Combination therapeutic methods for treating myeloproliferative tumors with diphtheria toxin-human interleukin-3 conjugates in combination with other agents

Abstract

The present invention provides a method for treating or inhibiting a myeloproliferative tumor (MPN) in a subject in need thereof, the method comprising administering to the subject a diphtheria toxin-human interleukin-3 conjugate (DT-IL3) and one or more Jak inhibitors and / or one or more hypomethylating agents.

Description

[0001] This invention application is a divisional application of the invention patent application filed on October 30, 2019, with application number 201980071179.0 (international application number PCT / US2019 / 058769) entitled "Combined treatment method for myeloproliferative tumors using diphtheria toxin-human interleukin-3 conjugate in combination with other agents".

[0002] Cross-references to related applications

[0003] This international application claims priority to U.S. Provisional Application Serial No. 62 / 753,145, filed October 31, 2018, and U.S. Provisional Application Serial No. 62 / 832,558, filed April 11, 2019, the contents of each of which are hereby incorporated in their entirety by reference.

[0004] sequence list

[0005] This application is submitted together with the electronic sequence listing. The sequence listing is provided in the form of a file titled “2019-10-21_01214-0017-00PCT_SEQ_List_ST25.txt”, created on October 21, 2019, with a file size of 12,288 bytes. Information about the electronic sequence listing is incorporated herein by reference in its entirety. Technical Field

[0006] This invention provides a method for treating or inhibiting myeloproliferative neoplasms (MPN) in a subject of need. The method comprises administering to the subject a diphtheria toxin-human interleukin-3 conjugate (DT-IL3) and one or more Jak inhibitors and / or one or more hypomethylating agents. Background Technology

[0007] Myeloproliferative neoplasm (MPN), also known as myeloproliferative disorder (MPD), is a blood disorder characterized by the overproduction of cells in the bone marrow. MPN is also characterized by the clonal expansion of one or more hematopoietic cell lines in the bone marrow. In some cases, genetic mutations, such as Jak2 protein mutations, may be present in the stem cells of MPN patients.

[0008] MPN can cause an increase in certain blood cells, abnormal blood cell counts, and splenomegaly (enlarged spleen). MPN can damage the bone marrow and lead to bone marrow fibrosis (myelofibrosis, or MF). Due to the changes in blood cell counts in MPN, anemia, fatigue, and weakness may occur. Thrombotic bleeding complications such as thrombosis are also a risk factor for MPN. Progression of MPN may lead to other conditions such as acute myeloid leukemia.

[0009] This application describes an improved method for treating MPN with diphtheria toxin-human interleukin-3 conjugate (DT-IL3) and one or more Jak inhibitors and / or one or more hypomethylating agents. Summary of the Invention

[0010] According to the specification, this application describes a method of treatment with DT-IL3 and (a) one or more Jak inhibitors and / or (b) one or more hypomethylating agents.

[0011] In one embodiment, this application provides a method for treating or inhibiting myeloproliferative neoplasms (MPN) in a subject of need, the method comprising administering to the subject a diphtheria toxin-human interleukin-3 conjugate (DT-IL3) and a) one or more Jak inhibitors and / or b) one or more hypomethylating agents.

[0012] In some embodiments, DT-IL3 is administered at a dose of 1 μg / kg or greater, 2 μg / kg or greater, or 4 μg / kg or greater. In some embodiments, DT-IL3 is administered at a dose of 2 μg / kg to 20 μg / kg or 9 μg / kg to 20 μg / kg. In some embodiments, DT-IL3 is administered at a dose of 4 μg / kg to 12 μg / kg or 9 μg / kg to 12 μg / kg. In some embodiments, DT-IL3 is administered at a dose of 5, 7, 9, or 12 μg / kg. In some embodiments, the administered dose of DT-IL3 is the maximum tolerated dose.

[0013] In some embodiments, the DT-IL3 and one or more Jak inhibitors and / or hypomethylating agents are applied at least once a week. In some embodiments, the DT-IL3 and one or more Jak inhibitors and / or hypomethylating agents are applied at least twice a week. In some embodiments, the DT-IL3 and one or more Jak inhibitors and / or hypomethylating agents are applied at least three times a week.

[0014] In some embodiments, the DT-IL3 and one or more Jak inhibitors (a) and / or one or more hypomethylating agents (b) are applied over a period of one week or longer. In some embodiments, the DT-IL3 and one or more Jak inhibitors (a) and / or one or more hypomethylating agents (b) are applied over a period of two weeks or longer.

[0015] In some embodiments, the DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents are administered for at least 3 consecutive days.

[0016] In some embodiments, the DT-IL3 and one or more Jak inhibitors (a) and / or one or more hypomethylating agents (b) are administered in multiple treatment cycles. In some embodiments, the treatment cycles are spaced at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, or a combination thereof.

[0017] In some embodiments, the DT-IL3 is administered for at least three consecutive days every 21 days over four cycles, followed by administration every 28 days over three cycles and then every 42 days.

[0018] In some embodiments, one or more of the Jak inhibitors may be administered at least once or at least twice daily.

[0019] In some embodiments, the one or more hypomethylating agents are applied for at least the first 3 days, at least the first 4 days, at least the first 5 days, at least the first 6 days, or at least the first 7 days of at least one cycle.

[0020] In some embodiments, after four 21-day cycles of DT-IL3 administration, the one or more hypomethylating agents are applied for at least the first 3, 4, 5, 6, or 7 days of a 28-day cycle for three cycles.

[0021] The method according to any one of the preceding claims, wherein the DT-IL3 and one or more Jak inhibitors (a) and / or one or more hypomethylating agents (b) are administered until disease progression and / or unacceptable toxicity is achieved.

[0022] In some embodiments, the subject is administered a pharmaceutical composition comprising the DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents as described in (a) or (b).

[0023] In some embodiments, the subject is a human being. In some embodiments, the human being has an adverse cytogenetic profile.

[0024] In some embodiments, DT-IL3 is a chemical conjugate.

[0025] In some embodiments, DT-IL3 is a recombinant expressed protein.

[0026] In some embodiments, the DT-IL3 is expressed as a single polypeptide comprising the catalytic domain and translocation domain of diphtheria toxin and human IL-3. In some embodiments, the DT-IL3 expressed as a single polypeptide comprising the catalytic domain and translocation domain of diphtheria toxin comprises the amino acid sequence SEQ ID NO:3, and / or the human IL-3 comprises the amino acid sequence SEQ ID NO:1.

[0027] In some embodiments, the DT-IL3 comprises amino acid residues 1 to 388 of a diphtheria toxin linked to human IL-3 via peptide bonds. In some embodiments, the diphtheria toxin has the amino acid sequence SEQ ID NO:2, and / or human IL-3 has the amino acid sequence SEQ ID NO:1.

[0028] In some embodiments, inhibition reduces MPN cell proliferation, stabilizes MPN cell numbers, and / or reduces MPN cell numbers.

[0029] In some embodiments, inhibition reduces the size of the spleen and / or liver.

[0030] In some embodiments, the MPN is polycythemia vera, essential thrombocythemia, myelofibrosis, chronic myeloid monocytic leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, systemic mastocytosis, symptomatic hypereosinophilic syndrome, or other bone marrow disorders that cause excessive production of red blood cells, white blood cells, and / or platelets. In some embodiments, the MPN is myelofibrosis. In some embodiments, the myelofibrosis is primary myelofibrosis, post-polycythemia vera myelofibrosis, post-esophageal thrombocythemia vera myelofibrosis, blast crisis primary myelofibrosis, blast crisis post-polycythemia vera myelofibrosis, or blast crisis ET post-blast crisis myelofibrosis.

[0031] In some embodiments, the MPN is in a rapid change phase.

[0032] In some embodiments, the MPN is primary myelofibrosis in the blast crisis, myelofibrosis after polycythemia vera in the blast crisis, or myelofibrosis after ET in the blast crisis.

[0033] In some embodiments, the one or more Jak inhibitors include ruxolitinib.

[0034] In some embodiments, the one or more hypomethylating agents include azacitidine, decitabine, and / or SGI-110.

[0035] In some embodiments, at least one Jak inhibitor and at least one hypomethylating agent are applied.

[0036] In some embodiments, the at least one Jak inhibitor includes ruxotinib, and the at least one hypomethylating agent includes decitabine, azacitidine, and / or SGI-110.

[0037] In some embodiments, the subject is unresponsive to prior treatment with one or more Jak inhibitors and / or one or more hypomethylating agents. In some embodiments, the subject is unresponsive to prior treatment with the Jak inhibitor ruxotinib. In some embodiments, the subject has myxomatosis (MF).

[0038] In some embodiments, the subject cannot tolerate a full dose of pretreatment with one or more Jak inhibitors and / or one or more hypomethylating agents. In some embodiments, the subject cannot tolerate a full dose of pretreatment with the Jak inhibitor ruxotinib. In some embodiments, the subject has myxomatosis (MF).

[0039] In some embodiments, the subject has a low platelet count or is unsuitable for treatment with ruxotinib. In some embodiments, the subject has myocardial infarction (MF).

