WO2020112642A1 - Improved methods of treating myeloproliferative neoplasms with a diphtheria toxin-human interleukin-3 conjugate - Google Patents

Abstract

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), wherein the subject has monocytosis.

Description

IMPROVED METHODS OF TREATING MYELOPROLIFERATIVE NEOPLASMS WITH A DIPHTHERIA TOXIN -HUMAN

INTERLEUKIN-3 CONJUGATE

DESCRIPTION

CROSS REFERENCE TO RELATED APPLICATION

[001] This international application claims priority to US Provisional Application Serial No. 62/773,530, filed November 30, 2018, the contents of which are hereby incorporated by reference in its entirety.

FIELD

[002] The present invention provides methods for treating or inhibiting a myeloproliferative neoplasm (MPN), such as myelofibrosis (MF) or polycythemia vera (PV), in a subject in need thereof, wherein the subject has monocytosis. The method comprises administering to the subject a diphtheria toxin-human interleukin- 3 conjugate (DT-IL3).

BACKGROUND

[003] Myeloproliferative neoplasms (MPNs), also known as

myeloproliferative diseases (MPDs), are hematological diseases characterized by excess production of bone marrow cells. MPNs are also characterized by clonal expansion of one or more hematopoietic cell lineages in the bone marrow. In some cases, a genetic mutation, such as mutation in the Jak2 protein, may be present in stem cells from a patient with an MPN.

[004] MPNs may lead to increases in certain blood cells, abnormal blood cells, and enlargement of the spleen (splenomegaly). MPNs may damage the bone marrow and result in bone marrow fibrosis (myelofibrosis or MF). Anemia, fatigue, and weakness may occur due to changes in blood cell counts in MPN.

Thrombohemorrhagic complications, such as thrombosis, are also a risk of MPN. Progression of MPNs can lead to development of further conditions, such as acute myeloid leukemia.

[005] Types of MPN include polycythemia vera (PV), essential

thrombocythemia (ET), myelofibrosis (MF), chronic myelomonocytic leukemia (CMML), chronic neutrophilic leukemia, chronic eosinophilic leukemia, systemic mastocytosis (SM), symptomatic hypereosinophilic disorder, other bone marrow disorders that causes the production of excess red blood cells, white blood cells, and/ or platelets, and primary eosinophilic disorder (PED).

[006] Myelofibrosis (MF) is a BCR- ABL1 -negative myeloproliferative neoplasm characterized by stem cell-derived clonal myeloproliferation, dysregulated kinase signaling, and release of abnormal cytokines. Myelofibrosis may occur as a sequela of polycythemia vera (PV) or essential thrombocythemia (ET), or may develop in the absence of these associated myeloproliferative conditions (e.g., primary MF (PMF)). The clonal myeloproliferation is associated with reactive bone marrow fibrosis, osteosclerosis, aberrant cytokine expression and extramedullary

hematopoiesis (EMH). Prominent clinical manifestations of MF include severe anemia, marked hepatosplenomegaly, and constitutional symptoms (including fatigue, fever, and night sweats). Additional symptoms include cachexia and bone pain;

patients are also susceptible to splenic infarcts, and thrombotic and hemorrhagic complications. Ineffective hematopoiesis results in anemia (and fatigue); EMH results in organomegaly. Hepatic complications may be accompanied by portal hypertension, which can result in upper gastrointestinal varices, bleeding, and/ or ascites. EMH may result in pulmonary hypertension, pleural effusions, ascites, bone pain and even spinal cord compression. It is likely that aberrant cytokine production contributes to bone marrow fibrosis, diminished erythropoiesis, cachexia, and other constitutional symptoms (Tefferi, A., N. Engl. J. Med. 366:844-846 (2012); Tefferi, A., Am. J.

Hematol. 88:142-150 (2013)).

[007] Leukemic progression occurs in approximately 20% of PMF, and also occurs in MF secondary to PV or ET. The multiple comorbidities associated with MF also contribute to mortality, most notably cardiovascular events, severe infections, and hemorrhage.

[008] Primary myelofibrosis (PMF) is charactered by progressive bone marrow failure with worsening cytopenia and, in a subset of patients, progression to acute leukemia.

[009] Published data have shown that the development of monocytosis in patients with PMF is associated with a poor prognosis. For example, in a study which included 129 patients, on multivariable analysis, an absolute monocyte count of > 1 x 10⁹/L carried an independent predictive value (p=0.02), for an inferior survival. See Elliott, et al., Leuk. Res. 31 (11): 1503-1509 (2007). Another study, Boiocchi et al, Modern Pathology 26:204—212 (2013), identified 10 out of 237 cases of PMF that developed persistent absolute monocytosis (>1 x 10⁹/L) during the course of disease. Five patients died after developing monocytosis and two experienced worsening disease and became transfusion dependent. No change in JAK2 mutational status or cytogenetic evolution was associated with the development of monocytosis.

