Combination therapy method of treating myeloproliferative neoplasm with diphtheria toxin-human interleukin-3 conjugate in combination with other agents

Abstract

To provide improved methods for treating a myeloproliferative neoplasm (MPN).SOLUTION: The present invention provides methods for treating or inhibiting a myeloproliferative neoplasm (MPN) in a subject in need thereof, 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.SELECTED DRAWING: Figure 3B

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This international application is a continuation of U.S. Provisional Application No. 62 / 753,145, filed October 31, 2018, and and the benefit of priority to U.S. Provisional Application No. 62 / 832,558, filed April 11, 2019. No. 6,399,423, filed on Dec. 13, 2006, the contents of which are hereby incorporated by reference in their entirety.

[0002] Sequence Listing This application is submitted in electronic format along with a sequence listing. The sequence listing will be filed on October 21, 2019. Created "2019-10-21-01214-001" with size 12,288 bytes The file is provided as "7-00PCT_SEQ_List_ST25.txt". The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. .

[0003] explanation Field The present invention relates to a method for treating or inhibiting myeloproliferative neoplasms (MPNs) in a subject in need of such treatment. A method for treating or inhibiting a MPN is provided, the method comprising administering to a subject , diphtheria toxin-human interleukin-3 conjugate (DT-IL3), and one or more The present invention relates to a method for treating cancer, comprising administering one or more Jak inhibitors and / or one or more hypomethylating agents. [Background technology]

[0004] Myeloproliferative neoplasms (MPNs), also known as myeloproliferative disorders (MPDs), are myeloproliferative disorders caused by myeloid cells. MPNs are blood disorders characterized by the overproduction of one or more hematopoiesis proteins in the bone marrow. It is also characterized by clonal expansion of cell lineages. In some cases, genetic mutations (e.g., Jak 2 protein mutations) may be present in stem cells from MPN patients.

[0005] MPNs can lead to an increase in certain blood cells, an increase in abnormal blood cells, and an enlarged spleen (splenomegaly). MPNs can damage the bone marrow, causing fibrosis of the bone marrow (myelofibrosis or MF). Changes in blood counts in MPNs can lead to anemia, fatigue, and weakness. Thrombotic and hemorrhagic complications, such as thrombosis, are also a risk for MPN. This can lead to the development of further conditions such as acute myeloid leukemia.

[0006] The present application relates to a diphtheria toxin-human interleukin-3 conjugate (DT-IL3) and Treatment of MPNs with one or more Jak inhibitors and / or one or more hypomethylating agents This paper describes an improved method for Summary of the Invention

[0007] According to the present specification, the present application relates to a method for the treatment of a DT-IL3 infection comprising administering to a subject a therapeutically effective amount of at least one of a DT-IL3 inhibitor and a therapeutically effective amount of at least one of a Jak ... and / or (b) describing methods of treatment with one or more hypomethylating agents.

[0008] In one embodiment, the present application relates to a method for treating or inhibiting myeloproliferative neoplasms (MPNs). and a method for treating or inhibiting said myeloproliferative neoplasm (MPN) in a subject in need thereof. The subject is administered a diphtheria toxin-human interleukin-3 conjugate (DT-IL3 ) and administering one or more Jak inhibitors and / or one or more hypomethylating agents. The present invention provides a method comprising:

[0009] In some embodiments, DT-IL3 is at least 1 μg / kg, at least 2 μg / kg In some embodiments, the DT-I is administered at a dose of 4 μg / kg or more. L3 was 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. In some embodiments, the antibody is administered at a dose of 9 μg / kg to 12 μg / kg. In some cases, DT-IL3 is administered at doses of 5, 7, 9, or 12 μg / kg. In this embodiment, DT-IL3 is administered at the maximum tolerated dose.

[0010] In some embodiments, DT-IL3 and one or more Jak inhibitors and / or In some embodiments, the DT or hypomethylating agent is administered at least once a week. - IL3 and one or more Jak inhibitors and / or hypomethylating agents at least twice weekly In some embodiments, DT-IL3 and one or more Jak inhibitors are administered once. The agonist and / or hypomethylating agent is administered at least three times per week.

[0011] In some embodiments, DT-IL3 and (a) one or more Jak inhibitors and and / or (b) the one or more hypomethylating agents are administered for a period of one week or more. In some embodiments, a combination of DT-IL3 and (a) one or more Jak inhibitors, and and / or (b) the one or more hypomethylating agents are administered for a period of two weeks or more.

[0012] In some embodiments, DT-IL3 and (a) one or more Jak inhibitors and and / or (b) the one or more hypomethylating agents are administered for at least three consecutive days.

[0013] In some embodiments, DT-IL3 and (a) one or more Jak inhibitors and and / or (b) one or more hypomethylating agents are administered in multiple treatment cycles. In some embodiments, treatment cycles are at least one week apart, at least two weeks apart. , at least 3 weeks apart, at least 4 weeks apart, at least 5 weeks apart, or This is a combination of:

[0014] In some embodiments, DT-IL3 is administered every 21 days for at least 3 consecutive days. It is given for 4 cycles, then every 28 days, then every 42 days for 3 cycles.

[0015] In some embodiments, the one or more Jak inhibitors are administered at least once a day, and is administered at least twice daily.

[0016] In some embodiments, the one or more hypomethylating agents are administered in at least one cycle. At least the first 3 days, at least the first 4 days, at least the first 5 days, Both are administered for the first 6 days, or at least the first 7 days.

[0017] In some embodiments, the one or more hypomethylating agents are administered for 21 days over four cycles. on at least the first 3 days of a 28-day cycle for 3 cycles after administration of DT-IL3 At least the first 4 days, at least the first 5 days, at least the first 6 days, or at least is also given for the first 7 days.

[0018] 13. The method according to any one of the preceding claims, comprising: (a) DT-IL3 and one or more (b) one or more hypomethylating agents, and / or Administered until unacceptable toxicity occurs.

[0019] In some embodiments, the subject is treated with DT-IL3 and (a) one or more Jak The patient is administered (a) an inhibitor and / or (b) a pharmaceutical composition comprising one or more hypomethylating agents.

[0020] In some embodiments, the subject is a human. have unfavorable cytogenetic findings.

[0021] In some embodiments, the DT-IL3 is a chemical conjugate.

[0022] In some embodiments, the DT-IL3 is a recombinantly expressed protein.

[0023] In some embodiments, DT-IL3 comprises the catalytic domain and the transcription factor of diphtheria toxin. It is expressed as a single polypeptide containing the IL-3 locus and human IL-3. In one embodiment, a single polypeptide comprising the catalytic domain and the translocation domain of diphtheria toxin is provided. The DT-IL3 expressed as a peptide comprises the amino acid sequence of SEQ ID NO:3, and / or Human IL-3 comprises the amino acid sequence of SEQ ID NO:1.

[0024] In some embodiments, DT-IL3 is linked to human IL-3 by a peptide bond. In some embodiments, the amino acid residues comprise amino acid residues 1-388 of diphtheria toxin. The diphtheria toxin comprises the amino acid sequence of SEQ ID NO:2, and / or the human IL-3 comprises It comprises the amino acid sequence of SEQ ID NO:1.

[0025] In some embodiments, inhibition includes a reduction in proliferation of MPN cells, a stabilization of MPN cell mass, This results in the stabilization and / or reduction of the amount of MPN cells.

[0026] In some embodiments, the inhibition results in a reduction in spleen and / or liver size. vinegar.

[0027] In some embodiments, the MPN is polycythemia vera, essential thrombocytosis, osteoporosis, Myelofibrosis, chronic myelomonocytic leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, systemic hypertension Mantle cell purpura, symptomatic eosinophilic disorder, or excess red blood cells, white blood cells, and / or platelets In some embodiments, the MPN is a bone marrow disorder that causes the production of bone marrow fibroblasts. In some embodiments, the myelofibrosis is primary myelofibrosis, myelofibrosis Post-polycythemia, Post-essential thrombocytosis, Primary myelofibrosis in the blast phase Myelofibrosis, postpolycythemia vera myelofibrosis in the blast crisis, or myelofibrosis in the blast crisis This is post-ET myelofibrosis.

[0028] In some embodiments, the MPN is in blast crisis phase.

[0029] In some embodiments, MPN is primary myelofibrosis in the blast crisis phase, acute Post-polycythemia vera myelofibrosis in the blast phase or post-ET myelofibrosis in the blast phase It is fibrosis.

[0030] In some embodiments, the one or more Jak inhibitors comprises ruxolitinib.

[0031] In some embodiments, the one or more hypomethylating agents are azacitidine, decitabine , and / or SGI-110.

[0032] In some embodiments, at least one Jak inhibitor and at least one low A methylating agent is administered.

[0033] In some embodiments, the at least one Jak inhibitor comprises ruxolitinib. , at least one hypomethylating agent is decitabine, azacitidine, and / or SGI- Includes 110.