[0040] In some embodiments, the subject has previously responded to a Jak inhibitor and / or a hypomethylating agent. In some embodiments, the subject has MF.

[0041] In some embodiments, the subject has a mutation in the JAK2, MPL, ASXL1, TET2, or CALR gene. In some embodiments, the subject has a JAK2V617F mutation.

[0042] This document also provides pharmaceutical compositions for treating or inhibiting myeloproliferative neoplasms (MPN) in subjects of need, said pharmaceutical compositions comprising an effective amount of diphtheria toxin-human interleukin-3 conjugate (DT-IL3) and one or more Jak inhibitors and / or one or more hypomethylating agents. In some embodiments, said pharmaceutical compositions further comprise pharmaceutically acceptable excipients.

[0043] This article also provides a method for treating myeloproliferative neoplasms (MPNs) in subjects of need, the method comprising: a) identifying the MPN, and b) administering to the subject of need an effective amount of diphtheria toxin-human interleukin-3 conjugate (DT-IL3) and one or more Jak inhibitors and / or one or more hypomethylating agents.

[0044] In some embodiments, the MPN is identified by physical examination, blood tests, bone marrow aspiration and biopsy, cytogenetic analysis, testing for mutations in the JAK2, MPL, ASXL1, TET2 or CALR genes, arterial oxygen saturation and carboxyhemoglobin levels, neutrophil alkaline phosphatase levels, vitamin B12 or B12 binding capacity or serum urate.

[0045] In some embodiments, the MPN is identified by testing for the JAK2V617F mutation.

[0046] Further objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this disclosure. These objects and advantages will be realized and obtained by means of the elements and combinations particularly pointed out in the appended claims.

[0047] It should be understood that the foregoing general description and the following detailed description are merely exemplary and illustrative, and do not limit the claims.

[0048] The accompanying drawings, which are incorporated in and form part of this specification, illustrate one or more embodiments and, together with the description, serve to explain the principles described herein. Attached Figure Description

[0049] Figures 1A-1E The expression of CD123 on blasts and monocytes in patients with chronic myeloid monocytic leukemia (CMML) (N=20) is shown by flow cytometry. Lymphocyte CD123 expression was used as a control. Figure 1A The mean fluorescence intensity of CD123 on CMML blast cells, monocytes, and lymphocytes is shown. Figure 1B The mean fluorescence intensity of CD123 on CMML blast cells and monocytes is shown as normalized relative to the mean fluorescence intensity of CD123 on lymphocytes. Figure 1C and Figure 1D The blastoblasts of different CMML subtypes (CMML-0, CMML-1, and CMML-2) are shown. Figure 1C ) and monocytes ( Figure 1D The mean fluorescence intensity of CD123 on lymphocytes was calculated. The results were normalized relative to the mean fluorescence intensity of CD123 on lymphocytes. Figure 1EThe percentage of CD123-positive blasts in CMML subtypes CMML-1 and CMML-2 is shown. Overall, Figures 1A-1E This indicates that CD123 is highly expressed in blast cells and monocytes of CMML patients, relative to lymphocytes.

[0050] Figures 2A-2C This demonstrates the effect of using increased concentrations of azacitidine (AZA) in vitro. Figure 2A ), DT-IL-3 (SL-401, Figure 2B ) or a combination of AZA and DT-IL3 ( Figure 2C Results of cell viability assays for CD123-expressing K562 cell lines after treatment. AZA and DT-IL3 significantly reduced K562 cell viability compared to AZA alone.

[0051] Figures 3A-3B The results of cell viability assays of fresh peripheral blood mononuclear cells from CMML patients following in vitro treatment with AZA, DT-IL3, or a combination of AZA and DT-IL3 (SL-401) are shown. Figure 3A In this process, cells are exposed to increased concentrations of AZA or a combination of AZA and DT-IL3. Figure 3B Cells were exposed to 1 μM AZA, 4.8 nM DT-IL3, or a combination of 1 μM AZA and 4.8 nM DT-IL3. Overall, Figures 3A-3B This indicates that, compared with AZA alone, AZA and DT-IL3 significantly reduced the cell viability of primary CMML mononuclear cells.

[0052] Figures 4A-4D The results of a colony formation assay using fresh mononuclear cells from peripheral blood of CMML patients are shown. Figures 4A-4D The results for a single patient's cells are shown. Figures 4A-4C In this study, cells were exposed to AZA alone (1, 5, or 10 μM) or a combination of 1 μM AZA and DT-IL-3 (2.5 or 10 nM). Adding 10 nM DT-IL3 to 1 μM AZA significantly reduced colony formation. Figure 4D In this study, cells were exposed to DT-IL3 alone (2.5, 10, or 20 nM) or a combination of 10 nM DT-IL3 and AZA (1 or 2.5 μM). Adding AZA to DT-IL3 significantly reduced colony formation. Overall, Figures 4A-4D This indicates that AZA and DT-IL3 significantly reduced CMML monocyte colony formation compared to AZA or DT-IL3 alone.

[0053] Sequence Description

[0054] Table 1 provides a list of some of the sequences cited in this paper.

[0055] Detailed Implementation

[0056] I. Treatment drugs

[0057] This application provides a method for treating myeloproliferative neoplasms (MPN), the method comprising administering to a subject a diphtheria toxin-human interleukin-3 conjugate (DT-IL3) and (a) one or more Jak inhibitors and / or (b) one or more hypomethylating agents.

[0058] In some embodiments, DT-IL3 and (a) one or more Jak inhibitors and / or (b) one or more hypomethylating agents are administered simultaneously or on the same day. In some embodiments, the pharmaceutical composition comprises DT-IL3 and (a) one or more Jak inhibitors and / or (b) one or more hypomethylating agents.

[0059] In some embodiments, DT-IL3 and (a) one or more Jak inhibitors and / or (b) one or more hypomethylating agents are applied separately. In some embodiments, DT-IL3 and (a) one or more Jak inhibitors and / or (b) one or more hypomethylating agents are applied at different times or on different dates.

[0060] A. Diphtheria toxin-human interleukin-3 conjugate (DT-IL3)

[0061] "DT-IL3" refers to a conjugate of human interleukin-3 (IL-3) and diphtheria toxin (DT). DT-IL-3 conjugates are known in the art, and their application according to the methods of this disclosure is contemplated herein. For example, DT-IL-3 conjugates described in U.S. Patent Nos. 7,763,242, 8,470,307, 9,181,317, 9,631,006, and WO2008 / 030539 can be used according to the methods disclosed herein. These references are incorporated herein by reference in their entirety for their disclosures concerning DT-IL3. See also the conjugates in the following literature, for example: Liu et al., Experimental Hematology 32:277-281 (2004); Hogge et al., Clin. Cancer Res. 12:1284-1291 (2006); Testa et al., Blood 106:2527-2529 (2005); and Klein et al., Biochem. Biophys. Res. Comm. 288:1244-1249 (2001), which are also incorporated herein by reference in their entirety.

[0062] In some embodiments, the conjugate comprises a catalytic domain and a translocation domain of diphtheria toxin fused to human IL-3 via covalent bonding. In some embodiments, the diphtheria toxin is linked to the human IL-3 moiety of the conjugate via a peptide linker. The length of the linker can be, for example, two, three, four, five, ten, up to fifteen amino acids. The length of the linker can be varied to provide optimal binding of the conjugate. In some embodiments, the length of the peptide linker is two to four amino acids. The peptide linker can be a His-Met linker. Although not intended to be limited to a specific mechanism of action, flexible peptide linkers facilitate chain pairing and minimize possible refolding. Linker molecules are well known in the art and have been described in the following references: Denardo et al., 1998, Clinical Cancer Research 4:2483-90; Peterson et al., 1999, Bioconjug. Chem. 10:553; and Zimmerman et al., 1999, Nucleic Medicine and Biology 26:943-50, each of which is incorporated herein by reference in its entirety.

[0063] In some embodiments, this application provides pharmaceutical compositions comprising the DT-IL3 of the present invention and a pharmaceutically acceptable carrier. According to the present invention, the conjugate may comprise any domain of DT linked to any domain of IL-3 via any linker known in the art. In some embodiments, the conjugate is DT. 388 IL-3 is a fusion protein consisting of N-terminal methionine and subsequent amino acids 1-388 of the DT of full-length mature human IL-3, fused to the DT via the His-Met amino acid linker.

[0064] In some embodiments, DT-IL3 selectively targets and mediates the expression of interleukin-3 receptor (IL-3 receptor). In some embodiments, DT-IL3 targets MPN cells expressing IL-3 receptor. In some embodiments, DT-IL3 targets tumor-promoting cells (such as IL-3R+ plasmacytoid dendritic cells) in the tumor microenvironment.

[0065] 1.IL-3

[0066] Interleukin-3 (IL-3) is a cytokine that supports the proliferation and differentiation of pluripotent and directed bone marrow and lymphoid progenitor cells. See, for example, Nitsche et al., Stem Cells 21:236-244 (2003). IL-3 is also known as hematopoietic growth factor, mast cell growth factor (MCGF), pluripotent colony-stimulating factor, or P cell stimulating factor.