[0010] Another study presented clinical and laboratory features of 454 patients with PMF stratified by absolute monocyte count (AMC) and showed a higher incidence of monocytosis in PMF (65/454 patients). Tefferi et al., Br. J. Flaematol. (2017), available at https:/ / doi.org/10.1111/bjh.l5061. After a median follow-up of 3.7 years, 289 (64%) deaths, 38 (8.4%) leukaemic transformations, and 28 (6%) allogeneic stem cell transplants were documented. In univariate analysis, AMC > 3 x 10⁹/1 (ha2ard ratio (FIR) 5.6, 95% confidence interval (Cl) 3.2—10.0) and AMC 1—3 x 10⁹/1 (HR 2.1, 95% Cl F5— 2.8) were both associated with inferior overall survival. Consistent with Elliott et al. (2007) and Boiocchi et al. (2013) noted above, this study demonstrated a dose-dependent adverse prognostic effect of monocytosis in PMF.

[0011] In some cases, morphological and/ or molecular (e.g., ASXL1, TET2, SRSF2, NRAS or KRAS mutations) characteristic of an intermediate between primary MF and chronic myelomonocytic leukemia (CMML) are observed,

(mutations also often present in blastic plasmacytoid dendritic cell neoplasm, BPDCN). See, e.g., Pardanani et al., Blood 132(5):492-500 (2018); Boiocchi et al., Modem Pathology 26:204—212 (2013); and Chapman et al., Mod. Pathol. 31(3):429- 441 (2018).

[0012] In current clinical practice, and following the 2008 World Health Organi2ation (WHO) criteria (See, e.g, Swerdlow et al, eds., WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, Lyon, France: IARC (2008), and Vardiman et al., Blood 114(5):937-951 (2009)), while monocytosis can develop during disease course in PMF, it does not lead to disease reclassification. Cases identified either: (1) fulfilled the 2008 WHO criteria for PMF but had absolute monocytosis and, when available, CMML-related mutations (ASXL1, SRSF2, TET2, NRAS or KRAS) or (2) fulfilled criteria of CMML but had megakaryocytic proliferation and atypia, marrow fibrosis, and myeloproliferative-type driver mutations (JAK2, MPL, CALR). Biologically, these cases likely represent PMF with monocytosis, dysplasia, and secondary (non-driver) mutations at presentation. Alternatively, they may represent a true gray 2one of neoplasms. Regardless of the specific subtype, their clinical behavior is aggressive and innovative therapeutic approaches may be beneficial in this particular subset. See Chapman et al., Mod. Pathol. 31(3):429-441 (2018).

[0013] In order to address concerns regarding confounding patients with chronic myelomonocytic leukemia (CMML) and MF with monocytosis, a

multiparametric flow cytometry-based approach was employed and demonstrated differences in monocytes between CMML and MPN-associated monocytosis (including MF and PV patients). The classic monocyte fraction (CD14-positive and CD 16-negative) which constitutes the major monocyte population (85%) in healthy conditions was <92% in all MPN cases (mean 77%) versus >92% in all CMML patients (mean 95.6%). (Patnaik et al., Blood Cancer J. 7:e584 (2017)). Thus, the classic monocyte fraction (CD14-positive and CD 16 -negative) could potentially be used as a biomarker predicting response. [0014] In conclusion, development of monocytosis in patients with

established PMF is associated with rapid disease progression and these patients should be considered as a high-risk group associated with inferior survival. See, e.g., Boiocchi et al., Mod. Pathol. 26(2):204-12 (2013); Elliott et al., Leuk. Res.

31(ll):1503-9 (2007); and Tefferi et al., Br. J. Haematol. (2017), available at https:/ / doi.org/10.1111/bjh.l5061.

[0015] The association between monocytosis and worse prognosis has been shown in additional malignancies. For example, a higher than expected incidence (21%) of monocytosis has also been reported in polycythemia vera (PV), along with an association with older age, higher leucocyte count and worse prognosis. Barraco et al., Am. J Hematol. 92:640—645 (2017). In addition, in a retrospective analysis of 202 patients with newly diagnosed Diffuse Large B Cell Lymphoma (DLBCL) between 2002 and 2017, a high absolute monocyte count (AMC) was associated with inferior complete remission (CR) rates, relapse free survival (RFS) and overall survival (OS). Irigoin et al., Blood 130(Suppl 1) :4161 (2017). This difference was statistically significant in patients with a very good or good prognosis score under the Revised International Prognostic Index (R-IPI), and patients in this group with high AMC had similar OS than adverse prognostic RIPI patients.

[0016] The present application describes improved methods for treating a subject with monocytosis and an MPN with a diphtheria toxin-human interleukin-3 conjugate (DT-IL3).

SUMMARY

[0017] In accordance with the description, this application describes a method for inhibiting or treating monocytosis in a subject in need thereof, comprising administering to the subject a diphtheria toxin-human interleukin-3 conjugate (DT- IL3).

[0018] This application also describes a method for treating or inhibiting myeloproliferative neoplasm (MPN) cells in a subject having an MPN and monocytosis, comprising administering an effective amount of a diphtheria toxin- human interleukin-3 conjugate (DT-IL3) to the subject.

[0019] This application also describes a method for treating or inhibiting an MPN in a subject in need thereof, comprising administering to the subject a diphtheria toxin-human interleukin-3 conjugate (DT-IL3), wherein the subject has monocytosis.

[0020] This application also describes a method for treating an MPN in a subject in need thereof, comprising: determining whether a subject with the MPN has monocytosis, and administering an effective amount of a diphtheria toxin-human interleukin-3 conjugate (DT-IL3) to the subject if the subject is determined to have monocytosis.

