The method of embodiment 10. (Aspect 12) 12. The method of aspect 10 or 11, wherein the nucleoside metabolism inhibitor is azacitidine. (Aspect 13) The nucleoside metabolic inhibitor is administered at a dose of 50 to 100 mg / m per day. 2 It is administered in doses ranging from The method according to any one of embodiments 10 to 12. (Aspect 14) In one embodiment, the nucleoside metabolism inhibitor is administered for a daily dosing period of 5 to 9 days. 14. The method of any one of claims 13. (Aspect 15) wherein the nucleoside metabolic inhibitor is administered according to a dosing regimen of repeated dosing periods, 15. The method of claim 14, wherein the end of one dosing period and the beginning of the next dosing period are separated by 18 to 25 days. How to do it. (Aspect 16) The initial dose of the nucleoside metabolic inhibitor is 16. The method of any one of embodiments 10 to 15, wherein the single dose is administered 7 to 21 days after the single dose. (Aspect 17) One of the daily doses of the nucleoside metabolic inhibitor is administered daily after the administration of an anti-CD70 antibody or antigen-binding fragment thereof. 17. The method of any one of embodiments 10 to 16, wherein the dose is administered on the same day as the administration. (Aspect 18) The method comprising: i) administering an anti-CD70 antibody according to any one of aspects 10 to 17 and a nucleoside metabolism inhibitor, Including, the first stage, and ii) administration of an anti-CD70 antibody according to any one of embodiments 1 to 9, and administration of a lower dose of a nucleoside metabolic inhibitor than that of a nucleoside metabolic inhibitor; The second stage includes: 18. The method of any one of embodiments 10 to 17, comprising: (Aspect 19) 19. The method of any one of aspects 1 to 18, wherein the subject is ineligible for standard intensive chemotherapy prior to treatment. How to post. (Aspect 20) 20. Any one of aspects 1 to 19, further comprising performing a hematopoietic stem cell transplant in said subject. The method described in section. (Aspect 21) The method of any one of aspects 1 to 20, wherein the subject is 60 years of age or older, optionally 75 years of age or older. . (Aspect 22) The subject is administered an anti-CD33 antibody, an anti-CD123 antibody, an E-selectin inhibitor, an FLT3 inhibitor, a cyclin- Phosphokinase inhibitors, BCL-2 inhibitors, aminopeptidase inhibitors and JAK / STAT inhibitors 22. Any one of embodiments 1-21, further comprising administering one or more active agents selected from the group consisting of: The method described in section. (Aspect 23) 23. The method of any one of embodiments 1 to 22, further comprising monitoring the subject's blast count. How to do it. (Aspect 24) including partial response (PR), complete response with incomplete hematologic recovery (CRi), or complete response (CR), The method according to any one of embodiments 1 to 23. (Aspect 25) The method of any one of embodiments 1 to 24, wherein the method induces a negative minimal residual disease state. (Aspect 26) 26. Any of embodiments 1 to 25, wherein the subject is a candidate for a standard of care agent for which the subject is eligible, and the therapeutic agent increases survival relative to the standard of care agent for which the subject is eligible. or the method described in any one of claims 1 to 5. (Aspect 27) 27. Any of aspects 1 to 26, wherein the anti-CD70 antibody or antigen-binding fragment thereof inhibits CD70-CD27 binding. The method according to any one of claims 1 to 4. (Aspect 28) 28. Any of aspects 1 to 27, wherein the anti-CD70 antibody or antigen-binding fragment thereof depletes CD70-expressing cells. The method according to any one of claims 1 to 4. (Aspect 29) the anti-CD70 antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), The VH and VL domains have the following CDRs: SEQ ID NO:3 (chemical 1) HCDR3 containing or consisting of TIFF0007822424000020.tif7170 SEQ ID NO:2 (Case 2) HCDR2 containing or consisting of TIFF0007822424000021.tif6170 SEQ ID NO:1 (C3) HCDR1 containing or consisting of TIFF0007822424000022.tif7170 SEQ ID NO:7 (C4) LCDR3 containing or consisting of TIFF0007822424000023.tif7170 SEQ ID NO:6 (C5) LCDR2 comprising or consisting of TIFF0007822424000024.tif7170, and SEQ ID NO:5 (6) LCDR1 contains or consists of TIFF0007822424000025.tif7170: The method of any one of embodiments 1 to 28, comprising: (Aspect 30) The anti-CD70 antibody or antigen-binding fragment has a VH domain that is at least 80% identical to SEQ ID NO:4. and / or a VL domain that is at least 80% identical to SEQ ID NO: 8. 