[0067] In some embodiments, the DT-IL3 conjugate comprises a full-length mature (signal peptide-deficient) interleukin-3 protein (IL-3) or a portion, analogue, or derivative thereof conjugated to diphtheria toxin (DT) or a portion thereof, an analogue, or a derivative thereof by recombinant technology or by chemical (covalent) bond to a portion thereof, an analogue, or a derivative thereof, which is bound to an interleukin-3 receptor or a subunit thereof expressed on the cell surface, wherein the toxin lacks a natural cell-binding domain.

[0068] Fragments, analogues, and derivatives of IL-3 can be used in this invention, provided that when fused to the DT portion of the conjugate, such fragments, analogues, and derivatives maintain the ability to bind to the IL-3 receptor or subunits of the native IL-3 receptor expressed on the cell surface. The binding kinetics of said fragments, analogues, and derivatives may remain unchanged or vary by no more than 25%. IL-3 peptides can be derived from any species. In some embodiments, IL-3 is mammalian IL-3, for example, IL-3 peptides are human IL-3, its analogues, derivatives, or fragments.

[0069] In some embodiments, IL-3 is human IL-3. An exemplary amino acid sequence of human IL-3 can be found in the GenBank database (see, for example, accession number AAC08706) or UniProt#P08700. An exemplary amino acid sequence of human IL-3 is as follows:

[0070] msrlpvllll qllvrpglqa pmtqttslkt swvncsnmid eiithlkqpp lplldfnnln

[0071] gedqdilmen nlrrpnleaf nravkslqna saiesilknl lpclplataa ptrhpihikd

[0072] gdwnefrrkl tfylktlena qaqqttlsla if[SEQ ID NO:1].

[0073] In some embodiments, IL-3 is at least 50%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1.

[0074] 2. Diphtheria toxin

[0075] Diphtheria toxin (DT) is a protein with three domains: a catalytic domain (amino acids 26-112; the underlined sequence in SEQ ID NO:1 below), which is connected by an arginine-rich disulfide ring to a translocation domain (amino acids 225-404; the italicized sequence in SEQ ID NO:1), followed by a cell-binding domain (amino acids 406-559). An exemplary amino acid sequence of DT obtained from GenBank accession number AOU74567.1 or UniProt#A0A142BWN1 is as follows:

[0076]

[0077]

[0078] Fragments, analogues, and derivatives of DT can be used in this application. In some embodiments, DT comprises a catalytic domain, a translocation domain, and a cell-binding domain of DT. In some embodiments, DT comprises a cell-binding domain and a catalytic domain of DT. In some embodiments, DT comprises a cell-binding domain and a translocation domain of DT. In some embodiments, DT comprises a catalytic domain and a translocation domain of DT. In some embodiments, DT includes one of a translocation domain, a catalytic domain, or a cell-binding domain.

[0079] The DT fragment conjugated to IL-3 consists of the catalytic and translocation domains of DT, as exemplified by SEQ ID NO:3:

[0080] GADDVVDSSK SFVMENFSSY HGTKPGYVDS IQKGIQKPKS GTQGNYDDDW

[0081] KGFYSTDNKY DAAGYSVDNE NPLSGKAGGV VKVTYPGLTK VLALKVDNAE

[0082] TIKKELGLSL TEPLMEQVGT EEFIKRFGDG ASRVVLSLPF AEGSSSVEYI

[0083] NNWEQAKALS VELEINFETR GKRGQDAMYE YMAQACAGNR VRRSVGSSLS

[0084] CINLDWDVIR DKTKTKIESL KEHGPIKNKM SESPNKTVSE EKAKQYLEEF

[0085] HQTALEHPEL SELKTVTGTN PVFAGANYAA WAVNVAQVID SETADNLEKT

[0086] TAALSILPGIGSVMGIADGAVHHNTEEIVA QSIALSSLMV AQAIPLVGEL

[0087] VDIGFAAYNF VESIINLFQV VHNSYNRPAY SPGHKTRP [SEQ ID NO:3].

[0088] In some embodiments, the DT fragment is at least 50%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3.

[0089] 3. Method for generating DT-IL3 conjugates

[0090] The DT-IL3 conjugates of this invention can be prepared using standard recombinant DNA techniques or protein synthesis techniques (e.g., by using a peptide synthesizer). For example, nucleic acid molecules encoding the conjugates of this invention can be synthesized using conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that generate complementary overhangs between two consecutive gene fragments, which are then annealed and re-amplified to generate chimeric gene sequences (see, for example, *Current Protocols in Molecular Biology*, eds. Ausubel et al., John Wiley & Sons, 1992).

[0091] The nucleotide sequences encoding the conjugates (IL-3 and diphtheria toxin sequences) of the present invention can be obtained from any information available to those skilled in the art (i.e., from GenBank, literature, or through conventional cloning). The nucleotide sequences encoding the conjugates can be inserted into suitable expression vectors, i.e., vectors containing elements necessary for transcription and translation of the inserted protein-coding sequence. In some cases, the diphtheria toxin sequence can be truncated to remove specific domains, such as targeting domains. Techniques for modifying or truncating DNA are well known to those skilled in the art of molecular biology. Furthermore, the IL-3 and diphtheria toxin sequences can be linked in such a way that the resulting DNA sequence, upon translation, produces a polypeptide that is a compound of the present invention. In some embodiments, a linker sequence is introduced into a recombinant sequence linking the IL-3 and diphtheria toxin sequences. Various host-vector systems can be used to express protein-coding sequences in the present invention. These include, but are not limited to, mammalian cell systems infected with viruses (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with viruses (e.g., baculovirus); microorganisms containing yeast vectors, such as yeast (e.g., Pichia); or bacteria transformed with bacteriophages, DNA, plasmid DNA, or copious DNA (e.g., Escherichia coli). The expression elements of the vectors vary in strength and specificity. Depending on the host-vector system used, any of a number of suitable transcriptional and translational elements can be used. In some embodiments, the protein is expressed in Escherichia coli. In some embodiments, the protein is expressed in Pichia.

[0092] The expression of the conjugates of the present invention can be controlled by any promoter or enhancer element known in the art. In some embodiments, the expression of the conjugates of the present invention is regulated by a constitutive promoter. In another embodiment, expression is regulated by an inducible promoter. In yet another embodiment, expression is regulated by a tissue-specific promoter.

[0093] In some embodiments, a vector is used that includes a promoter operatively linked to a conjugate encoding a nucleic acid, one or more origins of replication, and optionally one or more optional markers (e.g., an antibiotic resistance gene).

[0094] Expression vectors containing an insert gene encoding a conjugate can be identified using three general methods: (a) nucleic acid hybridization, (b) the presence or absence of a “marker” gene function, and (c) expression of the insert sequence. In the first method, the presence of the gene encoding the conjugate in the expression vector can be detected by nucleic acid hybridization using probes comprising sequences homologous to the insert gene encoding the conjugate. In the second method, recombinant vector / host systems can be identified and selected based on the presence or absence of certain “marker” gene functions (e.g., thymidine kinase activity, antibiotic resistance, transformation phenotype, inclusion body formation in baculoviruses, etc.) resulting from the insertion of a nucleotide sequence encoding the conjugate into the vector. For example, if a nucleotide sequence encoding the conjugate is inserted into a marker gene sequence in the vector, the recombinant containing the gene encoding the conjugate insert can be identified by the absence of marker gene function. In the third method, recombinant expression vectors can be identified by measuring the gene product (e.g., the conjugate) expressed by the recombinant. Such measurements can be based on, for example, the physical or functional properties of the conjugate in an in vitro assay system, such as binding to antibodies or IL-3 receptors.

[0095] The recombinant conjugates can be stably expressed for long-term, high-yield production. For example, cell lines stably expressing the conjugates of this invention can be engineered. Host cells can be transformed using DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and optional markers without using expression vectors containing viral origins of replication. After introducing the foreign DNA, engineered cells can be grown in enriched media for 1-2 days, then switched to selective media. Optional markers in the recombinant plasmid confer resistance to selection and allow cells to stably integrate the plasmid into their chromosomes and grow to form lesions, which can then be cloned and expanded into cell lines. This method can be advantageously used for engineering cell lines expressing the conjugates of this invention.

[0096] As mentioned above, DT-IL3 is typically produced by recombinant bacterial, insect, or mammalian cells containing nucleic acids engineered to express conjugate proteins.

[0097] Once the conjugates of the present invention are produced by recombinant expression or chemical synthesis, they can be purified by any method known in the art for purifying proteins, such as chromatography (e.g., ion exchange, affinity (especially affinity of protein A for specific antigens) and fractionation column chromatography), centrifugation, differential dissolution, or any other standard technique for protein purification.

[0098] B.Jak inhibitors

[0099] As used herein, a "Jak inhibitor" is an agent that inhibits one or more Janus kinases (Jak). A Jak inhibitor can be any agent that inhibits one or more Jaks, regardless of whether the agent has other pharmacological effects. Jaks include Jak1, Jak2, Jak3, and Tyk2. In some embodiments, a Jak inhibitor inhibits Jak1 or Jak2. In some embodiments, a Jak inhibitor is a Jak2 / Jak1 inhibitor. In some embodiments, a Jak inhibitor may also inhibit other kinases.