[0034] In some embodiments, the subject is receiving one or more Jak inhibitors and / or one or more In some embodiments, the subject has a refractory disease to prior treatment with a hypomethylating agent. The patient was refractory to previous treatment with the Jak inhibitor ruxolitinib. In the present invention, the subject has MF.

[0035] In some embodiments, the subject is receiving one or more Jak inhibitors and / or one or more He was not able to tolerate the full dose of previous treatment with hypomethylating agents. In one embodiment, the subject has completed all doses of prior treatment with the Jak inhibitor ruxolitinib. It has not been tolerated. In some embodiments, the subject has MF.

[0036] In some embodiments, the subject has a decreased platelet count or is receiving ruxolitinib. In some embodiments, the subject has MF.

[0037] In some embodiments, the subject has previously been treated with a Jak inhibitor and / or a hypomethylating agent. In some embodiments, the subject has MF.

[0038] In some embodiments, the subject has a gene encoding JAK2, MPL, ASXL1, TET2, or In some embodiments, the subject has a mutation in the JAK2V6 It has the 17F mutation.

[0039] Also provided herein is a method for treating or inhibiting myeloproliferative neoplasms (MPNs) in a subject in need of such treatment. A pharmaceutical composition for treating or inhibiting said myeloproliferative neoplasm (MPN) in Diphtheria toxin-human interleukin-3 conjugate (DT-IL3) and one or more Pharmaceutical compositions are also provided that include an effective amount of a Jak inhibitor and / or one or more hypomethylating agents. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutical acceptable excipient. include.

[0040] Also provided herein is the use of the compound in a subject in need of treatment for myeloproliferative neoplasms (MPNs). A method for treating a myeloproliferative neoplasm (MPN), comprising: a) identifying the MPN; and b) diphtheria toxin-human interleukin-3 conjugate (DT-IL3) and Administering an effective amount of one or more Jak inhibitors and / or one or more hypomethylating agents to a subject in need of such treatment. and administering to a subject a compound comprising the steps of:

[0041] In some embodiments, MPNs are assessed by physical exam, blood tests, bone marrow aspirate and biopsy, Cytogenetic analysis, JAK2, MPL, ASXL1, TET2, or CALR genes Examination of the mutation of β-amyloides, arterial blood oxygen saturation and carboxyhemoglobin levels, neutrophil alkaline phosphatase by sphatase levels, vitamin B12 or B12 binding capacity, or serum uric acid levels be identified.

[0042] In some embodiments, the MPN is identified by testing for the JAK2V617F mutation. will be done.

[0043] Further objects and advantages will be set forth in part in the detailed description that follows. These will be apparent in part from the detailed description or may be learned by practice of the invention. The objects and advantages thereof will become apparent from the following detailed description taken in conjunction with the accompanying drawings, in which: This is realized and achieved by the elements and combinations.

[0044] The foregoing general description and the following detailed description are given only as examples and are explanatory only. It should be understood that these are merely illustrative and do not limit the scope of the claims.

[0045] The accompanying drawings, which are incorporated in and constitute a part of this specification, include one or more ) and, together with the detailed description of the invention, the principles described herein. It serves the function of clarifying. [Brief description of the drawings]

[0046] [Figure 1] Figures 1A-1E show CD123 expression on blasts and monocytes in patients with chronic myelomonocytic leukemia (CMML) (N=20) as assessed by flow cytometry. Lymphocyte CD123 expression was used as a control. Figure 1A shows the mean fluorescence intensity of CD123 on CMML blasts, monocytes, and lymphocytes. Figure 1B shows the mean fluorescence intensity of CD123 on CMML blasts and monocytes normalized to the mean fluorescence intensity of CD123 on lymphocytes. Figures 1C and 1D show the mean fluorescence intensity of CD123 on blasts (Figure 1C) and monocytes (Figure 1D) from different CMML subtypes, CMML-0, CMML-1, and CMML-2. Results are normalized to the mean fluorescence intensity of CD123 on lymphocytes. Figure 1E shows the percentage of CD123 positive blasts in CMML subtypes, CMML-1 and CMML-2. Overall, Figures 1A-1E demonstrate that CD123 is more highly expressed on blasts and monocytes than on lymphocytes in CMML patients.

[0047] [Diagram 2] Figures 2A-2C show the results of cell viability assays using the CD123-expressing K562 cell line after in vitro treatment with increasing concentrations of azacytidine (AZA, Figure 2A), DT-IL-3 (SL-401, Figure 2B), or a combination of AZA and DT-IL3 (Figure 2C). AZA and DT-IL3 significantly reduced K562 cell viability compared to AZA alone.

[0048] [Diagram 3] Figures 3A-3B show the results of a cell viability assay using fresh mononuclear cells from peripheral blood of a CMML patient after in vitro treatment with AZA, DT-IL3, or a combination of AZA and DT-IL3 (SL-401). In Figure 3A, cells were exposed to increasing concentrations of AZA or a combination of AZA and DT-IL3. In 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 show that AZA and DT-IL3 significantly reduced cell viability of primary CMML mononuclear cells compared to AZA alone.

[0049] [Figure 4]Figures 4A-4D show the results of colony formation assays using fresh mononuclear cells from peripheral blood of CMML patients. Figures 4A-4D show the results from cells of individual patients. In Figures 4A-4C, cells were exposed to AZA (1, 5, or 10 μM) alone or a combination of 1 μM AZA and DT-IL-3 (2.5 or 10 nM). Addition of 10 nM DT-IL3 to 1 μM AZA significantly reduced colony formation. In Figure 4D, cells were exposed to DT-IL3 (2.5, 10, or 20 nM) alone or a combination of 10 nM DT-IL3 and AZA (1 or 2.5 μM). Addition of AZA to DT-IL3 significantly reduced colony formation. Overall, Figures 4A-4D show that AZA and DT-IL3 significantly reduced colony formation of CMML mononuclear cells compared to AZA or DT-IL3 alone.

[0050] Array Description Table 1 provides a list of the specific sequences referred to herein. [Table 1] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] I. Medication for Treatment The present application relates to a method for treating myeloproliferative neoplasms (MPNs), comprising administering diphtheria toxin-human Interleukin-3 conjugate (DT-IL3) and one or more Jak inhibitors and / or or one or more hypomethylating agents to a subject.

[0052] In some embodiments, DT-IL3 and (a) one or more Jak inhibitors and 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.

[0053] In some embodiments, DT-IL3 and (a) one or more Jak inhibitors and / or (b) the one or more hypomethylating agents are administered separately. and (a) one or more Jak inhibitors and / or (b) one The hypomethylating agents are administered at different times or on different days.

[0054] A. Diphtheria toxin-human interleukin-3 conjugate (DT-IL3) "DT-IL3" is a combination of human interleukin-3 (IL-3) and diphtheria toxin (D DT-IL-3 conjugates are known in the art and are described herein. The documents contemplate administration of such conjugates according to the methods of the present disclosure. ,763,242, U.S. Pat. No. 8,470,307, U.S. Pat. No. 9,181,317 No. 9,631,006, and WO2008 / 030539. T-IL-3 conjugates can be used according to the methods disclosed by the present invention. These references are incorporated by reference in their entirety for their disclosure of DT-IL3. For example, Liu et al. Exp. Hematol. 32:277-281 (2004 );Hogge et al.Clin.Cancer Res.12:1284-12 91(2006);Testa et al.Blood 106:2527-2529 (2005); and Klein et al. Biochem. Biophys. Res. See also the combination of (Also incorporated by reference in their entireties).

[0055] In some embodiments, the conjugate comprises a difenyl IL-3 covalently fused to human IL-3. In another embodiment, the catalytic domain and translocation domain of diphtheria toxin are included. The toxin is attached to the human IL-3 portion of the conjugate via a peptide linker. The anchors may be, for example, 2, 3, 4, 5, 10, up to 15 amino acids in length, or The length of the linker can be varied to optimize binding of the conjugate. In some embodiments, the peptide linker is 2 to 4 amino acids in length. The linker can be a His-Met linker. Although not essential, flexible peptide linkers facilitate chain pairing and allow for possible refolding. Linker molecules are generally known in the art and are disclosed in Denali rdo et al.,1998,Clin.Cancer Res.4:2483-9 0;Peterson et al.,1999,Bioconjug.Chem.10 :553; and Zimmerman et al., 1999, Nucl.Med.Bi Ol. 26:943-50, each of which is incorporated by reference in its entirety. (I).

[0056] In some embodiments, the present application relates to a method for the preparation of a DT-IL3 of the present invention and a pharma- ceutical acceptable salt thereof. According to the present invention, the conjugate is a conjugate having a structure as known in the art. Any domain of DT linked to any domain of IL-3 via any linker molecule In certain embodiments, the conjugate comprises the fusion protein DT 3 88IL-3, which has amino acids 1-388 of DT following the N-terminal methionine , fused to full-length mature human IL-3 via a His-Met amino acid linker.