[0021] In some embodiments, the method results in a reduction in the proliferation of MPN cells, a stabilization in the amount of MPN cells, a reduction in the amount of MPN cells, and/ or a reduction in spleen and/ or liver size.

[0022] In some embodiments, said stabilization or reduction is measured by blood tests; blast count; blast percentage; physical examination; complete blood count; flow cytometric analyses; bone marrow analyses; hematopoietic function; marrow blast index; the amount of normal white blood cells, red blood cells, and/ or platelets; histology; immunohistochemistry; frequency of transfusion; and/ or bone marrow biopsy.

[0023] In some embodiments, the method results in the inhibition and/ or treatment of the monocytosis.

[0024] In some embodiments, the cells of the MPN express the IL-3 receptor.

[0025] In some embodiments, the growth of the MPN cells is inhibited.

[0026] In some embodiments, the DT-IL3 is administered at a dose of 0.1 Pg/kg to 50 pg/kg.

[0027] In some embodiments, the DT-IL3 is administered at a dose of 4 pg/kg to 50 pg/kg. [0028] In some embodiments, the DT-IL3 is administered at a dose of 4 i¾/kg to 20 gg/kg.

[0029] In some embodiments, wherein the DT-IL3 is administered at a dose of 4 gg/kg to 12 gg/kg.

[0030] In some embodiments, the DT-IL3 is administered at a dose of 5 gg/kg, 7 gg/kg, 9 gg/kg, or 12 gg/kg.

[0031] In some embodiments, the DT-IL3 is administered at a dose that is the maximum tolerated dose.

[0032] In some embodiments, the DT-IL3 is administered at least once a week.

[0033] In some embodiments, the DT-IL3 is administered at least two times a week.

[0034] In some embodiments, the DT-IL3 is administered at least three times a week.

[0035] In some embodiments, the DT-IL3 is administered over a period of one week or more.

[0036] In some embodiments, the DT-IL3 is administered over a period of two weeks or more.

[0037] In some embodiments, the conjugate is administered once every day for three days.

[0038] In some embodiments, the conjugate is administered once every day for five days.

[0039] In some embodiments, the DT-IL3 is administered in multiple treatment cycles.

[0040] In some embodiments, 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.

[0041] In some embodiments, the DT-IL3 is administered for at least 3 consecutive days of a 21 -day cycle for four cycles, followed by at least three consecutive days of a 28-day cycle for 3 cycles. In some embodiments, the DT-IL3 is also administered at least three consecutive days of one or more 42-day cycles following the three 28-day cycles.

[0042] In some embodiments, the DT-IL3 is administered until disease progression and/ or unacceptable toxicity is obtained.

[0043] In some embodiments, the subject is human.

[0044] In some embodiments, the human has unfavorable cytogenetics.

[0045] In some embodiments, the DT-IL3 is a chemical conjugate.

[0046] In some embodiments, the DT-IL3 is a recombinantly expressed protein.

[0047] In some embodiments, the DT-IL3 is expressed as a single polypeptide comprising the catalytic and translocation domains of diphtheria toxin and human IL-

3.

[0048] In some embodiments, the DT-IL3 comprises amino acid residues 1 to 388 of diphtheria toxin linked via a peptide bond to human IL-3.

[0049] In some embodiments, the MPN is polycythemia vera (PV), essential thrombocythemia (ET), myelofibrosis (MF), chronic myelomonocytic leukemia (CMML), chronic neutrophilic leukemia, chronic eosinophilic leukemia, systemic mastocytosis (SM), symptomatic hypereosinophilic disorder, or other bone marrow disorder that causes the production of excess red blood cells, white blood cells, and / or platelets, or a primary eosinophilic disorder (PED).

[0050] In some embodiments, the MPN is polycythemia vera (PV)

[0051] In some embodiments, the MPN is myelofibrosis (MF). In some embodiments, the myelofibrosis is primary myelofibrosis, post-polycythemia vera myelofibrosis, post-essential thrombocythemia myelofibrosis, blast phase primary myelofibrosis, post-polycythemia vera myelofibrosis in blast phase, or post-ET myelofibrosis in blast phase.

[0052] In some embodiments, the MPN is refractory to at least one prior MPN treatment. [0053] In some embodiments, the subject is in a state of remission from the

MPN.

[0054] In some embodiments, the subject has been previously treated with a therapeutic agent and/ or has undergone radiation therapy.

[0055] In some embodiments, the subject is currentiy being administered a therapeutic agent other than a human IL-3-diphtheria toxin conjugate and/ or is undergoing radiation therapy.

[0056] In some embodiments, the subject has relapsed from prior MPN treatment.

[0057] In some embodiments, the subject has failed prior MPN treatment.

[0058] In some embodiments, the subject is susceptible to adverse reactions from other MPN therapies.

[0059] In some embodiments, the subject is refractory to chemotherapy.

[0060] In some embodiments, the subject has not been previously treated for the MPN.

[0061] In some embodiments, the method further comprises administering one or more Jak inhibitors and/ or one or more hypomethylating agents. In some embodiments, at least one Jak inhibitor and at least one hypomethylating agent is administered. In some embodiments, the one or more Jak inhibitors comprises ruxolitinib. In some embodiments, the one or more hypomethylating agents comprise a2acitidine, decitabine, and/or SGI-110.