[0100] Jak mediates the signaling of certain cytokines and growth factors that are crucial for hematopoiesis and immune function. In some embodiments, inhibition of one or more Jaks reduces splenomegaly and lowers circulating cytokine levels.

[0101] In some embodiments, one or more Jak inhibitors include ruxotinib, gandotinib, momelotinib, pacritinib, CHZ868, NS-018, SRC, tofacitinib, or itacitinib. In some embodiments, the Jak inhibitor used in some of the methods provided herein is ruxotinib.

[0102] C. Hypomethylating agents

[0103] As used herein, a "hypomethylating agent" is any drug that inhibits DNA methylation (i.e., the addition of methyl groups to DNA nucleotides). A hypomethylating agent can be any agent that inhibits DNA methylation, regardless of whether the agent has other pharmacological effects.

[0104] In some embodiments, the hypomethylating agent blocks the activity of DNA methyltransferases (i.e., the compound is a DNA methyltransferase inhibitor or a DNMT inhibitor). In some embodiments, the hypomethylating agent reduces DNA methylation without causing substantial inhibition of DNA synthesis. In some embodiments, the hypomethylating agent restores normal gene function. In some embodiments, the hypomethylating agent induces rapid cell division death.

[0105] In some embodiments, one or more hypomethylating agents include azacitidine, decitabine, and / or SGI-110.

[0106] D. Administration

[0107] The pharmaceutical agents of this invention can be administered at any clinically relevant dose. Clinically relevant means that the dose of the agent has an effect on the subject. In some embodiments, the combination of pharmaceutical agents disclosed herein allows administration of one or more agents at a dose level lower than the dose at which the agent would be effective when administered as a single agent. For example, treatment using DT-IL3 with one or more Jak inhibitors and / or one or more hypomethylating agents provides a clinically relevant effect that would not be observed when administered as a single agent with the same dose of a Jak inhibitor or hypomethylating agent.

[0108] In some embodiments, one or more agents are administered at the maximum tolerated dose. As used herein, “maximum tolerated dose” refers to the highest dose of an agent that a single patient can tolerate. In other words, side effects for a given patient may determine the maximum tolerated dose. Side effects may limit the ability to administer a therapeutic dose higher than the maximum tolerated dose. Therefore, the maximum tolerated dose for a given patient may be lower than the dose indicated in the treatment prescribing information or the dose commonly used in clinical practice. The maximum tolerated dose may have limited or no clinical efficacy for the patient.

[0109] 1. Exemplary dosage of DT-IL3

[0110] In some embodiments, DT-IL3 is administered at a dose of 1 μg / kg or greater, 2 μg / kg or greater, or 4 μg / kg or greater. In some embodiments, DT-IL3 is administered at a dose of 2 μg / kg to 20 μg / kg or 9 μg / kg to 20 μg / kg. In some embodiments, DT-IL3 is administered at a dose of 4 μg / kg to 12 μg / kg or 9 μg / kg to 12 μg / kg. In some embodiments, DT-IL3 is administered at a dose of 5 μg / kg. In some embodiments, DT-IL3 is administered at a dose of 7 μg / kg. In some embodiments, DT-IL3 is administered at a dose of 9 μg / kg. In some embodiments, DT-IL3 is administered at a dose of 12 μg / kg.

[0111] In some embodiments, the dose of DT-IL3 administered is the maximum tolerated dose.

[0112] 2. Exemplary dosage of Jak inhibitors

[0113] In some embodiments, one or more Jak inhibitors include ruxotinib. In some embodiments, the oral dose of ruxotinib is 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, or 25 mg.

[0114] In some embodiments, the oral dose of gandolinib is 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 30 mg, 60 mg, 90 mg, or 120 mg.

[0115] In some embodiments, the oral dose of molotinib is 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 300 mg, or 400 mg.

[0116] In some embodiments, the oral dose of pactinib is 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 300 mg, or 400 mg.

[0117] In some embodiments, the oral dose of CHZ868 is 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 300 mg, or 400 mg.

[0118] In some embodiments, the oral dose of NS-018 is 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 300 mg, or 400 mg.

[0119] In some embodiments, the oral dose of SRC is 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 300 mg, or 400 mg.

[0120] In some embodiments, the oral dose of tofacitinib is 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, or 30 mg.

[0121] In some embodiments, the oral dose of etatinib is 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 300 mg, or 400 mg.

[0122] In some embodiments, the Jak inhibitor is administered at the maximum tolerated dose.

[0123] 3. Exemplary dosage of hypomethylating agent

[0124] In some embodiments, azacitidine Administered at a dose of 75 mg / m2 or less. In some embodiments, azacitidine is administered by continuous intravenous infusion or subcutaneous injection at doses of 1 mg / m2, 2.5 mg / m2, 5 mg / m2, 10 mg / m2, 15 mg / m2, 20 mg / m2, 25 mg / m2, 37.5 mg / m2, 50 mg / m2, 75 mg / m2, or 100 mg / m2.

[0125] In some embodiments, decitabine Administer at a dose of 45 mg / m² / day or less. In some embodiments, decitabine... Administer at doses of 1 mg / m² / day, 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 33 mg / m² / day, or 45 mg / m² / day. Infusion may be a once-daily continuous intravenous infusion or multiple consecutive intravenous infusions per day.

[0126] In some embodiments, SGI-110 (guadecitabine) is administered subcutaneously at doses of 1 mg / m2, 3 mg / m2, 5 mg / m2, 10 mg / m2, 15 mg / m2, 20 mg / m2, 25 mg / m2, 30 mg / m2, or 60 mg / m2.

[0127] In some embodiments, the dose of the hypomethylating agent is the maximum tolerated dose.

[0128] In some embodiments, subjects are pre-medicated against nausea and vomiting prior to administration of the hypomethylating agent.

[0129] II. Treatment methods

[0130] In some embodiments, DT-IL3 is administered together with (a) one or more Jak inhibitors and / or (b) one or more hypomethylating agents to treat MPN.

[0131] In some embodiments, the treatment method includes administering DT-IL3 and one or more Jak inhibitors to the subject.

[0132] In some embodiments, the treatment method includes administering DT-IL3 and one or more hypomethylating agents to the subject.

[0133] In some embodiments, the treatment method includes administering DT-IL3, one or more Jak inhibitors, and one or more hypomethylating agents to a subject. In some embodiments, at least one Jak inhibitor and at least one hypomethylating agent are administered. In some embodiments, the at least one Jak inhibitor includes ruxotinib, and the at least one hypomethylating agent includes decitabine, azacitidine, and / or SGI-110.

[0134] In some embodiments, the treatment method includes administering DT-IL3 and decitabine to the subject.

[0135] In some embodiments, the treatment method includes administering DT-IL3 and azacitidine to the subject.

[0136] In some embodiments, the treatment method includes administering DT-IL3 and SGI-110 to the subject.

[0137] As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, the term “subject” refers to an animal. In some embodiments, the animal is a mammal, such as a nonprimate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human). In some embodiments, the subject is a human. In some embodiments, the subject is a nonhuman animal, such as a farm animal (e.g., horse, pig, or cow) or a pet (e.g., dog or cat). In some embodiments, the subject is an elderly person. In some embodiments, the subject is an adult. In some embodiments, the subject is a human child. In some embodiments, the subject is a human infant (i.e., a human under 18 years of age). In some embodiments, the subject is a human baby.

[0138] In some embodiments, a subject has an unfavorable cytogenetic profile. Subjects are determined to have a favorable or unfavorable cytogenetic profile based on survival outcomes. An unfavorable cytogenetic profile refers to a cytogenetic risk profile associated with an unfavorable outcome. An unfavorable cytogenetic profile in a subject with MF may include, for example, a complex karyotype (≥3 rearrangements) or one or two abnormalities, including trisomy 8, deletion 7 / 7q, inversion 17q, inversion 3, deletion 5 / 5q, deletion 12p, or 11q23 rearrangement, and a favorable cytogenetic profile includes all other cases, including a normal karyotype.

[0139] In some embodiments, the subject is unresponsive to pretreatment using one or more Jak inhibitors and / or one or more hypomethylating agents. As used herein, a subject is considered “unresponsive” to pretreatment if a patient fails to achieve a response to therapy, thus determining that the therapy is therapeutically ineffective, such as by failing to meet clinical endpoints, including any response, prolonged duration of response, prolonged disease-free status, survival, relapse-free survival, progression-free survival, and overall survival. In some embodiments, the subject is unresponsive to pretreatment using ruxotinib. In some embodiments, the subject who is unresponsive to pretreatment using ruxotinib has myelofibrosis (MF).