[0057] In some embodiments, DT-IL3 binds to the interleukin-3 receptor (IL- 3 receptor). In some embodiments, DT-IL3 targets MPN cells that express the IL-3 receptor. In embodiments, DT-IL3 inhibits tumor-promoting cells in the tumor microenvironment (e.g., IL-1, -3R+ plasmacytoid dendritic cells).

[0058] 1.IL-3 Interleukin-3 (IL-3) regulates the differentiation of multipotent and committed myeloid and lymphoid progenitor cells. It is a cytokine that supports cell proliferation and differentiation. IL-3 is a cytotoxic agent that inhibits the production of IL-3. Blood growth factor, mast cell growth factor (MCGF), pluripotent colony-stimulating factor, or P-cell It is also sometimes called a stimulatory factor.

[0059] In some embodiments, the DT-IL3 conjugate is produced by recombinant techniques or chemical (covalent) ) binding to interleukin-3 receptor expressed on the cell surface or Full-length mature (lacking signal peptide) interleukin-3 protein bound to the subunit The present invention relates to a method for the preparation of a human IL-3 antibody, or a portion, analog, or derivative thereof, and a native cellular Diphtheria toxin (DT) lacking the cytoplasmic binding domain, or a portion, analog or includes derivatives.

[0060] Fragments, analogs, and derivatives of IL-3 include those fragments, analogs, and derivatives of IL-3. When the IL-3 receptor agonist and its derivatives are fused to the DT portion of the conjugate, they bind to the IL-3 receptor agonist expressed on the surface of cells. If it retains the ability to bind to the receptor subunits or the native IL-3 receptor, , may be useful in the present invention. The binding kinetics of the fragments, analogs, or derivatives are The IL-3 polypeptide may remain the same or may vary by no more than 25%. The peptide may be from any species. In certain embodiments, the IL-3 is a mammalian IL-3. For example, the IL-3 polypeptide is human IL-3, its analogs, derivatives, Or a fragment.

[0061] In some embodiments, the IL-3 is human IL-3. The amino acid sequence can be found in the GenBank database (e.g., accession number AAC0 8706) or UniProt number P08700. Human IL-3 An exemplary amino acid sequence of is as follows: msrlpvllll qllvrpglqa pmtqttslkt swvncsn mid eiithlkqpp lplldfnnln gedqdilmen nlrrpnleaf nravkslqna saiesil knl lpclplataa ptrhpihikd gdwnefrrkl tfylktlena qaqqttlsla([SEQ ID NO:1] in the case of)

[0062] In some embodiments, the IL-3 is at least 50% less than SEQ ID NO:1. at least 65%, at least 70%, at least 75%, at least 80%, at least 8 5%, at least 90%, or at least 95%, at least 96%, at least 97 %, at least 98%, or at least 99% identical.

[0063] 2. Diphtheria toxin Diphtheria toxin (DT) is a three-domain protein, the catalytic domain (amino acids 26 to 112; the underlined sequence in SEQ ID NO: 1 below) is an arginine-rich The disulfide loop binds the translocation domain (amino acids 225 to 404; The thallic sequence is followed by the cell-binding domain (amino acids 406 to 559). An exemplary amino acid sequence of DT is as follows (GenBank accession no. Accessed via no. AOU74567.1 or UniProt no. A0A142BWN1 Noh). [ka]

[0064] Fragments, analogs, and derivatives of DT may be useful in the present application. In an embodiment, DT comprises the catalytic domain, the translocation domain, and the cell binding domain of DT. In some embodiments, DT comprises the cell-binding domain and the catalytic domain of DT. In some embodiments, the DT comprises the cell binding domain and the translocation domain of DT. In some embodiments, DT comprises the catalytic domain and the translocation domain of DT. In some embodiments, the DT comprises a translocation domain, a catalytic domain, or a cell-binding domain.

[0065] The DT fragment bound to IL-3 is the catalytic and translocation domains of DT. and is represented by exemplary SEQ ID NO:3. [ka]

[0066] In some embodiments, the DT fragment is at least 50% identical to SEQ ID NO:3. %, at least 65%, at least 70%, at least 75%, at least 80%, at least at least 85%, at least 90%, or at least 95%, at least 96%, are 97%, at least 98%, or at least 99% identical.

[0067] 3. Methods for Producing DT-IL3 Conjugates The DT-IL3 conjugates of the invention can be prepared by standard recombinant DNA techniques or by protein synthesis. It can be made by synthetic techniques (e.g., using a peptide synthesizer). For example, nucleic acid molecules encoding the conjugates of the invention can be synthesized by conventional DNA synthesis, including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using the This may be done using anchor primers, which are complementary between two consecutive gene fragments. This generates suitable overhangs which are then annealed and reamplified to produce the chimeric gene sequence. You can generate columns (for example, Current Protocols in Mo lecular Biology,Ausubel et al.,eds.,John (see Wiley & Sons, 1992).

[0068] The nucleotide sequences encoding the conjugates of the invention (IL-3 and diphtheria toxin sequences) are , from any information available to one of skill in the art (i.e., GenBank, the literature, or conventional The nucleotide sequence encoding the conjugate can be obtained by suitable A suitable expression vector, i.e., a vector that contains the necessary genes for the transcription and translation of the inserted protein-coding sequence. In some cases, the targeting domain or other elements may be inserted into a vector that contains the targeting domain or other elements. The diphtheria toxin sequence can be truncated to remove specific domains of the DNase. Techniques for modifying or truncating A are well known to those skilled in the art of molecular biology. and the diphtheria toxin sequence are DNA sequences which, when translated, produce the polypeptides of the compounds of the invention. They can also be ligated to generate strings. An anchor sequence is introduced into the recombinant sequence linking the IL-3 sequence and the diphtheria toxin sequence. A variety of host-vector systems can be utilized in the present invention to express the protein coding sequence. Such systems include, but are not limited to, mammalian cells infected with a virus. Cell lines (e.g., vaccinia virus, adenovirus, etc.); virus-infected insects Insect cell systems (e.g., baculovirus); microorganisms, e.g., yeast containing yeast vectors (e.g., e.g., Pichia); or bacteriophage, DNA, plasmid DNA, or Examples of vectors include bacteria (e.g., E. coli) transformed with cosmid DNA. Expression elements vary in strength and specificity. Depending on the host-vector system utilized, Any one of several suitable transcription and translation elements can be used. In some embodiments, the protein is expressed in E. coli. The protein is expressed in Pichia.

[0069] Expression of the conjugates of the invention can be achieved using any promoter or enhancer known in the art. In some embodiments, the present invention provides a method for controlling the expression of a stimuli. In another embodiment, the expression of the conjugate is regulated by a constitutive promoter. In another embodiment, expression is regulated by an inducible promoter. It is regulated by a heterologous promoter.

[0070] In some embodiments, a promoter operably linked to the nucleic acid encoding the conjugate. , one or more origins of replication, and optionally one or more selection markers (e.g., antibiotics A vector containing a gene for resistance to the vector is used.

[0071] Expression vectors containing inserts of genes encoding conjugates can be prepared using three general approaches: (a) nucleic acid hybridization; (b) the presence or absence of a "marker" gene function. The first approach can be identified by (a) the presence of the inserted sequence, and (b) the expression of the inserted sequence. In the present invention, the presence of a gene encoding a conjugate in an expression vector indicates that an insert encoding the conjugate By nucleic acid hybridization using a probe containing a sequence that is homologous to the inserted gene, In the second approach, the recombinant vector / host system can be detected by: Certain specific binding sites are generated by inserting a nucleotide sequence encoding the conjugate into a vector. "marker" gene function (e.g., thymidine kinase activity, antibiotic resistance, transformation Based on the presence or absence of recombinant phenotypes, granule formation in baculovirus, etc. For example, a nucleotide sequence encoding the conjugate can be identified and selected. When inserted within the marker gene sequence of the Recombinants can be identified by the absence of marker gene function. In this approach, a recombinant expression vector is used to express a gene product (e.g., Such an assay can be used to identify the binding site of the antibody. can be determined, for example, by measuring the physical or functional properties of the conjugate in an in vitro assay system (e.g., For example, the binding may be based on an antibody or on binding to the IL-3 receptor.

[0072] The recombinant conjugates can be stably expressed for long-term, high-yield production. Alternatively, cell lines can be engineered that stably express the conjugates of the invention. Instead of using expression vectors containing the replication origin of the vector, we used expression control elements ( For example, promoters, enhancers, sequences, transcription terminators, polyadenylation sites The vector can be transformed using DNA controlled by a selection marker such as those described above. After introducing the foreign DNA, engineered cells can be grown in enriched medium for 1-2 days and then Switch to selective medium immediately after the recombinant plasmid is inserted. The selective marker in the recombinant plasmid confers resistance to the selection. This allows cells to stably integrate the plasmid into their chromosomes and grow to form foci. These foci can then be cloned and expanded into cell lines. The method can be advantageously used to engineer cell lines that express the conjugates of the invention. do.