[0062] In some embodiments, the subject is administered a pharmaceutical composition comprising the DT-IL3 and the (a) one or more Jak inhibitors and/ or (b) one or more hypomethylating agents.

[0063] In some embodiments, the subject with monocytosis has a monocyte count equal to or greater than 1 x 10⁹/L.

[0064] The application also describes a pharmaceutical composition for treating or inhibiting an MPN in a subject in need thereof comprising an effective amount of a diphtheria toxin-human interleukin-3 conjugate (DT-IL3), wherein the subject has monocytosis. In some embodiments, the MPN is myelofibrosis. In some embodiments, the MPN is polycythemia vera. In some embodiments, the subject with monocytosis has a monocyte count equal to or greater than 1 x 10⁹/L.

[0065] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0066] In some embodiments, the pharmaceutical composition further comprises one or more Jak inhibitors and/ or one or more hypomethylating agents.

[0067] Additional 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. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0068] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0069] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description, serve to explain the principles described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 shows the change (%) in splenomegaly (best response at any time point) compared to baseline (pre-treatment) for all patients treated with DT-IL3 that had complete splenomegaly data.

[0071] Figure 2 shows the change (%) in splenomegaly (best response at any time point) compared to baseline (pre-treatment) for patients treated with DT-IL3 that had monocytosis (monocyte count > 1 x 10⁹/L) at baseline and complete splenomegaly data.

DESCRIPTION OF THE SEQUENCES

[0072] Table 1 provides a listing of certain sequences referenced herein.

DESCRIPTION OF THE EMBODIMENTS

I. Agents for treatment

[0073] This application provides methods of treatment of an MPN, such as MF, in a subject with monocytosis, comprising administering a diphtheria toxin- human interleukin-3 conjugate (DT-IL3) to the subject.

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

[0074]“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

administration in accordance with the methods of the present disclosure are contemplated herein. For example, DT-IL-3 conjugates described in US Patent No. 7,763,242; US Patent No. 8,470,307; US Patent No. 9,181,317; US Patent No.

9,631,006, and W02008/ 030539 may be used in accordance with the methods disclosed by the present invention. These references are incorporated by reference in their entirety for their disclosure of DT-IL3. See also, e.g., the conjugates of Liu et al. Exp. Hematol. 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)), also incorporated by reference in their entirety for their disclosure of DT-IL3.

[0075] In certain embodiments, the conjugate comprises the catalytic and translocation domains of diphtheria toxin fused via a covalent bond to human IL-3. In other embodiments, the diphtheria toxin is linked via a peptide linker to the human IL-3 portion of the conjugate. The linker for the conjugate may be, for example, two, three, four, five, ten, up to fifteen, or fifteen amino acids in length. The length of the linker may vary to provide optimal binding of the conjugate. In some embodiments, the peptide linker is two to four amino acids long. The peptide linker may be a His-Met linker. Although not intending to be bound by a particular mechanism of action, the flexible peptide linker facilitates chain pairing and minimizes possible refolding. Linker molecules are commonly known in the art and described in Denardo et al., 1998, Clin. Cancer Res. 4:2483-90; Peterson et al., 1999, Bioconjug. Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943-50 each incorporated by reference in their entireties.

[0076] In some embodiments, the application provides pharmaceutical compositions that include a DT-IL3 of the invention and a pharmaceutically acceptable carrier. In accordance with the present invention, the conjugate can include any domain of DT linked via any linker molecule known in the art to any domain of IL-3. In certain embodiments, the conjugate is DT₃₈sIL-3, which is a fusion protein of an N-terminal methionine, followed by amino acids 1-388 of DT fused to full-length, mature, human IL-3 via a His-Met amino acid linker.

[0077] In some embodiments, DT-IL3 mediates selective targeting to cells expressing the interleukin-3 receptor (IL-3 receptor). In some embodiments, DT-IL3 targets to myelofibrosis cells expressing the IL-3 receptor. In some embodiments, DT-IL3 targets tumor-promoting cells (such as IL-3R+ plasmacytoid dendritic cells) in the tumor microenvironment.

1. IL-3

[0078] Interleukin-3 (IL-3) is a cytokine that supports the proliferation and differentiation of multi-potential and committed myeloid and lymphoid progenitors. See, e.g., Nitsche et al. Stem Cells 21: 236-244 (2003). IL-3 may also be referred to as hematopoietic growth factor, mast cell growth factor (MCGF), multipotential colony- stimulating factor, or P-cell-stimulating factor.

[0079] In some embodiments, the DT-IL3 conjugates include the full-length, mature (lacking the signal peptide) interleukin-3 protein (IL-3), or a portion, analog or derivative thereof that binds to the interleukin-3 receptor or a subunit thereof expressed on a cell surface, conjugated through a recombinant technology or through chemical (covalent) bond to diphtheria toxin (DT), or a portion, analog or derivative thereof, which toxin lacks the native cell binding domain.