[0140] In some embodiments, the subject cannot tolerate the full dose of pretreatment with one or more Jak inhibitors and / or one or more hypomethylating agents. As used herein, “full dose” means one or more doses indicated in the prescribing information and / or a dose commonly used in clinical practice. In some embodiments, the maximum tolerated dose for a given patient is less than the full dose. In other words, the subject cannot tolerate the full dose of pretreatment due to side effects. In some embodiments, the subject cannot tolerate the full dose of pretreatment with ruxotinib. In some embodiments, the subject who cannot tolerate the full dose of pretreatment with ruxotinib has MF.

[0141] In some embodiments, the subject has a low platelet count or is unsuitable for treatment with ruxotinib. In some embodiments, the subject with a low platelet count or unsuitable for treatment with ruxotinib has mycotic leukemia (MF). In some embodiments, a platelet count low is equal to or less than 5 × 10⁻⁶. 9 / L, equal to or less than 10×10 9 / L, equal to or less than 20×10 9 / L, equal to or less than 30×10 9 / L, equal to or less than 40×10 9 / L or equal to or less than 50×10 9 Platelet count is measured in / L. In some embodiments, patients may not be suitable for treatment with ruxolitinib due to kidney or liver damage.

[0142] In some embodiments, the subject has previously responded to a Jak inhibitor and / or a hypomethylating agent. In some embodiments, the subject who has previously responded to a Jak inhibitor and / or a hypomethylating agent has MF. In some embodiments, the subject has previously responded to ruxotinib. Many reasons for failure of previous treatment for MF have been characterized, such as myelosuppression, resistance and the persistence of potentially malignant clones (see Pettit and Odenike, Curr Hematol Malig Rep., 12(6):611-624(2017)).

[0143] A. Myeloproliferative neoplasm

[0144] In some embodiments, MPN refers to polycythemia vera (PV), essential thrombocythemia (ET), myelofibrosis (MF), chronic myeloid monocytic leukemia (CMML), chronic neutrophilic leukemia, chronic eosinophilic leukemia, systemic mastocytosis (SM), symptomatic hypereosinophilic syndrome, or other bone marrow disorders that cause excessive production of red blood cells, white blood cells, and / or platelets. In some embodiments, MPN refers to primary eosinophilic syndrome (PED).

[0145] In some embodiments, MPN stands for myelofibrosis (MF). MF is characterized by the replacement of bone marrow with scar tissue. Due to bone marrow scarring, the number of blood cells produced may be insufficient, potentially leading to anemia, bleeding problems, and increased risk of infection. The liver and spleen may enlarge in an attempt to produce additional blood cells.

[0146] In some embodiments, MF is primary MF (PMF), post-PV MF, post-ET MF, primary blast crisis primary MF (PMF-BP), post-PV MF, or post-ET MF.

[0147] In some embodiments, the MPN is in a rapid change phase.

[0148] In some embodiments, MPN is PMF-BP, MF after PV in the blast phase, or MF after ET in the blast phase.

[0149] B. Treatment and identification of subjects with MPN

[0150] In some embodiments, a method for treating an MPN in a subject in need includes identifying the MPN and administering an effective amount of DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents to the subject in need.

[0151] In some embodiments, the MPN is identified by physical examination, blood tests, bone marrow aspiration and biopsy, cytogenetic analysis, testing for mutations in the JAK2, MPL, ASXL1, TET2, or CALR genes, arterial oxygen saturation and carboxyhemoglobin levels, neutrophil alkaline phosphatase levels, vitamin B12 or B12 binding capacity, or serum urate. In some embodiments, the mutation in the JAK2 gene is the JAK2V617F mutation.

[0152] In some embodiments, the patient has a mutation in the JAK2, MPL, ASXL1, TET2, or CALR gene. In some embodiments, the patient has a JAK2V617F mutation.

[0153] C. Methods for measuring clinical efficacy

[0154] In some embodiments, inhibition of MPN results in reduced MPN cell proliferation, stable MPN cell numbers, and / or a decrease in the number of MPN cells. As used herein, "MPN cell" refers to any abnormal bone marrow cell that causes MPN.

[0155] In some embodiments, inhibition of MPN reduces the size of the spleen and / or liver. In some embodiments, spleen size is measured by MRI or CT at different time points before and after treatment. In some embodiments, subjects with MPN have an abnormally large spleen (i.e., splenomegaly) or an abnormally large liver. In some embodiments, splenomegaly is defined by physical examination as ≥5 cm below the costal margin (BCM).

[0156] In some embodiments, subjects with MPN do not have an abnormally large spleen or liver. In some embodiments, subjects with MPN do not have baseline splenomegaly (≤5 cm BCM).

[0157] In some embodiments, a reduction in spleen size is a reduction of at least 25%, at least 29%, at least 33%, at least 35%, at least 40%, or at least 50%. In some embodiments, a reduction in spleen size is a reduction of ≥33% in the spleen size of a subject with baseline splenomegaly. Subjects who meet the parameters for spleen size reduction are treatment “spleen responders.”

[0158] In some embodiments, clinical efficacy is measured by improvement in splenomegaly. In some embodiments, improvement in splenomegaly in a group of subjects is measured by the percentage of spleen responders.

[0159] In some embodiments, improvement in splenomegaly is measured by a percentage change in the amount of splenomegaly palpable below the left costal margin.

[0160] In some embodiments, the subject has MF. Response criteria for MF include, but are not limited to, the exemplary response criteria in Table 2 below from the International Working Group for MF - MRT and ELN (ELN for MF).

[0161] Table 2

[0162]

[0163]

[0164]

[0165] Adapted from Tefferi et al., Blood 122(6):1395-98 (2013). In Table 2, CR = complete response or remission, PR = partial response or remission, EMH = extramedullary hematopoiesis, LCM = left costal margin, and UNL = upper limit of normal. *Baseline and post-treatment bone marrow sections will be interpreted once through a central review process. CR allocation does not require cytogenetic and molecular responses. The classification of MF is based on the European classification (Thiele et al., Haematologica 90:1128 (2005)). Immature myeloid cells consist of blastocytes + promyelocytes + myelocytes + metamyelocytes + nucleated erythrocytes. In splenectomized patients, <5% of immature myeloid cells are permissible. § Definitions of anemia response, splenic response, and progressive disease are given above. Increased severity of anemia includes the occurrence of new transfusion dependence or a decrease in hemoglobin level from pretreatment baseline ≥20 g / L lasting at least 12 weeks. According to the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0, an increase in the severity of thrombocytopenia or neutropenia is defined as a grade 2 decrease in platelet count or absolute neutrophil count from pretreatment baseline. Additionally, allocation to CI requires a minimum platelet count ≥25000 × 10⁹ / L and an absolute neutrophil count ≥0.5 × 10⁹ / L. || Only applicable to patients with baseline hemoglobin <100 g / L. For patients who do not meet the strict criteria for transfusion dependence at the time of study registration but have received a transfusion within the previous month, the pre-transfusion hemoglobin level should be used as the baseline. Pre-enrollment transfusion dependence is defined as a transfusion of at least 6 units of PRBCs within 12 weeks prior to enrollment, without bleeding or treatment-induced anemia, for a hemoglobin level <85 g / L. Additionally, the most recent transfusion event must have occurred within 28 days prior to enrollment. Response in transfusion-dependent patients requires no PRBC transfusions during any consecutive 12-week "rolling" intervals in the treatment phase, with a hemoglobin level ≥85 g / L as the upper limit. #In patients who have undergone splenectomy, palpable hepatomegaly is substituted using the same measurement strategy. **Splenic or hepatic response must be confirmed by imaging studies, requiring a spleen volume reduction of ≥35% as assessed by MRI or CT. Furthermore, a spleen or liver volume reduction of ≥35% by MRI or CT constitutes a response regardless of the physical examination report. Symptoms are assessed using the MPN-SAF TSS.17. The MPN-SAF TSS is self-assessed by the patient and includes fatigue, concentration, early satiety, inactivity, night sweats, itching, bone pain, abdominal discomfort, weight loss, and fever. Each item is scored from 0 (absent / excellent) to 10 (worst imaginable / very poor). The MPN-SAF TSS is the sum of all individual scores (0-100). Symptom response requires a reduction of ≥50% in the MPN-SAF TSS. Disease-assigned classification for splenomegaly requires confirmation of a ≥25% increase in spleen volume from baseline on MRI or computed tomography. Baseline values ​​for physical examinations and imaging studies refer to pre-treatment baselines, not post-treatment measurements.

[0166] III. Drug Application

[0167] These methods include multiple administration regimens of DT-IL3 and one or more Jak inhibitors and / or hypomethylating agents.

[0168] In some embodiments, DT-IL3 is applied at least once a week. In some embodiments, DT-IL3 is applied at least twice a week. In some embodiments, DT-IL3 is applied at least three times a week.

[0169] In some embodiments, one or more Jak inhibitors are administered at least once a week. In some embodiments, one or more Jak inhibitors are administered at least twice a week. In some embodiments, one or more Jak inhibitors are administered at least three times a week. In some embodiments, one or more Jak inhibitors are administered every other day. In some embodiments, one or more Jak inhibitors are administered daily. In some embodiments, one or more Jak inhibitors are administered more than once a day. In some embodiments, one or more Jak inhibitors are administered twice a day.