30. The method of any one of 1 to 29. (Aspect 31) The method according to any one of aspects 1 to 30, wherein the anti-CD70 antibody is an IgG1 antibody. (Aspect 32) The method of any one of aspects 1 to 31, wherein the anti-CD70 antibody is ARGX-110. (Aspect 33) An anti-CD70 antibody for use in the method according to any one of embodiments 1 to 32. (Aspect 34) A pharmaceutical composition for use in the method according to any one of aspects 1 to 33, comprising The pharmaceutical composition comprises an anti-CD70 antibody and a pharmaceutically acceptable excipient or carrier. The pharmaceutical composition. (Aspect 35) 1. An anti-CD70 antibody or antigen-binding fragment thereof for use in the treatment of AML, wherein The antibody or antigen-binding fragment is administered in combination with a hypomethylating agent, preferably azacytidine. The anti-CD70 antibody or antigen-binding fragment thereof. (Aspect 36) 36. The method of claim 33, wherein the anti-CD70 antibody or antigen-binding fragment thereof inhibits CD70-CD27 binding. An antibody or pharmaceutical composition for use according to any one of claims 1 to 4. (Aspect 37) 37. The method of claim 33, wherein the anti-CD70 antibody or antigen-binding fragment thereof depletes CD70-expressing cells. An antibody or pharmaceutical composition for use according to any one of claims 1 to 4. (Aspect 38) The anti-CD70 antibody or antigen-binding fragment comprises a heavy chain variable domain (VH) and a light chain variable domain (V and L), wherein the VH and VL domains comprise the following CDRs: SEQ ID NO:3 (C7) HCDR3 containing or consisting of TIFF0007822424000026.tif7170 SEQ ID NO:2 (8) HCDR2 containing or consisting of TIFF0007822424000027.tif7170 SEQ ID NO:1 (9) HCDR1 containing or consisting of TIFF0007822424000028.tif7170 SEQ ID NO:7 (C10) LCDR3 containing or consisting of TIFF0007822424000029.tif7170 SEQ ID NO:6 (Chem.11) LCDR2 comprising or consisting of TIFF0007822424000030.tif8170, and SEQ ID NO:5 (C12) LCDR1 contains or consists of TIFF0007822424000031.tif7170: 38. The antibody or pharmaceutical composition for use according to any one of aspects 33 to 37, comprising: (Aspect 39) The anti-CD70 antibody or antigen-binding fragment has a VH domain that is at least 80% identical to SEQ ID NO:4. and / or a VL domain that is at least 80% identical to SEQ ID NO: 8. 39. The antibody or pharmaceutical composition for use according to any one of claims 33 to 38. (Aspect 40) 40. The method of claim 39, wherein the anti-CD70 antibody is an IgG1 antibody. An antibody or pharmaceutical composition. (Aspect 41) 41. The method for use according to any one of aspects 33 to 40, wherein the anti-CD70 antibody is ARGX-110. An antibody or pharmaceutical composition. (Aspect 42) A combination comprising an anti-CD70 antibody or antigen-binding fragment thereof and an NMI. (Aspect 43) The combination of embodiment 42, wherein the NMI is a hypomethylating agent. (Aspect 44) 44. The combination of embodiment 43, wherein said hypomethylating agent is azacitidine. (Aspect 45) 45. The method of claim 42, wherein the anti-CD70 antibody or antigen-binding fragment thereof inhibits CD70-CD27 binding. The combination according to any one of claims 1 to 4. (Aspect 46) 46. ​​The method of claim 42, wherein the anti-CD70 antibody or antigen-binding fragment thereof depletes CD70-expressing cells. The combination according to any one of claims 1 to 4. (Aspect 47) the anti-CD70 antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), The VH and VL domains have the following CDRs: SEQ ID NO:3 (C13) HCDR3 containing or consisting of TIFF0007822424000032.tif8170 SEQ ID NO:2 (C14) HCDR2 containing or consisting of TIFF0007822424000033.tif8170 SEQ ID NO:1 (C15) HCDR1 containing or consisting of TIFF0007822424000034.tif7170 SEQ ID NO:7 (C16) LCDR3 containing or consisting of TIFF0007822424000035.tif8170 SEQ ID NO:6 (C17) LCDR2 comprising or consisting of TIFF0007822424000036.tif7170, and SEQ ID NO:5 (C18) LCDR1 contains or consists of TIFF0007822424000037.tif7170: 47. The combination according to any one of embodiments 42 to 46, comprising: (Aspect 48) The anti-CD70 antibody or antigen-binding fragment has a VH domain that is at least 80% identical to SEQ ID NO:4. and / or a VL domain that is at least 80% identical to SEQ ID NO: 8. 48. The combination according to any one of claims 42 to 47. (Aspect 49) A combination according to any one of aspects 42 to 48, wherein the anti-CD70 antibody is an IgG1 antibody. (Aspect 50) 50. The combination according to any one of aspects 42 to 49, wherein the anti-CD70 antibody is ARGX-110. (Aspect 51) 51. The combination of any one of embodiments 42 to 50 for use in a method of treating AML or MDS. Match. (Aspect 52) Any of embodiments 42 to 50 for use in the method of any of embodiments 1 to 32. The combination according to claim 1.