[0080] Fragments, analogs, and derivatives of IL-3 can be useful in the present invention provided that when fused to the DT portion of the conjugate, such fragments, analogs and derivatives maintain the ability to bind a subunit of the IL-3 receptor or the native IL-3 receptor expressed on the surface of a cell. The binding kinetics of the fragments, analogs or derivatives may remain the same or vary only by not more than 25%. The IL-3 polypeptide may be from any species. In certain embodiments, the IL-3 is a mammalian IL-3, e.g., an IL-3 polypeptide is human IL-3, an analog, derivative, or a fragment thereof.

[0081] In some embodiments, the IL-3 is human IL-3. An exemplary amino acid sequence of human IL-3 can be found in the GenBank database (see, e.g., Accession No. AAC08706) or UniProt #P08700. An exemplary amino acid sequence of human IL-3 is:

MSRLPVLLLL QLLVRPGLQA PMTQTTSLKT SWVNCSNMID EIITHLKQPP LPLLDFNNLN GEDQDILMEN NLRRPNLEAF NRAVKSLQNA SAIESILKNL LPCLPLATAA PTRHPIHIKD GDWNEFRRKL TF YLKTLEN A QAQQTTLSLA IF (SEQ ID NO: 1)

[0082] 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.

2. Diphtheria toxin

[0083] Diphtheria toxin (DT) is a protein with three domains: a catalytic domain (amino acids 26-112; bold sequence within SEQ ID NO: 2 below) connected by an arginine-rich disulfide loop to a translocation domain (amino acids 225-404; italicized sequence within SEQ ID NO:2) followed by a cell binding domain (amino acids 406-559). An exemplary amino acid sequence of DT, accessible from GenBank Accession No. AOU74567.1 or UniProt # A0A142BWN1 is:

MSRKLFASIL IGALLGIGAP PSAHAGADDY VDSSKSFVME NFSSYHGTKP GYVDSIQKGIQKPKSGTQGN YDDDWKGFYS TDNKYDAAGY SVDNENPLSG KAGGVVKVTY PGLTKVLALK VDN AETIKKE LGLSLTEPLM EQVGTEEFIK RFGDGASRVY

LSLPFAEGSS SVEYINNWEQ AKALSVELEI NFETRGKRGQ DAMYEYMAQA CAGNRVRRSV GSS SCINLD WD VIRDKTKT KIESLKEHGP IKNKMSESPN KTVSEEKAKQ YLEEFHQTAL EHPELSELKT VTGTNPVFAG ANYAAWAVNV AQVIDSETAD NLEKTTAALS ILPGIGSVMG L4DGAVHHNT EEIVAQSLAL

SSLMVAgAIP LVGELVDIGF AAYNFVESII NEFQWHNSY

NKPA YTPGHK TQPFLHDGYA VSWNTVEDSI IRTGFQGESG HDIKITAENT PLPIAGVLLP TIPGKLDVNK SKTHISYNGR KIRMRCRAID GDVTFCRPKS PVYVGNGVHA NLHVAFHRSS SEKIHSNEIS SDSIGVLGYQ KTVDHTKVNS KLSLFFEIKS

(SEQ ID NO: 2)

[0084] Fragments, analogs and derivatives of DT can be useful in the present application. In some embodiments, DT consists of the catalytic, the translocation, and the cell binding domains of DT. In some embodiments, DT consists of the cell binding and the catalytic domains of DT. In some embodiments, DT consists of the cell binding and the translocation domains of DT. In some embodiments, DT consists of the catalytic and translocation domains of DT. In some embodiments, DT comprises one of the translocation, catalytic, or cell binding domain.

[0085] The DT fragment conjugated to the IL-3 is the catalytic domain and the translocation domain of DT, represented by exemplary SEQ ID NO: 3:

GADDVVDSSK SFVMENFSSY HGTKPGYVD S IQKGIQKPKS GTQGNYDDDW KGFYSTDNKY DAAGYSVDNE NPLSGKAGGV VKVTYPGLTK VLALKVDNAE TIKKELGLSL TEPLMEQVGT EEFIKRFGDG ASRVVLSLPF AEGSSSVEYI NNWEQAKALS VELEINFETR GKRGQDAMYE YMAQACAGNR VRRSVGSSLS CINLDWDVIR DKTKTKIESL KEHGPIKNKM SESPNKTVSE EKAKQYLEEF HQTALEHPEL SELKTVTGTN PVF AGANYAA WAVNVAQVID SETADNLEKT TAALSILPGI GSVMGIADGA VHHNTEEI V A QSIALSSLMV AQAIPLVGEL VDIGFAAYNF VESIINLFQV VHNSYNRPAY SPGHKTRP (SEQ ID NO: 3)

[0086] 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.

3. Methods for producing DT-IL3 conjugates

[0087] The DT-IL3 conjugates of the present invention can be made by standard recombinant DNA techniques or by protein synthetic techniques, e.g., by use of a peptide synthesizer. For example, a nucleic acid molecule encoding a conjugate of the invention can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequentiy be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992).