[0170] In some embodiments, one or more hypomethylating agents are applied at least once a week. In some embodiments, one or more hypomethylating agents are applied at least twice a week. In some embodiments, one or more hypomethylating agents are applied at least three times a week. In some embodiments, one or more hypomethylating agents are applied daily. In some embodiments, one or more hypomethylating agents are applied twice a day. In some embodiments, one or more hypomethylating agents are applied three times a day. In some embodiments, one or more hypomethylating agents are applied daily for a period of time, followed by a period of non-application of the one or more hypomethylating agents. In some embodiments, one or more hypomethylating agents are applied daily for a period of up to 7 days.

[0171] In some embodiments, DT-IL3 and one or more Jak inhibitors (a) and / or one or more hypomethylating agents (b) are applied over a period of one week or longer. In some embodiments, DT-IL3 and one or more Jak inhibitors (a) and / or one or more hypomethylating agents (b) are applied over a period of two weeks or longer. In some embodiments, DT-IL3 and one or more Jak inhibitors (a) and / or one or more hypomethylating agents (b) are applied for at least three consecutive days.

[0172] A. Treatment cycle

[0173] In some embodiments, the DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents are administered over multiple treatment cycles.

[0174] As used herein, a “treatment cycle” refers to a period of time during which DT-IL3 and (a) one or more Jak inhibitors and / or (b) one or more hypomethylating agents are administered. In some embodiments, there is a single treatment cycle. In some embodiments, there are multiple treatment cycles.

[0175] In some embodiments, when DT-IL3 and (a) one or more Jak inhibitors and / or (b) one or more hypomethylating agents are not administered, there are time intervals between multiple treatment cycles. In some embodiments, the treatment cycles are spaced at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, or a combination thereof.

[0176] In some embodiments, the DT-IL3 is administered for at least 3 consecutive days every 21 days over four cycles, followed by administration every 28 days over three cycles, and then every 42 days. In some embodiments, one or more Jak inhibitors and / or one or more hypomethylating agents are administered concurrently with the DT-IL3.

[0177] In some embodiments, one or more Jak inhibitors and / or one or more hypomethylating agents are administered at least once or twice daily.

[0178] In some embodiments, one or more Jak inhibitors and / or one or more hypomethylating agents are administered for at least the first 3 days, at least the first 4 days, at least the first 5 days, at least the 6th day, or at least the 7th day of at least one cycle.

[0179] In some embodiments, one or more Jak inhibitors and / or one or more hypomethylating agents are administered at least for the first 3, 4, 5, 6, or 7 days of three cycles of 28-day intervals. In some embodiments, DT-IL3 is administered concurrently with one or more Jak inhibitors and / or one or more hypomethylating agents. In some embodiments, one or more Jak inhibitors and / or one or more hypomethylating agents are administered after four 21-day cycles of DT-IL3 administration.

[0180] In some embodiments, DT-IL3 and (a) one or more Jak inhibitors and / or (b) one or more hypomethylating agents are administered until, for example, disease progression and / or unacceptable toxicity are determined by the treating physician.

[0181] IV. Pharmaceutical Compositions

[0182] In some embodiments, a pharmaceutical composition for treating or inhibiting myeloproliferative neoplasms (MPN) in a subject of need comprises an effective amount of DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents. In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0183] In some embodiments, the present invention provides a pharmaceutical composition comprising an effective amount of the conjugate of the present invention and a pharmaceutically acceptable carrier or mediator. In some embodiments, the pharmaceutical composition comprises an effective amount of the conjugate of the present invention and a pharmaceutically acceptable carrier or mediator. The pharmaceutical composition is suitable for administration to animals and / or humans.

[0184] The pharmaceutical compositions of the present invention may be in any form permissible for administration to a subject.

[0185] The materials used to prepare the pharmaceutical composition may be non-toxic in the amounts used. It will be apparent to those skilled in the art that the optimal dosage of one or more active ingredients in the pharmaceutical composition will depend on a variety of factors. These factors include, but are not limited to, the type of subject (e.g., human), the subject's overall health level, the type of cancer the subject has, the purpose of the composition as part of a multi-drug regimen, the specific form of the composition of the invention, and the method of administration.

[0186] The term "carrier" refers to a diluent, adjuvant, or excipient administered with the compositions of the present invention. Any adjuvant, stabilizer, thickener, lubricant, and colorant may be used. In one embodiment, the compositions of the present invention and the pharmaceutically acceptable carrier are sterile when administered to a subject. When the compositions of the present invention are administered intravenously, water may be used as a carrier. Saline solutions and aqueous solutions of dextran and glycerol may also be used as liquid carriers, particularly for injectable solutions. If desired, the compositions of the present invention may also contain a small amount of pH buffer.

[0187] The liquid compositions of the present invention, whether in solution, suspension, or other similar form, may also contain one or more of the following: sterile diluents such as water for injection, saline solution, physiological saline, Ringer's solution, isotonic sodium chloride, etc.; fixing oils such as synthetic mono- or diglycerides of glycerol, polyethylene glycol, glycerol, cyclodextrin, propylene glycol, or other solvents that can be used as solvents or suspension culture media; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for regulating tension such as sodium chloride or dextrose. The parenteral compositions may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass, plastic, or other materials. In some embodiments, physiological saline is an adjuvant. The injectable compositions may be sterile.

[0188] The pharmaceutical composition comprises an effective amount of the composition of the present invention to obtain an appropriate dosage. Typically, this amount is at least 0.01% of the composition of the present invention by weight. When intended for oral administration, this amount can vary between 0.1% by weight and 80% by weight of the pharmaceutical composition. An oral pharmaceutical composition may comprise between 4% and 50% of the composition of the present invention by weight. Pharmaceutical compositions can be prepared such that a parenteral dose unit contains between 0.01% by weight and 2% by weight of the composition of the present invention.

[0189] The pharmaceutical composition can be administered via any convenient route (e.g., by infusion or rapid concentration, absorption through the epithelial or mucosal layers of the skin (e.g., oral mucosa, rectal and intestinal mucosa, etc.)). Administration can be systemic or local. Various delivery systems are known, such as microparticles, microcapsules, capsules, etc., and can be used to administer the compositions of the present invention. Administration methods may include, but are not limited to, oral and parenteral administration; parenteral administration includes, but is not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous; intranasal, epidural, sublingual, intranasal, intracerebral, intraventricular, intrathecal, intravaginal, intravaginal, vaginal, percutaneous, transrectal, by inhalation, or local application to the ear, nose, eye, or skin. The method of administration is determined by the physician and depends in part on the site of the medical condition (e.g., the site of cancer, cancerous tumor, or precancerous lesions).

[0190] In some embodiments, the compositions of the present invention are administered parenterally. In some embodiments, the compositions of the present invention are administered intravenously. In another embodiment, the compositions of the present invention are administered by continuous infusion. In specific embodiments, the compositions of the present invention are administered by infusion over a period of 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, or 2 hours.

[0191] In some embodiments, it may be desirable to apply one or more compositions of the present invention topically to the area requiring treatment. This can be achieved, for example, but not limited to, local infusion during surgery; local application, such as in conjunction with postoperative wound dressings; by injection; by catheter; by suppository; or by implantation, said implant being a porous, non-porous, or gel material comprising a membrane (such as a silicone rubber membrane) or fiber. In some embodiments, one or more compositions of the present invention may be injected intraperitoneally.

[0192] In yet another embodiment, the composition of the present invention can be delivered in a controlled release system.

[0193] In some embodiments, the pump may be used to deliver the compositions of the present invention (see, for example, Sefton, CRC Crit. Ref. Biomed. Eng. 1987, 14, 201; Buchwald et al., Surgery 1980, 88: 507; Saudek et al., New England Journal of Medicine 1989, 321: 574). In some embodiments, the pump may be, but is not limited to, an insulin pump.

[0194] The compositions of this invention can be in the form of solutions, suspensions, tablets, pills, granules, capsules, liquid-containing capsules, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other suitable form. Examples of suitable drug carriers are described in EWMartin's Remington's Pharmaceutical Sciences.

[0195] In some embodiments, the compositions of the invention are formulated according to conventional procedures to be pharmaceutical compositions suitable for intravenous administration to animals, particularly humans. Typically, the carrier or mediator for intravenous administration is a sterile isotonic buffered aqueous solution. If necessary, the composition may also contain a solubilizer. The composition for intravenous administration may optionally include a local anesthetic, such as lidocaine for relieving injection site pain. Generally, the components are provided separately or mixed together in unit dosage forms, for example, as a dry lyophilized powder or anhydrous concentrate in an hermetically sealed container, such as an ampoule or capsule indicating the amount of active agent. When the compositions of the invention are administered by infusion, they may be dispensed, for example, using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the compositions of the invention are administered by injection, ampoules of sterile water for injection or saline may be provided so that the components can be mixed prior to administration.