[0073] DT-IL3 was engineered to express the conjugate protein generally as described above. It is produced recombinantly using bacterial, insect, or mammalian cells that contain the nucleic acid.

[0074] Once a conjugate of the invention has been produced by recombinant expression or chemical synthesis, the conjugate can be used to The protein can be purified by any method known in the art, for example, by chromatography. affinity (e.g., ion exchange, affinity (especially for specific antigens after Protein A) (by sizing column chromatography), centrifugation, and solubility differences or any other standard technique for protein purification. .

[0075] B. Jak inhibitors As used herein, a "Jak inhibitor" refers to an agent that inhibits one or more Janus kinases (Jak Jak inhibitors are drugs that inhibit the mitochondrial endothelial cell cycle (MTC) and the mitochondrial endothelial cell cycle (MCC). The Jak inhibitor can be any agent that inhibits one or more Jaks, regardless of whether they are Jak or not. In some embodiments, the AK1, Jak2, Jak3, and Tyk2 are included. The Jak inhibitor inhibits Jak1 or Jak2. In some embodiments, the Jak inhibitor is a Jak2 / Jak1 inhibitor. The drugs may also inhibit other kinases.

[0076] Jaks signal certain cytokines and growth factors essential for hematopoiesis and immune function. In some embodiments, inhibition of one or more Jaks reduces splenomegaly. and reduces circulating cytokine levels.

[0077] In some embodiments, the one or more Jak inhibitors are ruxolitinib, gandotidinib, nib, momelotinib, pacritinib, CHZ868, NS-018, SRC, or ita In some embodiments, some of the methods provided herein include citinib. The Jak inhibitor used therefore is ruxolitinib.

[0078] C. Hypomethylating Agents As used herein, a "hypomethylating agent" refers to an agent that reduces DNA methylation (i.e., DNA A hypomethylating agent is any drug that inhibits the addition of a methyl group to a nucleotide. Any agent that inhibits DNA methylation, regardless of whether the agent has other medicinal effects. It can be a drug.

[0079] In some embodiments, the hypomethylating agent inhibits the activity of DNA methyltransferase. block the activity of the In some embodiments, the hypomethylating agent is a DNMT inhibitor. In some embodiments, the method reduces DNA methylation without causing substantial inhibition. In some embodiments, the hypomethylating agent restores normal function to the gene. In this case, hypomethylating agents cause the death of rapidly dividing cells.

[0080] In some embodiments, the one or more hypomethylating agents are azacitidine, decitabine , and / or SGI-110.

[0081] D. Administration The agents of the invention can be administered in any clinically relevant dose. By "meaningful" it is meant that the dose of the drug has an effect in the subject. In embodiments, the drug combinations disclosed herein include combinations in which one or more of the drugs is is administered at a lower dose than would be effective when administered as a single agent. For example, one or more Jak inhibitors and / or one or more hypomethylating agents Treatment with DT-IL3 in combination with IL-16 produced the same dose of Jak inhibitors as when administered as single agents. This provides clinically meaningful benefits not seen with drugs or hypomethylating agents.

[0082] In some embodiments, one or more agents are administered at the maximum tolerated dose. When used, "maximum tolerated dose" means the highest dose of a drug that an individual patient can tolerate. In other words, side effects in a given patient may determine the maximum tolerated dose. This may limit the ability to administer higher doses of therapy than can be tolerated for a given patient. The maximum tolerated dose is the dose indicated in the prescribing information for a treatment or commonly used in clinical practice. The maximum tolerated dose may be lower than the maximum tolerated dose for some patients with limited efficacy or clinically significant adverse events. may not have any practical effectiveness.

[0083] 1. Exemplary Doses of DT-IL3 In some embodiments, DT-IL3 is at least 1 μg / kg, at least 2 μg / kg In some embodiments, the DT-I is administered at a dose of 4 μg / kg or more. L3 was 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. In some embodiments, the antibody is administered at a dose of 9 μg / kg to 12 μg / kg. In some embodiments, DT-IL3 is administered at a dose of 5 μg / kg. DT-IL3 is administered at a dose of 7 μg / kg. -IL3 is administered at a dose of 9 μg / kg. L3 is administered at a dose of 12 μg / kg.

[0084] In some embodiments, DT-IL3 is administered at the maximum tolerated dose.

[0085] 2. Exemplary Doses of Jak Inhibitors In some embodiments, the one or more Jak inhibitors include ruxolitinib. In some embodiments, ruxolitinib is administered at a dose of 0.5 mg, 1 mg, 2.5 mg, 5 mg , 10 mg, 15 mg, 20 mg, or 25 mg given orally.

[0086] In some embodiments, gandotinib is administered at 0.5 mg, 1 mg, 2.5 mg, 5 mg, mg, 10 mg, 15 mg, 30 mg, 60 mg, 90 mg, or 120 mg orally It is given.

[0087] In some embodiments, momelotinib is administered at 0.5 mg, 1 mg, 2.5 mg, 5 mg, mg, 10mg, 25mg, 50mg, 75mg, 100mg, 150mg, 200mg , 300 mg, or 400 mg given orally.

[0088] In some embodiments, pacritinib is administered at 0.5 mg, 1 mg, 2.5 mg, 5 mg, mg, 10mg, 25mg, 50mg, 75mg, 100mg, 150mg, 200mg , 300 mg, or 400 mg given orally.

[0089] In some embodiments, CHZ868 is administered at 0.5 mg, 1 mg, 2.5 mg, 5 mg, mg, 10mg, 25mg, 50mg, 75mg, 100mg, 150mg, 200mg , 300 mg, or 400 mg given orally.

[0090] In some embodiments, NS-018 is administered at 0.5 mg, 1 mg, 2.5 mg, 5 mg, mg, 10mg, 15mg, 25mg, 50mg, 75mg, 100mg, 150mg, It is given orally at 200 mg, 300 mg, or 400 mg.

[0091] In some embodiments, the SRC is 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10mg, 15mg, 20mg, 25mg, 50mg, 75mg, 100mg, 150m It is given orally at doses of 200 mg, 300 mg, or 400 mg.

[0092] In some embodiments, tofacitinib is administered at 0.5 mg, 1 mg, 2.5 mg, It is given orally at 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, or 30 mg.

[0093] In some embodiments, itacitinib is administered at 0.5 mg, 1 mg, 2.5 mg, 5 mg, mg, 10mg, 15mg, 20mg, 25mg, 50mg, 75mg, 100mg, 1 It is given orally at 50 mg, 200 mg, 300 mg, or 400 mg.

[0094] In some embodiments, the Jak inhibitor is administered at a maximum tolerated dose.

[0095] 3. Exemplary Doses of Hypomethylating Agents In some embodiments, azacitidine (Vidaza®) is administered at 75 mg g / m 2 In some embodiments, azacitidine is administered at 1 mg / m 2 , 2.5 mg / m 2 , 5 mg / m 2 , 10 mg / m2 , 15 mg / m 2 , 20 mg / m 2 , 25 mg / m 2 , 37.5 mg / m 2 , 50 mg / m 2 , 75 mg / m 2 , or 1 00mg / m 2 It is administered by continuous intravenous infusion or subcutaneous injection.

[0096] In some embodiments, decitabine (Dacogen®) is administered at 45 mg g / m 2 In some embodiments, decitabine (Dacog en®) is 1 mg / m 2 / day, 5mg / m 2 / day, 10mg / m 2 / day, 1 5 mg / m 2 / day, 20mg / m 2 / day, 33mg / m 2 / day, or 45 mg / m 2 / The infusion may be a single daily continuous intravenous infusion or multiple daily continuous intravenous infusions. It may also be by intravenous injection.

[0097] In some embodiments, SGI-110 (guadecitabine) is administered at 1 mg / m 2 , 3 mg / m 2 , 5 mg / m 2 , 10 mg / m 2 , 15 mg / m 2 , 20 mg / m 2 ,twenty five mg / m 2 , 30 mg / m 2 , or 60 mg / m 2 and is administered by subcutaneous injection.

[0098] In some embodiments, the hypomethylating agent is administered at a maximum tolerated dose.

[0099] In some embodiments, the subject is diagnosed with nausea and vomiting prior to administration of the hypomethylating agent. The patient is premedicated for this purpose.

[0100] II. Treatment method In some embodiments, DT-IL3 is administered to treat MPN by administering to: (a) one or more and / or (b) one or more hypomethylating agents.

[0101] In some embodiments, the method of treatment comprises administering to a subject DT-IL3 and one or more of Ja This involves administering a k inhibitor.

[0102] In some embodiments, the method of treatment comprises administering to a subject DT-IL3 and one or more low-molecular This includes administering a chilling agent.