[0088] The nucleotide sequences encoding a conjugate of the invention (IL-3 and diphtheria toxin sequences) may be obtained from any information available to those of skill in the art (i.e., from Genbank, the literature, or by routine cloning). The nucleotide sequence coding for a conjugate can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. In some instances, the diphtheria toxin sequence can be truncated in order to remove a specific domain, such as the targeting domain. The techniques for modifying or truncating DNA are well known to those of skill in the art of molecular biology. Also, the IL-3 and the diphtheria toxin sequences can be ligated in such a way as to generate a DNA sequence that, when translating, creates a polypeptide that is a compound of the invention. In some embodiments, a linker sequence is introduced into the recombinant sequence that links the IL-3 sequence and the diphtheria toxin sequence. A variety of host-vector systems may be utilized in the present invention to express the protein-coding sequence. These include but are not limited to mammalian cell systems infected with virus (e.g, vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g, baculo virus) ; microorganisms such as yeast (e.g,

Pichia ) containing yeast vectors; or bacteria (such as E. coli₎ transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used. In some embodiments, the protein is expressed in E. coli. In some embodiments, the protein is expressed in Pichia.

[0089] The expression of a conjugate of the invention may be controlled by any promoter or enhancer element known in the art. In some embodiments, the expression of a conjugate of the invention is regulated by a constitutive promoter. In another embodiment, the expression is regulated by an inducible promoter. In another embodiment, the expression is regulated by a tissue-specific promoter.

[0090] In some embodiments, a vector is used that comprises a promoter operably linked to a conjugate-encoding nucleic acid, one or more origins of replication and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene).

[0091] Expression vectors containing inserts of a gene encoding a conjugate can be identified by three general approaches: (a) nucleic acid hybridization,

(b) presence or absence of“marker” gene functions, and (c) expression of inserted sequences. In the first approach, the presence of a gene encoding a conjugate in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted gene encoding the conjugate. In the second approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain“marker” gene functions (e.g, thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of a nucleotide sequence encoding a conjugate in the vector. For example, if the nucleotide sequence encoding the conjugate is inserted within the marker gene sequence of the vector, recombinants containing the gene encoding the conjugate insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by assaying the gene product {e.g, conjugate) expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the conjugate in in vitro assay systems, e.g., binding to an antibody or the IL-3 receptor.

[0092] Recombinant conjugates may be stably expressed for long-term, high- yield production. For example, cell lines which stably express the conjugate of the invention may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements {e.g, promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.

Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched medium, and then are switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express a conjugate of the invention.

[0093] The DT-IL3 is generally produced recombinantly, using bacterial, insect, or mammalian cells containing a nucleic acid engineered to express the conjugate protein, as described above.

[0094] Once a conjugate of the invention has been produced by recombinant expression or by chemical synthesis, it may be purified by any method known in the art for purification of a protein, for example, by chromatography {e.g, ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and si2ing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

B. Additional Therapeutic Agents

[0095] In some embodiments, the method of treatment further comprises administering one or more additional therapeutic agents to the subject. In some embodiments, one or more Jak inhibitors and/ or one or more hypomethylating agents are administered.

1. Jak Inhibitors

[0096] As used herein, a“Jak inhibitor” is an agent that inhibits one or more Janus kinases (Jak). The Jak inhibitor can be any agent that inhibits one or more Jak, whether or not this agent has other pharmaceutical effects. Jak includes Jakl, Jak2, Jak3, and Tyk2. In some embodiments, the Jak inhibitor inhibits Jakl or Jak2. In some embodiments, the Jak inhibitor is a Jak2/Jakl inhibitor. In some embodiments, the Jak inhibitor may also inhibit other kinases.

[0097] Jaks mediate the signaling of certain cytokines and growth factors critical for hematopoiesis and immune function. In some embodiments, inhibition of one or more Jaks reduces splenomegaly and decreases circulating cytokine levels.

[0098] In some embodiments, the one or more Jak inhibitors comprises ruxolitinib, gandotinib, momelotinib, pacritinib, CHZ868, NS-018, SRC, tofacitinib, or itacitinib. In some embodiments, the Jak inhibitor used in accordance with some of the methods provided herein is ruxolitinib.

2. Hypomethylating agents

[0099] As used herein, a“hypomethylating agent” is any drug that inhibits DNA methylation (i.e., the addition of a methyl group to a DNA nucleotide). The hypomethylating agent can be any agent that inhibits DNA methylation, whether or not this agent has other pharmaceutical effects.

[00100] In some embodiments, the hypomethylating agent blocks the activity of a DNA methyltransferase (i.e., the compound is a DNA methyltransferase inhibitor or DNMT inhibitor). In some embodiments, a hypomethylating agent decreases DNA methylation without causing substantial suppression of DNA synthesis. In some embodiments, a hypomethylating agent restores normal function to genes. In some embodiments, a hypomethylating agent causes death of rapidly dividing cells.

[00101] In some embodiments, the one or more hypomethylating agents comprise a2acitidine, decitabine, and/ or SGI-110.

C. Dosing

[00102] The agents of this invention may be dosed at any clinically relevant dose. By clinically relevant, it is meant that the dose of the agent has an effect in the subject. In some embodiments, the combinations of agents disclosed herein allow one or more agents to be dosed at a lower dosage level than the dose at which said agent would have an effect when dosed as a single agent. For example, treatment with DT-IL3 together with one or more Jak inhibitors and/ or one or more hypomethylating agents provides clinically relevant effects that would not be seen for the same dose of Jak inhibitor or hypomethylating agent when dosed as a single agent.