[0196] The compositions of the present invention may include, but are not limited to, additional active agents selected from, but not limited to, additional prophylactic agents, additional therapeutic agents, antiemetics, hematopoietic colony-stimulating factors, adjunctive therapies, vaccines or other immunostimulants, antibody / antibody fragment-based agents, antisuppressants, and analgesics. In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier or mediator.

[0197] Pharmaceutical compositions can be prepared using methods well-known in the pharmaceutical field. For example, a composition intended for injection can be prepared by combining the composition of the present invention with water to form a solution. Surfactants can be added to promote the formation of a homogeneous solution or suspension. Surfactants are complexes that can non-covalently interact with the composition of the present invention to promote the dissolution or homogeneous suspension of the composition of the present invention in an aqueous delivery system.

[0198] Example

[0199] Example 1. In vitro testing of DT-IL3 and hypomethylating agents

[0200] Preclinical studies were conducted using human leukemia cell lines and primary mononuclear cells from peripheral blood of CMML patients. To determine the activity concentrations of the hypomethylating agent (HMA) azacitidine (AZA) and DT-IL3 (SL-401) in vitro, cells were exposed to increased concentrations of AZA, DT-IL3, or combinations thereof, and cell viability was measured. In the CD123-expressing K562 cell line, the IC50 values ​​for AZA and DT-IL3 were 772 μM and 4.8 nM, respectively. Figures 2A-2C ). Figures 2A-2C This indicates that, compared with AZA alone, AZA and DT-IL3 significantly reduced K562 cell viability.

[0201] Next, cell viability was determined in vitro using fresh monocytes from peripheral blood of CMML patients exposed to increased concentrations of AZA and DT-IL3. The IC50 of AZA alone was 1442 μM, while the IC50 of the combination of AZA and DT-IL3 (at a concentration of 4.8 nM) was 725 μM. Figure 3A Compared to 1 μM AZA alone, the addition of 4.8 nMDT-IL3 to 1 μM AZA in this assay significantly reduced cell viability. Figure 3B (p<0.05).

[0202] In a colony formation assay using fresh mononuclear cells from peripheral blood of CMML patients, the combination of AZA and DT-IL3 significantly reduced colony formation compared to AZA alone. Figures 4A-4C (p<0.05). Similarly, compared with DT-IL3 alone, the addition of AZA to increasing concentrations of DT-IL3 significantly reduced colony formation. Figure 4D (p<0.05). Importantly, a combinatorial effect was observed in patient samples with high-risk genotypes, including those with NRAS / ASXL1 / TET2 mutations ( Figure 4C Mutations in SRSF2 / TET2 / STAG2 ( Figure 4A Those.

[0203] Currently, treatment options for CMML are limited, and its treatment remains a significant unmet medical need. While HMA therapy has been shown to be effective in CMML, its scope and duration of action are often limited. Preclinical data (including data from primary CMML samples) demonstrate the potential therapeutic role of the combination of HMA and DT-IL3 in CMML. See also Figures 2A-2C , Figures 3A-3B and Figures 4A-4D .

[0204] Example 2. Clinical trials of patients who have never received treatment

[0205] Clinical trials can evaluate the efficacy of DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents in treatment-naïve MPN patients. "Treatment-naïve" means that these patients have not previously received treatment for MPN. For example, these patients have not received prior treatment using Jak inhibitors or hypomethylating agents.

[0206] Patients in the trial may have a range of different types of MPN. Alternatively, all patients in the trial may have myelofibrosis.

[0207] Based on physical examination, blood tests, bone marrow aspiration and biopsy, cytogenetic analysis, testing for mutations in the JAK2, MPL, ASXL1, TET2, or CALR genes, arterial oxygen saturation and carboxyhemoglobin levels, neutrophil alkaline phosphatase levels, vitamin B12 or B12 binding capacity, or serum urate levels, a patient may be diagnosed with MPN.

[0208] In some embodiments, the patient has a mutation in the JAK2, MPL, ASXL1, TET2, or CALR gene. In some embodiments, the patient has a JAK2V617F mutation.

[0209] Trials can have adaptive designs. In other words, trials can allow for modifications to the trial and / or procedures during the trial period. Alternatively, a treatment group may be established that is maintained throughout the trial.

[0210] The treatment group may include comparisons of DT-IL3 with combinations of DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents.

[0211] The treatment group may include a comparison of one or more Jak inhibitors with a combination of DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents.

[0212] Treatment groups may include comparisons of one or more hypomethylating agents with DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents.

[0213] Using adaptive design, patients who have failed treatment with one or more hypomethylating agents or one or more Jak inhibitors can be treated with a combination of DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents.

[0214] Treatment success or failure can be assessed using standard measures employed in MPN trials, such as reduction in spleen and / or liver size. Changes in blood components, disease relapse, and patient survival will also be assessed during treatment. Toxicity and adverse events will be measured during treatment.

[0215] Any dosage and treatment cycle described in this application can be used. DT-IL3 can be DT... 388 IL-3. Jak inhibitors may include ruxotinib. One or more hypomethylating agents may be azacitidine, decitabine, and / or SGI-110.

[0216] The applicant hopes that patients who have undergone treatment will see clinical improvement.

[0217] Example 3. Clinical trials of treatment-resistant patients

[0218] Clinical trials can evaluate the efficacy of DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents in MPN patients who are unresponsive to or cannot tolerate full doses of prior treatment. Prior treatment can be one or more hypomethylating agents or one or more Jak inhibitors. Ruxotinib can be a form of prior treatment.

[0219] Clinical trials for treatment-resistant patients may not include a treatment group where the patient had received prior treatment that was unresponsive. Clinical trials for treatment-resistant patients may compare the efficacy of DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents to the efficacy of agents that the patient had not previously treated with. For example, a trial for patients unresponsive to ruxotinib may include a treatment group with hypomethylating agents and a treatment group with DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents.

[0220] Apart from patient selection, clinical trials for treatment-resistant patients can have similar or identical parameters to those for patients who have never received treatment.

[0221] The applicant hopes that patients who have undergone treatment will see clinical improvement.

[0222] Equivalent form

[0223] The foregoing written description is considered sufficient to enable those skilled in the art to practice the embodiments. The foregoing description and examples detail certain embodiments and describe the best mode conceived by the inventors. However, it should be understood that, however detailed the foregoing may appear in the text, the embodiments can be practiced in many ways and should be interpreted in accordance with the appended claims and any of their equivalents.

[0224] As used herein, all numerical values ​​are assumed to be “about”. As used herein, the term “about” refers to numerical values, including, for example, integers, fractions, and percentages, whether explicitly stated or not. The term “about” generally refers to a range of numerical values ​​(e.g., + / - 5% to 10% of the listed range) that would be considered equivalent to the listed values ​​(e.g., having the same function or result) by one of ordinary skill in the art. When terms such as “at least” and “about” precede a list of numerical values ​​or ranges, these terms modify all values ​​or ranges provided in the list. In some cases, the term “about” may include numerical values ​​rounded to the nearest significant figure.

[0225] This disclosure relates to the following implementation plan.

[0226] 1. A method for treating or inhibiting myeloproliferative neoplasm (MPN) in a subject of need, the method comprising administering diphtheria toxin-human interleukin-3 conjugate (DT-IL3) to the subject and

[0227] a. One or more Jak inhibitors and / or

[0228] b. One or more hypomethylating agents.

[0229] 2. The method according to embodiment 1, wherein the DT-IL3 is administered at a dose of 1 μg / kg or greater, 2 μg / kg or greater, or 4 μg / kg or greater.

[0230] 3. The method according to embodiment 1, wherein the DT-IL3 is administered at a dose of 2 μg / kg to 20 μg / kg or 9 μg / kg to 20 μg / kg.

[0231] 4. The method according to embodiment 3, wherein the DT-IL3 is administered at a dose of 4 μg / kg to 12 μg / kg or 9 μg / kg to 12 μg / kg.

[0232] 5. The method according to any one of embodiments 1 to 4, wherein the DT-IL3 is administered at a dose of 5 μg / kg.

[0233] 6. The method according to any one of embodiments 1 to 4, wherein the DT-IL3 is administered at a dose of 7 μg / kg.

[0234] 7. The method according to any one of embodiments 1 to 4, wherein the DT-IL3 is administered at a dose of 9 μg / kg.

[0235] 8. The method according to any one of embodiments 1 to 4, wherein the DT-IL3 is administered at a dose of 12 μg / kg.

[0236] 9. The method according to embodiment 1 or embodiment 2, wherein the dose of DT-IL3 administered is the maximum tolerated dose.

[0237] 10. The method according to any one of embodiments 1 to 9, wherein the DT-IL3 and one or more Jak inhibitors and / or hypomethylating agents are applied at least once a week.

[0238] 11. The method according to embodiment 10, wherein the DT-IL3 and one or more Jak inhibitors and / or hypomethylating agents are applied at least twice a week.

[0239] 12. The method according to embodiment 11, wherein the DT-IL3 and one or more Jak inhibitors and / or hypomethylating agents are applied at least three times a week.

[0240] 13. The method according to any one of embodiments 1 to 12, wherein the DT-IL3 and the (a) one or more Jak inhibitors and / or the (b) one or more hypomethylating agents are applied over a period of one week or longer.