[0103] In some embodiments, the treatment method comprises administering to a subject an antibody against DT-IL3, one or more Jak In some embodiments, the method further comprises administering a medicament for treating a tumor, the medicament for treating a tumor, and one or more hypomethylating agents. At least one Jak inhibitor and at least one hypomethylating agent are administered. In some embodiments, the at least one Jak inhibitor comprises ruxolitinib, At least one hypomethylating agent is selected from the group consisting of decitabine, azacitidine, and / or SGI-11. Including 0.

[0104] In some embodiments, the method of treatment comprises administering to a subject DT-IL3 and decitabine. This includes giving.

[0105] In some embodiments, the method of treatment comprises administering to a subject DT-IL3 and azacytidine. This includes administering.

[0106] In some embodiments, the method of treatment comprises administering to a subject DT-IL3 and SGI-110 The method includes administering

[0107] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the term "subject" means an animal. In this case, the animal is a mammal, e.g., a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.). In some embodiments, the mammal is a mammalian animal, such as a human or a mouse, or a primate (e.g., monkey or human). The subject is a human. In some embodiments, the subject is a non-human animal, such as a livestock animal (e.g., For example, a horse, pig, or cow) or a pet (for example, a dog or cat). In some embodiments, the subject is an elderly human. The subject is an adult human. In some embodiments, the subject is a child human. In some embodiments, the subject is a pediatric human (ie, a human under the age of 18). In some embodiments, the subject is an infant human.

[0108] In some embodiments, the subject has unfavorable cytogenetic findings. Based on survival outcomes, patients were determined to have favorable or unfavorable cytogenetic findings. An unfavorable cytogenetic finding is a cytogenetic risk factor that is associated with an unfavorable outcome. Unfavorable cytogenetic findings in subjects with MF and Examples include complex karyotypes (three or more rearrangements) or trisomy 8, 7 / 7q deletion, 17q inversion, 3 inversion, 5 / 5q deletion, 12p deletion, or 11q23 rearrangement. Favorable cytogenetic findings include a normal karyotype and / or 1 or 2 abnormalities. All other scenarios may be considered, including

[0109] In some embodiments, the subject is receiving one or more Jak inhibitors and / or one or more As used herein, a subject may have a history of refractory disease to prior treatment with a hypomethylating agent. "Refractory" to a past treatment means that the patient fails to achieve a response to the therapy, e.g., clinical Endpoints (any response, duration of response, disease-free status, survival time, recurrence-free survival, failure to achieve clinical outcomes (including progression-free survival, and overall survival) and thus the therapy is not curative In some embodiments, the subject is determined to be ineffective for the treatment of a ruxolitinib or ruxolitinib. In some embodiments, the patient has been refractory to prior treatment with ruxolitinib. Subjects who have been refractory to prior therapy with ribosomes have myelofibrosis (MF).

[0110] In some embodiments, the subject is receiving one or more Jak inhibitors and / or one or more The patient was not able to tolerate all doses of previous treatment with hypomethylating agents. As used herein, "total dose" means the dose(s) and / or or a dose commonly used in medical practice. The maximum tolerated dose for these patients was less than the full dose. In other words, the subjects were able to tolerate the drug without any side effects. For this reason, the patient was not able to tolerate the full dose of the previous treatment. In this study, subjects were unable to tolerate a full dose of prior treatment with ruxolitinib. In some embodiments, all doses of prior treatment with ruxolitinib were tolerated. Subjects who fail to demonstrate this have MF.

[0111] In some embodiments, the subject has a decreased platelet count or is receiving ruxolitinib. In some embodiments, the patient has a decreased platelet count and was not eligible for treatment with thrombocyte immunoglobulin (TIG). Subjects who were not eligible for treatment with ruxolitinib or had MF. In some embodiments, the platelet count reduction is greater than or equal to 5×10 9 / L or less, 10×10 9 / L or less, 20×10 9 / L or less, 30×10 9 / L or less, 40×10 9 / L or less, or 50 x 1 0 9 In some embodiments, the patient is diagnosed with renal failure. Patients may be ineligible for treatment with ruxolitinib due to hepatic toxicity or liver dysfunction.

[0112] In some embodiments, the subject has previously been treated with a Jak inhibitor and / or a hypomethylating agent. In some embodiments, the patient has responded to a Jak inhibitor and / or hypomethylation In some embodiments, the subject has previously undergone rheumatoid arthritis. Responded to solitinib. Many reasons for failure of previous treatments for MF (e.g., myelosuppression) The following characteristics have been characterized (Pettit et al., 2003; P ...). and Odenike Curr Hematol Malig Rep.12(6) :611-624(2017).

[0113] A. Myeloproliferative neoplasms In some embodiments, the MPN is polycythemia vera (PV), essential thrombocytosis Myelofibrosis (MF), chronic myelomonocytic leukemia (CMML), chronic neutrophilic leukemia (CLL) Leukemia, chronic eosinophilic leukemia, systemic mastocytosis (SM), symptomatic eosinophilic disorders, and is another bone marrow disorder that causes excess production of red blood cells, white blood cells, and / or platelets. In some embodiments, the MPN is a primary eosinophilic disorder (PED).

[0114] In some embodiments, the MPN is myelofibrosis (MF). MF is a condition in which bone marrow becomes scarred. Characterized by bone marrow scarring, which causes insufficient blood cell production. This can lead to anemia, bleeding problems, and risk of infection. The liver and spleen become enlarged. There may be an attempt to produce more blood cells.

[0115] In some embodiments, MF is primary MF (PMF), post-polycythemia vera MF, F (post-PV MF), post-essential thrombocytosis MF (post-ET MF), primary MF in the blast crisis phase PMF-BP, post-PV MF in the blast crisis phase, or EMF in the blast crisis phase T post MF.

[0116] In some embodiments, the MPN is in blast crisis phase.

[0117] In some embodiments, MPNs are PMF-BP, post-PV M in blast crisis phase. F, or post-ET MF in the blast crisis phase.

[0118] B. Treating and Identifying Subjects with MPNs In some embodiments, the present invention relates to a method for treating MPN in a subject in need of such treatment. The method for treating the MPN comprises identifying the MPN and administering DT-IL3 and one or more J administering an effective amount of an ak inhibitor and / or one or more hypomethylating agents to a subject in need of such treatment; and administering.

[0119] In some embodiments, MPNs are assessed by physical exam, blood tests, bone marrow aspirate and biopsy, Cytogenetic analysis, JAK2, MPL, ASXL1, TET2, or CALR genes Examination of the mutation of β-amyloides, arterial blood oxygen saturation and carboxyhemoglobin levels, neutrophil alkaline phosphatase by sphatase levels, vitamin B12 or B12 binding capacity, or serum uric acid levels In some embodiments, the mutation in the JAK2 gene is JAK2V617 This is the F mutation.

[0120] In some embodiments, the patient has a JAK2, MPL, ASXL1, TET2, or In some embodiments, the patient has a mutation in the JAK2V6 It has the 17F mutation.

[0121] C. Methods for Measuring Clinical Effectiveness In some embodiments, inhibition of MPNs includes reducing the proliferation of MPN cells, inhibiting ... As used herein, this results in a stabilization of the amount of, and / or a reduction in the amount of, MPN cells. By "MPN cell" is meant any abnormal cell in the bone marrow that gives rise to MPN.

[0122] In some embodiments, inhibition of MPNs results in a reduction in spleen and / or liver size. In some embodiments, the spleen size is measured at different times before and after treatment. In some embodiments, the MPN is measured by MRI or CT at the same time. A subject with has an abnormally large spleen (i.e., splenomegaly) or an abnormally large liver. In some embodiments, splenomegaly is greater than 5 cm below the costal margin (BCM) by physical examination. It is defined as:

[0123] In some embodiments, a subject with MPN also has an abnormally large spleen. In some embodiments, the subject with MPN has a baseline No splenomegaly (≤5cm BCM).

[0124] In some embodiments, the reduction in spleen size is at least 25% of spleen size, At least 29%, at least 33%, at least 35%, at least 40%, or less In some embodiments, the reduction in spleen size is at least a 50% reduction. A reduction of 33% or more in spleen size in subjects with splenomegaly. Subjects who meet the parameters for are "splenic responders" to the treatment.

[0125] In some embodiments, clinical efficacy is measured by improvement in splenomegaly. In some embodiments, the improvement in splenomegaly in a group of subjects is determined by the percentage of splenic responders. It is measured by.

[0126] In some embodiments, improvement in splenomegaly is measured by a palpable cm below the left costal margin. The change in splenomegaly is measured by the percentage change in splenomegaly determined by the spleen size.