[00103] In some embodiments, one or more agents is dosed at the maximum tolerated dose.“Maximum tolerated dose,” as used herein, refers to the highest dose of an agent that an individual patient can tolerate. In other words, side effects in a given patient can determine the maximum tolerated dose. Side effects may limit the ability to administer higher doses of a treatment than the maximum tolerated dose. Therefore, the maximum tolerated dose for a given patient may be lower than those indicated in the prescribing information for the treatment or those commonly used in clinical practice. The maximum tolerated dose may have limited or not clinical efficacy in a patient.

1. Exemplary doses of DT-IL3

[00104] In some embodiments, the DT-IL3 is administered at a dose of 1 pg/kg or greater, 2 pg/kg or greater, or 4 pg/kg or greater. In some embodiments, DT-IL3 is administered at a dose of 2 pg/kg to 20 pg/kg or 9 pg/kg to 20 pg/kg. In some embodiments, the DT-IL3 is administered at a dose of 4 mg/kg to 12 gg/kg or 9 gg/kg to 12 gg/kg. In some embodiments, the DT-IL3 is administered at a dose of 5 gg/kg. In some embodiments, the DT-IL3 is administered at a dose of 7 gg/kg. In some embodiments, the DT-IL3 is administered at a dose of 9 gg/kg. In some embodiments, the DT-IL3 is administered at a dose of 12 gg/kg.

[00105] In some embodiments, the DT-IL3 is administered at a dose that is the maximum tolerated dose.

2. Exemplary doses of Jak inhibitors

[00106] In some embodiments, the one or more Jak inhibitors comprises ruxolitinib. In some embodiments, ruxolitinib is dosed at 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, or 25 mg (for example, orally).

[00107] In some embodiments, gandotinib is dosed at 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 30 mg, 60 mg, 90 mg, or 120 mg (for example, orally).

[00108] In some embodiments, momelotinib is dosed at 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 (for example, orally).

[00109] In some embodiments, pacritinib is dosed at 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 (for example, orally).

[00110] In some embodiments, CHZ868 is dosed at 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 (for example, orally).

[00111] In some embodiments, NS-018 is dosed at 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 (for example, orally).

[00112] In some embodiments, SRC is dosed at 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 (for example, orally). [00113] In some embodiments, tofacitinib is dosed at 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, or 30 mg (for example, orally).

[00114] In some embodiments, itacitinib is dosed at 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 (for example, orally).

[00115] In some embodiments, the Jak inhibitor is administered at a dose that is the maximum tolerated dose.

3. Exemplary doses of hypomethylating agents

[00116] In some embodiments, a2acitidine (Vida2a®) is dosed at 75 mg/ m² or less. In some embodiments, a2acitidine is dosed at 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/m/ or 100 mg/m² by continuous intravenous infusion or subcutaneous injection.

[00117] In some embodiments, decitabine (Dacogen®) is dosed at 45 mg/m²/day or less. In some embodiments, decitabine (Dacogen®) is dosed at 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. The infusion may be a single daily continuous intravenous infusion or multiple continuous intravenous infusions in a day.

[00118] In some embodiments, SGI-110 (guadecitabine) is dosed at 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 by subcutaneous injection.

[00119] In some embodiments, the hypomethylating agent is

administered at a dose that is the maximum tolerated dose.

[00120] In some embodiments, the subject is premedicated for nausea and vomiting before administration of the hypomethylating agent.

II. Methods of treatment

[00121] In some embodiments, DT-IL3 is administered for treatment of monocytosis in a subject. In some embodiments, DT-IL3 is administered for treatment of an MPN in a subject with monocytosis. In some embodiments, the MPN is polycythemia vera (PV), essential thrombocythemia (ET), myelofibrosis (MF), chronic myelomonocytic leukemia (CMML), chronic neutrophilic leukemia, chronic eosinophilic leukemia, systemic mastocytosis (SM), symptomatic

hypereosinophilic disorder, or other bone marrow disorder that causes the

production of excess red blood cells, white blood cells, and/ or platelets, or a primary eosinophilic disorder (PED). Types of MPNs are discussed in more detail below in section II.B and monocytosis is discussed in more detail below in section II.A.

[00122] In some embodiments, a DT-IL3 is administered for treatment of myelofibrosis (MF) or polycythemia vera (PV) in a subject with monocytosis.

[00123] In some embodiments, a DT-IL3 is administered with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents comprise (a) one or more Jak inhibitors and/or (b) one more hypomethylating agents. In some embodiments, at least one Jak inhibitor and at least one hypomethylating agent is administered. In some embodiments, the at least one Jak inhibitor comprises ruxolitinib and the at least one hypomethylating agent comprises decitabine, a2acitidine, and/ or SGI-110.

[00124] 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 non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkey and human). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal such as a farm animal (e.g., a horse, pig, or cow) or a pet (e.g., a dog or cat). In some embodiments, the subject is an elderly human. In some embodiments, the subject is a human adult. In some embodiments, the subject is a human child. In some

embodiments, the subject is a pediatric human (i.e. human of less than 18 years of age). In some embodiments, the subject is a human infant.

[00125] In some embodiments, the subject was refractory to prior treatment with one or more Jak inhibitors and/ or one or more hypomethylating agents. As used herein, a subject is“refractory” to prior treatment if the patient has failed to achieve a response to a therapy such that the therapy is determined to not be therapeutically effective, such as: failure to reach clinical endpoint, including any of response, extended duration of response, extended disease free, survival, relapse free survival, progression free survival, and overall survival. In some embodiments, the subject was refractory to prior treatment with ruxolitinib.