Claims

1. 1. A pharmaceutical composition comprising an anti-CD70 antibody or antigen-binding fragment thereof for use in a method for treating acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) in a human subject, wherein the antibody or antigen-binding fragment thereof is administered in combination with azacitidine or decitabine, wherein the anti-CD70 antibody or antigen-binding fragment thereof is administered at a dose ranging from 0.1 mg / kg to 25 mg / kg per dose, and wherein each dose of the anti-CD70 antibody or antigen-binding fragment thereof is separated by 12 to 18 days.

2. 10. The pharmaceutical composition of claim 1, wherein each dose of the anti-CD70 antibody or antigen-binding fragment thereof is separated by 14 to 17 days.

3. 10. The pharmaceutical composition of claim 1, wherein the anti-CD70 antibody or antigen-binding fragment thereof is administered at a dosage ranging from 0.1 mg / kg to 20 mg / kg.

4. 10. The pharmaceutical composition of claim 1, wherein the anti-CD70 antibody or antigen-binding fragment thereof is administered at a dosage ranging from 1 mg / kg to 20 mg / kg.

5. 10. The pharmaceutical composition of claim 1, wherein the anti-CD70 antibody or antigen-binding fragment thereof is administered at a dosage ranging from 0.1 to 15 mg / kg.

6. 10. The pharmaceutical composition of claim 1, wherein the anti-CD70 antibody or antigen-binding fragment thereof is administered at a dosage ranging from 0.5 to 2 mg / kg.

7. 10. The pharmaceutical composition of claim 1, wherein the anti-CD70 antibody or antigen-binding fragment thereof is administered at a dosage of 1 mg / kg.

8. 10. The pharmaceutical composition of claim 1, wherein the anti-CD70 antibody or antigen-binding fragment thereof is administered at a dosage of 3 mg / kg.

9. 10. The pharmaceutical composition of claim 1, wherein the anti-CD70 antibody or antigen-binding fragment thereof is administered at a dosage of 10 mg / kg.

10. 10. The pharmaceutical composition of claim 1, wherein the anti-CD70 antibody or antigen-binding fragment thereof is administered at a dosage of 20 mg / kg.

11. The azacitidine or decitabine is administered at a dose of 50 to 100 mg / m per day. 2 10. The pharmaceutical composition of claim 1, wherein the composition is administered at a dosage in the range of

12. The azacitidine or decitabine is administered at a dose of 70 to 80 mg / m per day. 2 10. The pharmaceutical composition of claim 1, wherein the composition is administered at a dosage in the range of

13. The azacitidine or decitabine is administered at a dose of 75 mg / m per day. 2 2. The pharmaceutical composition of claim 1, wherein the composition is administered at a dose of

14. 10. The pharmaceutical composition of claim 1, wherein the azacitidine or decitabine is administered for a dosing period of 5 to 9 days daily.

15. 10. The pharmaceutical composition of claim 1, wherein the azacitidine or decitabine is administered for a dosing period of 7 days daily.

16. 10. The pharmaceutical composition of claim 1, wherein the azacitidine or decitabine is administered according to a dosing regimen of multiple dosing periods, wherein the end of one dosing period and the beginning of the next dosing period are separated by 18 to 25 days.

17. 17. The pharmaceutical composition of claim 16, wherein the end of one dosing period and the beginning of the next dosing period are separated by 21 days.

18. 10. The pharmaceutical composition of claim 1, wherein the subject is ineligible for standard intensive chemotherapy.

19. 10. The pharmaceutical composition of claim 1, wherein the subject is at least 60 years old.

20. 20. The pharmaceutical composition of claim 19, wherein the subject is at least 70 years old.

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