[0127] In some embodiments, the subject has MF. Response criteria for MF include: Including but not limited to, International Working Papers for MF The following Table 2 provides exemplary response criteria from the Group -MRT and ELN: do. Table 2 [Table 2] TIFF2025032098000006.tif248170TIFF2025032098000007.tif156170

[0128] Tefferi et al.,Blood 122(6):1395-98(201 3) Adapted from Table 2. In Table 2, CR = complete response or remission, PR = partial response or remission, EM H=extramedullary hematopoiesis, LCM=left costal margin, and UNL=upper limit of normal. * Baseline and post-treatment Bone marrow slides will be interpreted in one go through a central review process. Assignment of CR Cytogenetic and molecular response is not required for MF. †MF grading is based on the European classification. (Thiele et al., Haematologica 90:1128(200 5)) Immature bone marrow cells are classified as blasts + promyelocytes + myelocytes + metamyelocytes + nucleated red blood cells. In splenectomized patients, less than 5% immature bone marrow cells are acceptable. § Anemic response, splenic See above for definitions of response and disease progression. Increased severity of anemia may indicate new transfusion dependency. Incidence of cerebrovascular disease or a decrease in hemoglobin level of ≥20 g / L from pretreatment baseline The severity of thrombocytopenia or neutropenia is The increase is due to the Common Terminology Criteria for Advancement Platelet count or absolute number of platelets was measured according to the Clinical Trials Events (CTCAE) version 4.0. Defined as a 2-grade reduction in neutrophil count from pretreatment baseline. For CI assignment, a minimum platelet count of ≥ 25 000 × 10(9) / L and absolute neutrophil count of ≥ 100 were required. A baseline hemoglobin count of 0.5×10(9) / L or greater is required. Applicable only to patients with blood glucose levels <100 g / L. Strict criteria for transfusion dependency were established at study entry. For patients who did not meet the criteria but had received a transfusion within the previous month, the pre-transfusion hemoglobin level was Transfusion dependency prior to study entry should be determined based on bleeding or In the absence of treatment-induced anemia, hemoglobin levels must be less than 85 g / L and at least 1% of the mean time of study entry. Defined as having received at least 6 units of packed red blood cells (PRBCs) in the previous 2 weeks. For inclusion, the most recent transfusion episode must have occurred within 28 days prior to study enrolment. Responses in transfusion-dependent patients were assessed over any consecutive "rolling" 12-week period during treatment. maintain hemoglobin levels above 85 g / L during the interval and avoid any PRBC transfusions In splenectomized patients, palpable hepatomegaly can be measured using the same measurement strategy. Use. ** Splenic or hepatic response must be confirmed by imaging studies, MR The requirement is a reduction of 35% or more of the spleen as assessed by MRI or CT. or spleen or liver volume reduction of 35% or more on CT scan as reported by physical examination ††Symptoms are assessed using MPN-SAF TSS.17. MPN-SAF TSS is assessed by the patient themselves and includes fatigue, concentration, and speed. Symptoms include abdominal fullness, inactivity, night sweats, itching, bone pain, abdominal discomfort, weight loss, and fever. Each item was scored from 0 (none / very good) to 10 (worst possible / very bad). The MPN-SAF TSS is the sum of all individual scores (on a 0-100 scale). Symptomatic response requires a 50% or greater reduction in MPN-SAF TSS. The assignment of disease progression to spleen was based on splenic volume vectors assessed by MRI or computed tomography. A minimum of 25% increase from baseline is required. Physical and imaging examinations All baseline values ​​refer to pre-treatment baselines and post-treatment measurements. It is not something that can be achieved.

[0129] III. Medication Administration These methods include administering DT-IL3 and one or more Jak inhibitors and / or hypomethylating agents. The present invention includes various dosing schedules for anticancer drugs.

[0130] In some embodiments, DT-IL3 is administered at least once a week. In some embodiments, DT-IL3 is administered at least twice a week. In one embodiment, DT-IL3 is administered at least three times a week.

[0131] In some embodiments, the one or more Jak inhibitors are administered at least once a week. In some embodiments, the one or more Jak inhibitors are administered at least twice a week. In some embodiments, the one or more Jak inhibitors are administered at least three times per week. In some embodiments, the one or more Jak inhibitors are administered every other day. In some embodiments, the one or more Jak inhibitors are administered daily. In some embodiments, the one or more Jak inhibitors are administered more than once per day. In one embodiment, the one or more Jak inhibitors are administered twice daily.

[0132] In some embodiments, the one or more hypomethylating agents are administered at least once a week. In some embodiments, the one or more hypomethylating agents are administered at least twice a week. In some embodiments, the one or more hypomethylating agents are administered at least three times per week. In some embodiments, the one or more hypomethylating agents are administered daily. In some embodiments, the one or more hypomethylating agents are administered twice daily. In some embodiments, the one or more hypomethylating agents are administered three times a day. In some embodiments, the one or more hypomethylating agents are administered for a period following a period in which no hypomethylating agent is administered. In some embodiments, the one or more hypomethylating agents are administered daily for up to Dosages are given daily for a period of 7 days.

[0133] In some embodiments, DT-IL3 and (a) one or more Jak inhibitors and and / or (b) the one or more hypomethylating agents are administered for a period of one week or more. In some embodiments, a combination of DT-IL3 and (a) one or more Jak inhibitors, and and / or (b) the one or more hypomethylating agents are administered for a period of two weeks or more. In some embodiments, DT-IL3 and (a) one or more Jak inhibitors and / or or (b) the one or more hypomethylating agents are administered for at least three consecutive days.

[0134] A. Treatment Cycle In some embodiments, DT-IL3 and (a) one or more Jak inhibitors and and / or (b) one or more hypomethylating agents are administered in multiple treatment cycles.

[0135] As used herein, a "treatment cycle" refers to a combination of DT-IL3 and (a) one or more (b) the period during which one or more Jak inhibitors and / or one or more hypomethylating agents are administered In some embodiments, there is a single treatment cycle. In this case, there are multiple cycles of treatment.

[0136] In some embodiments, during multiple treatment cycles, DT-IL3 and (a (a) one or more Jak inhibitors and / or (b) one or more hypomethylating agents are not administered In some embodiments, treatment cycles are at least one week apart. At least 2 weeks apart, at least 3 weeks apart, at least 4 weeks apart, at least 5 weeks apart interval, or a combination thereof.

[0137] In some embodiments, DT-IL3 is administered every 21 days for at least 3 consecutive days. It is administered every 42 days for 3 cycles, followed by every 28 days for 4 cycles. In some embodiments, the one or more Jak inhibitors and / or the one or more hypomethylating agents are It is given at the same time as IL3.

[0138] In some embodiments, one or more Jak inhibitors and / or one or more low methyl The lysing agent is administered at least once a day, or at least twice a day.

[0139] In some embodiments, one or more Jak inhibitors and / or one or more low methyl The regulating agent should be administered for at least the first 3 days and at least the first 4 days of at least one cycle. The dose is administered for at least the first 5 days, at least 6 days, or at least 7 days.

[0140] In some embodiments, one or more Jak inhibitors and / or one or more low methyl The inhibitor was administered for at least the first 3 days of each 28-day cycle for at least the maximum The first 4 days, at least the first 5 days, at least the first 6 days, or at least the first In some embodiments, DT-IL3 is administered to one or more of the Jak In some embodiments, the inhibitor and / or one or more hypomethylating agents are administered simultaneously. In this study, one or more Jak inhibitors and / or one or more hypomethylating agents were administered for 4 cycles. It will be administered over a 21-day cycle following administration of DT-IL3.

[0141] In some embodiments, DT-IL3 and (a) one or more Jak inhibitors and and / or (b) one or more hypomethylating agents, e.g., as determined by the treating physician, may be used to treat a disease Administered until progression and / or unacceptable toxicity.

[0142] IV. Pharmaceutical Compositions In some embodiments, the present invention relates to a method for treating or inhibiting a myeloproliferative neoplasm (MPN). The present invention relates to a pharmaceutical composition for treating or inhibiting a myeloproliferative neoplasm (MPN) in a subject. , DT-IL3 and one or more Jak inhibitors and / or one or more hypomethylating agents In some embodiments, the pharmaceutical composition further comprises a pharma- ceutical acceptable salt thereof. Contains excipients.

[0143] In some embodiments, the present invention provides a method for the preparation of a conjugate of the present invention comprising administering to a subject a subject an effective amount of a conjugate of the present invention and a pharma- ceutical In some embodiments, a pharmaceutical composition is provided that comprises a carrier or vehicle that is The pharmaceutical composition comprises an effective amount of the conjugate of the present invention and a pharma- ceutical acceptable carrier or vehicle. The pharmaceutical compositions are suitable for administration to animals and / or humans.

[0144] The pharmaceutical compositions of the present invention may be in any form that allows the composition to be administered to a subject. It is possible.