[0241] 14. The method according to embodiment 13, wherein the DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents are administered over a period of two weeks or longer.

[0242] 15. The method according to any one of embodiments 1 to 14, wherein the DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents are administered for at least 3 consecutive days.

[0243] 16. The method according to any one of embodiments 1 to 15, wherein the DT-IL3 and the (a) one or more Jak inhibitors and / or the (b) one or more hypomethylating agents are administered during multiple treatment cycles.

[0244] 17. The method according to embodiment 16, wherein the treatment cycles are spaced at least 1 week apart, at least 2 weeks apart, at least 3 weeks apart, at least 4 weeks apart, at least 5 weeks apart, or a combination thereof.

[0245] 18. The method according to embodiment 16 or embodiment 17, wherein the DT-IL3 is applied for at least 3 consecutive days every 21 days in four cycles, then every 28 days in three cycles, and then every 42 days.

[0246] 19. The method according to any one of embodiments 16 to 18, wherein one or more Jak inhibitors are administered at least once a day or at least twice a day.

[0247] 20. The method according to any one of embodiments 16 to 19, wherein the one or more hypomethylating agents are applied at least 3 days, at least 4 days, at least 5 days, at least 6 days or at least 7 days in at least one cycle.

[0248] 21. The method according to embodiment 20, wherein after four 21-day cycles of DT-IL3 application, the one or more hypomethylating agents are applied for at least the first 3 days, at least the first 4 days, at least the first 5 days, at least the first 6 days, or at least the first 7 days of a 28-day cycle, for a total of 3 cycles.

[0249] 22. The method according to any one of embodiments 1 to 21, wherein the DT-IL3 and one or more Jak inhibitors (a) and / or one or more hypomethylating agents (b) are administered until disease progression and / or unacceptable toxicity is achieved.

[0250] 23. The method according to any one of embodiments 1 to 22, wherein the subject is administered a pharmaceutical composition comprising the DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents as described in (a) or (b).

[0251] 24. The method according to any one of embodiments 1 to 23, wherein the subject is a human being.

[0252] 25. The method according to embodiment 24, wherein the person has adverse cytogenetics.

[0253] 26. The method according to any one of embodiments 1 to 25, wherein the DT-IL3 is a chemical conjugate.

[0254] 27. The method according to any one of embodiments 1 to 26, wherein the DT-IL3 is a recombinant expressed protein.

[0255] 28. The method according to embodiment 27, wherein the DT-IL3 is expressed as a single polypeptide comprising the catalytic domain and the translocation domain of diphtheria toxin and human IL-3.

[0256] 29. The method according to embodiment 28, wherein the DT-IL3 comprises amino acid residues 1 to 388 of diphtheria toxin linked to human IL-3 via peptide bonds.

[0257] 30. The method according to any one of embodiments 1 to 29, wherein inhibition reduces MPN cell proliferation, stabilizes the number of MPN cells, and / or reduces the number of MPN cells.

[0258] 31. The method according to any one of embodiments 1 to 30, wherein the inhibition reduces the size of the spleen and / or liver.

[0259] 32. The method according to any one of embodiments 1 to 31, wherein the MPN is polycythemia vera, essential thrombocythemia, myelofibrosis, chronic myeloid monocytic leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, systemic mastocytosis, symptomatic hypereosinophilic syndrome, or other bone marrow disorders that cause excessive production of red blood cells, white blood cells, and / or platelets.

[0260] 33. The method according to embodiment 32, wherein the MPN is myelofibrosis (MF).

[0261] 34. The method according to implementation scheme 33, wherein the myelofibrosis is primary myelofibrosis, post-polycythemia vera myelofibrosis, post-essential thrombocythemia vera myelofibrosis, blast crisis primary myelofibrosis, blast crisis post-polycythemia vera myelofibrosis, or blast crisis post-ET myelofibrosis.

[0262] 35. The method according to any one of embodiments 1 to 34, wherein the MPN is in a rapid change phase.

[0263] 36. The method according to embodiment 35, wherein the MPN is primary myelofibrosis in the blast crisis, myelofibrosis after polycythemia vera in the blast crisis, or myelofibrosis after ET in the blast crisis.

[0264] 37. The method according to any one of embodiments 1 to 36, wherein the one or more Jak inhibitors include ruxolitinib.

[0265] 38. The method according to any one of embodiments 1 to 37, wherein the one or more hypomethylating agents include azacitidine, decitabine, and / or SGI-110.

[0266] 39. The method according to any one of embodiments 1 to 38, wherein at least one Jak inhibitor and at least one hypomethylating agent are applied.

[0267] 40. The method according to embodiment 39, wherein the at least one Jak inhibitor comprises ruxotinib, and the at least one hypomethylating agent comprises decitabine, azacitidine, and / or SGI-110.

[0268] 41. The method according to any one of embodiments 1 to 40, wherein the subject is unresponsive to pretreatment with one or more Jak inhibitors and / or one or more hypomethylating agents.

[0269] 42. The method according to embodiment 41, wherein the subject is unresponsive to prior treatment with the Jak inhibitor ruxotinib.

[0270] 43. The method according to embodiment 42, wherein the subject suffers from MF.

[0271] 44. The method according to any one of embodiments 1 to 43, wherein the subject cannot tolerate a full dose of pretreatment with one or more Jak inhibitors and / or one or more hypomethylating agents.

[0272] 45. The method according to embodiment 44, wherein the subject cannot tolerate the full dose of pretreatment with the Jak inhibitor ruxotinib.

[0273] 46. ​​The method according to embodiment 45, wherein the subject suffers from MF.

[0274] 47. The method according to any one of embodiments 1 to 46, wherein the subject has a low platelet count or is unsuitable for treatment with ruxolitinib.

[0275] 48. The method according to embodiment 47, wherein the subject suffers from MF.

[0276] 49. The method according to any one of embodiments 1 to 48, wherein the subject has previously responded to a Jak inhibitor and / or a hypomethylating agent.

[0277] 50. The method according to embodiment 49, wherein the subject suffers from MF.

[0278] 51. The method according to any one of embodiments 1 to 50, wherein the subject has a mutation in the JAK2, MPL, ASXL1, TET2 or CALR gene.

[0279] 52. The method according to any one of embodiments 1 to 51, wherein the subject has the JAK2V617F mutation.

[0280] 53. A pharmaceutical composition for treating or inhibiting myeloproliferative neoplasms (MPN) in a subject of need, said pharmaceutical composition comprising an effective amount of diphtheria toxin-human interleukin-3 conjugate (DT-IL3) and one or more Jak inhibitors and / or one or more hypomethylating agents.

[0281] 54. The pharmaceutical composition according to embodiment 53, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0282] 55. A method for treating a subject with myeloproliferative neoplasm (MPN), the method comprising:

[0283] a. Identify the MPN, and

[0284] b. Administer to the subject in need an effective amount of diphtheria toxin-human interleukin-3 conjugate (DT-IL3) and one or more Jak inhibitors and / or one or more hypomethylating agents.

[0285] 56. The method according to embodiment 55, wherein the MPN is identified by physical examination, blood tests, bone marrow aspiration and biopsy, cytogenetic analysis, testing for mutations in the JAK2, MPL, ASXL1, TET2 or CALR genes, arterial blood oxygen saturation and carboxyhemoglobin levels, neutrophil alkaline phosphatase levels, vitamin B12 or B12 binding capacity or serum urate.

[0286] 57. The method according to embodiment 56, wherein the MPN is identified by testing for the JAK2V617F mutation.

Claims

1. A method for treating or inhibiting myeloproliferative neoplasm (MPN) in a subject of need, the method comprising administering diphtheria toxin-human interleukin-3 conjugate (DT-IL3) to the subject and a. One or more Jak inhibitors and / or b. One or more hypomethylating agents.

2. The method of claim 1, wherein the DT-IL3 is administered at a dose of 1 μg / kg or greater, 2 μg / kg or greater, or 4 μg / kg or greater.

3. The method of claim 1, wherein the DT-IL3 is administered at a dose of 2 μg / kg to 20 μg / kg or 9 μg / kg to 20 μg / kg.

4. The method of claim 3, wherein the DT-IL3 is administered at a dose of 4 μg / kg to 12 μg / kg or 9 μg / kg to 12 μg / kg.

5. The method according to any one of claims 1 to 4, wherein the DT-IL3 is administered at a dose of 5 μg / kg.

6. The method according to any one of claims 1 to 4, wherein the DT-IL3 is administered at a dose of 7 μg / kg.

7. The method according to any one of claims 1 to 4, wherein the DT-IL3 is administered at a dose of 9 μg / kg.

8. The method according to any one of claims 1 to 4, wherein the DT-IL3 is administered at a dose of 12 μg / kg.

9. The method according to claim 1 or claim 2, wherein the dose of DT-IL3 administered is the maximum tolerated dose.

10. The method according to any one of claims 1 to 9, wherein the DT-IL3 and one or more Jak inhibitors and / or hypomethylating agents are administered at least once a week.

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