[0145] Materials used in preparing pharmaceutical compositions may be non-toxic in the amounts used Those of skill in the art will recognize that optimal dosages of the active ingredient(s) in a pharmaceutical composition can be determined by a variety of factors. It will be apparent that the effect of the present invention will depend on the following factors: The type of subject (e.g., human), the overall health of the subject, the type of cancer the subject has, The use of the compositions as part of a therapeutic regimen, the particular forms and modes of administration of the compositions of the invention are Some examples include:

[0146] The term "carrier" refers to a diluent, adjuvant, or other substance with which a composition of the invention is administered. means excipients. Optional auxiliary substances, stabilizers, thickeners, lubricants, and colorants may be used. In one embodiment, the compositions and pharmaceutical compositions of the present invention may be administered to a subject. Water is a suitable carrier when the composition of the present invention is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are also suitable liquid carriers. The composition of the present invention can be used as a pharmaceutical, in particular as an injectable solution. In this case, a small amount of a pH buffer may be included.

[0147] The liquid compositions of the present invention, whether in the form of a solution, suspension, or other similar form, It may also include one or more of the following: a sterile diluent, such as water for injection, saline, normal saline. Water, Ringer's solution, isotonic sodium chloride, synthetic monoamines that can act as solvents or suspending media, or diglycerides, polyethylene glycol, glycerin, cyclodextrin phosphate, propylene glycol, or other solvents; antibacterial agents, e.g., benzyl alcohol; or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffering agents, e.g., acetate, citrate Salts, or phosphates; and tonicity adjusters, such as sodium chloride or dextrose. Parenteral compositions may be packaged in ampoules, disposable capsules made of glass, plastic, or other material. In some embodiments, the composition may be packaged in a syringe or a multi-dose vial. Physiological saline is an adjuvant. Injectable compositions can be sterile.

[0148] The pharmaceutical compositions contain an effective amount of the composition of the invention such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% by weight of the composition of the present invention. When intended for administration, this amount may vary between 0.1% and 80% by weight of the pharmaceutical composition. The oral pharmaceutical composition may contain between 4% and 50% by weight of the pharmaceutical composition. The pharmaceutical composition may include a parenteral dosage unit of 0.01 wt. % to 2% by weight of the composition of the present invention.

[0149] The pharmaceutical compositions can be administered by any convenient route, for example, by infusion or bolus injection. Absorption through epithelial or mucosal linings (e.g., oral, rectal, and intestinal mucosa) The administration can be systemic or local. Delivery systems, such as microparticles, microcapsules, capsules, etc., are known and are The above-mentioned compositions may be useful for administering the compositions. Methods of administration include, but are not limited to: These include oral administration and parenteral administration, including but not limited to: None, but intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous; intranasal, epidural, sublingual, intranasal, intracerebral, Intraventricular, intrathecal, intravaginal, transdermal, rectal, inhalation, or topical to the ears, nose, eyes, or skin The mode of administration is left to the discretion of the practitioner and will depend in part on the disease. The type of treatment will depend on the site of the cancer (eg, the site of the cancer, cancerous tumor, or pre-cancerous condition).

[0150] In some embodiments, the compositions of the invention are administered parenterally. In one embodiment, the compositions of the present invention are administered intravenously. The composition is administered by continuous infusion. In certain embodiments, the composition of the invention is Administered by infusion lasting 15, 20, 30, 45 minutes, 1 hour, or 2 hours. can be.

[0151] In some embodiments, one or more compositions of the present invention are administered to the area in need of treatment. This may be desirable, for example and not by way of limitation, during surgery. Local injection; topical application (e.g., after surgery with a wound dressing); by injection; through a catheter via a suppository; or via an implant (the implant is made of a silastic membrane Made of porous, non-porous, or gelatinous materials, including membranes, such as suction cups, or fibers. In certain embodiments, one or more compositions of the present invention may be used. can be injected intraperitoneally.

[0152] In yet another embodiment, the compositions of the invention can be delivered in a controlled release system. Cut.

[0153] In some embodiments, a pump can be used to deliver the compositions of the present invention. (See, e.g., Sefton, CRC Crit. Ref. Biomed. Eng. 1999). 87,14,201;Buchwald et al.,Surgery 1980,8 8:507;Saudek et al.,N.Engl.J.Med.1989,32 1:574). In some embodiments, the pump may be may be an insulin-like pump.

[0154] The compositions of the present invention may be used in the form of solutions, suspensions, tablets, pills, pellets, capsules, liquid-containing capsules, etc. Cells, powders, sustained release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or The pharmaceutical carrier may take any other suitable form for use. Examples of suitable pharmaceutical carriers are described in Rem. ington's Pharmaceutical Sciences(EWMar This is explained in .

[0155] In some embodiments, the compositions of the invention are suitable for intravenous administration to animals, particularly humans. The combined pharmaceutical composition is formulated according to the usual procedures. Typically, it is administered intravenously. The carrier or vehicle for use is a sterile, isotonic aqueous buffer solution. Compositions for intravenous administration may optionally contain an agent to ease pain at the site of the injection. The composition may include a local anesthetic such as lignocaine to aid in the administration of the active ingredient. In the form of, for example, a dry lyophilized powder or anhydrous concentrate, an ampoule or container may be provided indicating the amount of active agent. The ingredients may be supplied separately or mixed together in a sealed container such as a sachet. When the composition is administered by injection, for example, sterile pharmaceutical grade water or saline is added. When the composition of the present invention is administered by injection, it can be dispensed by an infusion bottle containing An ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed prior to administration. .

[0156] The compositions of the present invention may be used in combination with, but are not limited to, additional prophylactic agents, additional therapeutic agents, inhibitors, Emetics, hematopoietic colony stimulating factors, adjuvant therapy, vaccines or other immune stimulants, antibodies / Additional agents selected from those including antibody fragment-based drugs, antidepressants, and analgesics In some embodiments, the pharmaceutical composition can include an active agent of An acceptable carrier or vehicle is included.

[0157] The pharmaceutical compositions can be prepared using methods well known in the pharmaceutical art. For example, Compositions intended for administration by injection may be prepared by combining the composition of the invention with water to form a solution. A surfactant can be added to prepare a homogeneous solution or suspension. Surfactants can non-covalently interact with the compositions of the present invention. The present invention can be used to facilitate dissolution or uniform suspension of the compositions of the present invention in an aqueous delivery system. It is a complex that can EXAMPLES

[0158] Example 1. In vitro testing of DT-IL3 and hypomethylating agents Preclinical trials were conducted using human leukemia cell lines and primary mononuclear cells derived from peripheral blood of CMML patients. The hypomethylating agent (HMA) azacytidine (AZA) and DT-IL3 (SL-40 1) In order to measure the active concentration of AZA and DT-I in vitro, increasing concentrations were used. Cells were exposed to either CD123, L3, or the combination and cell viability assays were performed. In the 562 cell line, the IC50 values ​​of AZA and DT-IL3 were 772 μM and 772 μM, respectively. 4.8 nM (FIGS. 2A to 2C). These results show that ZA significantly reduced K562 cell viability compared to ZA alone.

[0159] Next, CMMs exposed in vitro to increasing concentrations of AZA and DT-IL3 were Cell viability assays were performed using fresh mononuclear cells from peripheral blood of patients with L. AZA. The IC50 of the single agents was 1442 μM, whereas the combination of AZA and DT-IL3 (4.8 nM) The IC50 of 1 μM of 1 μM in this assay was 725 μM (Figure 3A). The addition of 4.8 nM DT-IL3 to AZA significantly reduced cell survival compared to 1 μM AZA alone. This resulted in a significant decrease in the rate (Figure 3B; p<0.05).

[0160] In a colony-forming assay using fresh mononuclear cells from peripheral blood of CMML patients, The combination of AZA and DT-IL3 significantly reduced colony formation compared with AZA alone. Similarly, AZ treatment with increasing concentrations of DT-IL3 significantly increased the expression of DT-IL3 (Figures 4A-4C, p<0.05). The addition of A significantly reduced colony formation compared with DT-IL3 alone (Figure 4D, p < 0.01). Importantly, the combined effect was similar to that of NRAS / ASXL1 / TET2 (Figure 4C) and High-risk patients include those with mutations in SRSF2 / TET2 / STAG2 (Figure 4A) and SRSF2 / TET2 / STAG2 (Figure 4B). This was observed across patient samples with genotype.

[0161] Current treatment options for CMML are limited, and treatment of CMML remains challenging. This is an important unmet medical need. HMA therapy has demonstrated efficacy in CMML. However, the effects are often limited in extent and duration. Preclinical data support the potential treatment of the combination of HMA and DT-IL3 in CMML. See Figures 2A-2C, 3A-3B, and 4A-4D.

[0162] Example 2. Clinical trial in treatment-naive patients Clinical trials are evaluating the efficacy and safety of DT-IL3 and one or more Jak inhibitors in patients with previously untreated MPN. The efficacy of the drug and / or one or more hypomethylating agents can be evaluated. This means that such patients have not been previously treated for MPN. Patients with this condition will not have previously been treated with a Jak inhibitor or hypomethylating agent. .

[0163] Patients enrolled in the study may have different types of MPN. All patients may have myelofibrosis.

[0164] Patients underwent physical examination, blood tests, bone marrow aspirate and biopsy, cytogenetic analysis, JAK2, M Testing for mutations in the PL, ASXL1, TET2, or CALR genes, and arterial oxygen saturation and carboxyhemoglobin level, neutrophil alkaline phosphatase level, and vitamin B MPN can be diagnosed based on serum uric acid levels or B12 or B12 binding capacity. do.

[0165] In some embodiments, the patient has a JAK2, MPL, ASXL1, TET2, or In some embodiments, the patient has a mutation in the JAK2V6 It has the 17F mutation.

[0166] The test may have an adaptive design. In other words, the test may be adapted to the test and / or the Alternatively, the test may be maintained for the entire duration of the test. It is also possible to set up treatment groups for which the study is conducted.

[0167] Treatment arms were DT-IL3 and DT-IL3 plus one or more Jak inhibitors and / or can include a comparison with a combination of one or more hypomethylating agents.

[0168] Treatment arms were: one or more Jak inhibitors, and DT-IL3 and one or more Jak inhibitors and / or a comparison with a combination of one or more hypomethylating agents.

[0169] Treatment arms consisted of one or more hypomethylating agents plus DT-IL3 and one or more Jak inhibitors. and / or a comparison with a combination of one or more hypomethylating agents.

[0170] In adaptive designs, treatment with one or more hypomethylating agents or one or more Jak inhibitors Patients who failed DT-IL3 and one or more Jak inhibitors and / or one or more Treatment with a combination of hypomethylating agents can be initiated.

[0171] Treatment success or failure will be assessed using standard measures used in MPN trials, e.g., spleen and / or or reduction in liver size. Changes in blood composition, recurrence of disease The duration of treatment and patient survival will also be assessed. Toxicity and adverse events will be measured during treatment.

[0172] Any of the drug doses and treatment cycles described in this application may be used. DT 388 The Jak inhibitor can be ruxolitinib. The one or more hypomethylating agents may be azacitidine, decitabine, and / or SGI- It can be 110.

[0173] Applicants anticipate clinical improvement in treated patients.

[0174] Example 3. Clinical Trials in Refractory Patients In clinical trials, patients with MPN who were refractory to previous treatments or had completed a full course of previous therapy were DT-IL3 and one or more J in MPN patients who could not tolerate The efficacy of ak inhibitors and / or one or more hypomethylating agents can be evaluated. The treatment may be one or more hypomethylating agents or one or more Jak inhibitors. Tinib may be the previous treatment.

[0175] Clinical trials of refractory patients should not include treatment arms to which patients were refractory to previous treatments. Clinical trials in refractory patients have included DT-IL3 and one or more Jak inhibitors and and / or the efficacy of one or more hypomethylating agents and the availability of agents for which the patient has not been previously treated. For example, in a study of patients refractory to ruxolitinib, The treatment group was treated with hypomethylating agents and the other group was treated with DT-IL3 and one or more Jak inhibitors and / or or treatment with one or more hypomethylating agents.

[0176] In addition to patient selection, clinical trials for refractory patients may be similar or more cost-effective than clinical trials for untreated patients. The parameter may be one.

[0177] Applicants anticipate clinical improvement in treated patients.

[0178] Equivalent The above specification is believed to be sufficient to enable one skilled in the art to practice the embodiments. It is contemplated that the above description and examples detail certain specific embodiments contemplated by the inventors. However, the above description may differ in detail from the best mode for carrying out the present invention. Regardless of what appears in the text, the embodiments may be practiced in many ways and may be modified in various ways, as described in the accompanying patents. It is understood that the present invention should be construed in accordance with the appended claims and any equivalents thereof. Wax.

[0179] As used herein, all numerical values ​​are estimated to be "about." As used herein, The term "about" is used to refer to any number of numbers, whether or not expressly indicated, including, for example, whole numbers, fractions, In general, the term "about" refers to numerical values ​​that are within the skill of the art. A range of values ​​(e.g., For example, it means + / - 5% to 10% of the stated range. When terms such as "also" and "about" are used, they are intended to cover all values ​​or ranges listed. In some cases, the term about refers to numbers that are rounded to the nearest significant figure. It can include.

Claims

1. 1. A pharmaceutical composition for treating or inhibiting myeloproliferative neoplasms (MPNs) in a human subject comprising a diphtheria toxin-human interleukin-3 conjugate (DT-IL3), The DT-IL3 is a. one or more Jak inhibitors selected from ruxolitinib and pacritinib, and / or b. one or more hypomethylating agents selected from azacitidine and decitabine The pharmaceutical composition is adapted to be administered to the human subject simultaneously or at different times with

2. The DT-IL3 is a. 1 μg / kg or more, 2 μg / kg or more, or 4 μg / kg or more; b. 2 μg / kg to 20 μg / kg or 9 μg / kg to 20 μg / kg; c. 4 μg / kg to 12 μg / kg or 9 μg / kg to 12 μg / kg; d. 5 μg / kg, 7 μg / kg, 9 μg / kg or 12 μg / kg; or e. maximum tolerated dose; The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition is administered in a dose of

3. the DT-IL3 is in combination with the one or more Jak inhibitors and / or the one or more hypomethylating agents; a. at least once a week, at least twice a week, or at least three times a week; b. for a period of one week or more, or for a period of two weeks or more; c. For at least three consecutive days; and / or d. With multiple treatment cycles; The pharmaceutical composition according to claim 1 or 2,

4. 4. The pharmaceutical composition of claim 3, wherein the treatment cycles are 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.

5. a. the DT-IL3 is administered for at least 3 consecutive days, every 21 days for 4 cycles, then every 28 days for 3 cycles, then every 42 days; b. the one or more Jak inhibitors are administered at least once daily or at least twice daily; c. the 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 first 6 days, or at least the first 7 days of at least one cycle; d. the 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 first 6 days, or at least the first 7 days of a 28 day cycle for three cycles following administration of DT-IL3 for 21 days for four cycles; and / or e. the DT-IL3 is administered with the one or more Jak inhibitors and / or the one or more hypomethylating agents until disease progression and / or unacceptable toxicity occurs; The pharmaceutical composition according to claim 3 or 4.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the human subject has an unfavorable cytogenetic finding.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the DT-IL3 is a chemical conjugate and / or a recombinantly expressed protein.

8. 8. The pharmaceutical composition of claim 7, wherein the DT-IL3 is a recombinant protein expressed as a single polypeptide comprising the catalytic and translocation domains of diphtheria toxin and human IL-3.

9. 9. The pharmaceutical composition of claim 8, wherein said DT-IL3 comprises amino acid residues 1-388 of diphtheria toxin bound to human IL-3 by a peptide bond.

10. 10. The pharmaceutical composition of any one of claims 1 to 9, wherein the inhibition results in reduced proliferation of MPN cells, stabilization of MPN cell mass, reduction of MPN cell mass, and / or reduction in spleen and / or liver size.

11. 11. The pharmaceutical composition of any one of claims 1 to 10, wherein the MPN is polycythemia vera, essential thrombocytosis, myelofibrosis, chronic myelomonocytic leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, systemic mastocytosis, symptomatic eosinophilic disorder, or other bone marrow disorder that causes excess production of red blood cells, white blood cells, and / or platelets.

12. The pharmaceutical composition of claim 11, wherein the MPN is myelofibrosis (MF).

13. 13. The pharmaceutical composition of claim 12, wherein the myelofibrosis is primary myelofibrosis, post-polycythemia vera myelofibrosis, post-essential thrombocythemia myelofibrosis, primary myelofibrosis in blast crisis phase, post-polycythemia vera myelofibrosis in blast crisis phase, or post-ET myelofibrosis in blast crisis phase.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the MPN is in blast crisis phase.

15. 15. The pharmaceutical composition of claim 14, wherein the MPN is primary myelofibrosis in blast crisis phase, post-polycythemia vera myelofibrosis in blast crisis phase, or post-ET myelofibrosis in blast crisis phase.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the DT-IL3 is administered in combination with at least one Jak inhibitor and at least one hypomethylating agent.

17. a. the human subject has been refractory to prior treatment with one or more Jak inhibitors and / or one or more hypomethylating agents; b. The human subject has failed to tolerate full doses of prior treatment with one or more Jak inhibitors and / or one or more hypomethylating agents; c. The human subject has a decreased platelet count or was ineligible for treatment with ruxolitinib or pacritinib; d. the human subject has previously responded to a Jak inhibitor and / or a hypomethylating agent; and / or e. the human subject has a mutation in the JAK2, MPL, ASXL1, TET2, or CALR gene; The pharmaceutical composition according to any one of claims 1 to 16.

18. 18. The pharmaceutical composition of claim 17, wherein the human subject has a JAK2V617F mutation.

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