[00126] In some embodiments, the subject could not tolerate the full dose of a prior treatment with one or more Jak inhibitors and/ or one or more hypomethylating agents.“Full dose,” as used herein, refers to the dose(s) 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 was below the full dose. In other words, the subject could not tolerate the full dose of a prior treatment because of side effects. In some embodiments, the subject has

myelofibrosis and could not tolerate the full dose of a prior treatment with

ruxolitinib.

[00127] In some embodiments, the subject has myelofibrosis and low platelet counts or was not eligible for treatment with ruxolitinib. In some

embodiments, low platelet counts are measured as a platelet count at or below 5 X 10⁹/L, at or below 10 X 10⁹/L, at or below 20 X 10⁹/L, at or below 30 X 10⁹/L, at or below 40 X 10⁹/L, or at or below 50 X 10⁹/L. In some embodiments, patients may be ineligible for treatment with ruxolitinib due to renal impairment or hepatic impairment.

[00128] In some embodiments, the subject previously responded to a Jak inhibitor and/ or a hypomethylating agent. In some embodiments, the subject who previously responded to a Jak inhibitor and/ or a hypomethylating agent has MF. In some embodiments, the subject previously responded to ruxolitinib. A number of reasons for failure of prior therapy for MF have been characterized, such as myelosuppression, drug resistance, and persistence of the underlying malignant clone (See Pettit and Odenike, Curr. Hematol. Malig. Rep. 12(6):611-624 (2017)). A. Monocytosis

[00129] Monocytosis is characterEed by an increase in the number of monocytes circulating in the blood. Monocytes are white blood cells that give rise to macrophages and dendritic cells in the immune system. Monocytosis is found where the subject has a monocyte count > 1 x 10⁹/L. In some embodiments, the peripheral blood monocytosis (e.g., > 1 x 10⁹/L) cannot be attributed to infectious,

autoimmune/ connective-tissue, or other non-neoplastic etiologies.

[00130] Monocytosis may be determined by manual or automated procedures. Blood is drawn into a test tube containing an anticoagulant (for example, EDTA, sometimes citrate). Before the advent of automated hematology analy2ers, complete blood count (CBC) tests were performed manually, by counting cells in a diluted sample of blood on a device called a hemocytometer, and by viewing a slide prepared with a sample of the patient's blood (a peripheral smear) under a

microscope. However, manual blood cell counts are becoming less common, and instead this process is generally performed by the use of an automated analy2er.

[00131] With an automatic procedure, typically analysis begins when a well-mixed whole blood sample is placed on a rack in the analy2er. The instrument utilEes flow cells, photometers, and apertures in order to analy2e different elements in the blood. The cell counting component counts the numbers and types of different cells within the blood. Blood cell counting occurs by flow cytometry when a very small amount of the specimen is aspirated, diluted, and passes through an aperture and a laser flow cell. Sensors count and identify the number of cells passing through the aperture. The instrument determines the type of blood cell by analy2ing data about the si/e and aspects of light as they pass through the cells. Some instruments measure different characteristics of the cells in order to catcgon/c them.

Sophisticated modern analy2ers can provide extended white blood cell (WBC) differential counts, which include, for example, hematopoietic progenitor cells, immature granulocytes, and erythroblasts. [00132] Manual methods typically utilize hemocytometers, counting chambers that hold a specified volume of diluted blood to enable enumeration with a microscope. The hemocytometers are used to calculate the number of red and white blood cells per volume of blood. To identify the numbers of different white blood cells, a blood film is made on a slide, and a large number of white blood cells (at least 100) are counted using a microscope. This gives the percentage of cells that are of each type. By multiplying these percentages by the total number of white blood cells, the absolute number of each type of white cell can be obtained.

[00133] Manual microscopic counting is useful in cases where automated analyzers cannot reliably count abnormal cells, such as those immature or atypical cells (that are not present in normal patients) and are only seen in peripheral blood with certain haematological conditions. Manual counting may be subject to sampling error because so few cells are counted compared with automated analysis.

[00134] In some embodiments, clinical efficacy is measured by the inhibition or treatment of monocytosis. In some embodiments, inhibition in monocytosis in a subject is measured by a stabilization or decrease in the monocyte count from baseline monocyte count, such as a reduction to less than 1 x 10⁹/L.

B. Myeloproliferative neoplasm

[00135] In some embodiments, the MPN is polycythemia vera (FV), essential thrombocythemia (ET), myelofibrosis (MF), chronic myelomonocytic leukemia (CMML), chronic neutrophilic leukemia, chronic eosinophilic leukemia, systemic mastocytosis (SM), symptomatic hypereosinophilic disorder, or other bone marrow disorder that causes the production of excess red blood cells, white blood cells, and / or platelets. In some embodiments, the MPN is a primary eosinophilic disorder (PED).

[00136] In some embodiments, the MPN is myelofibrosis (MF). MF is characterized by replacement of the bone marrow with scar tissue. With bone marrow scarring, insufficient number of blood cells may be produced and lead to anemia, bleeding problems, and infection risks. The liver and spleen may enlarge to attempt to produce additional blood cells.

[00137] In some embodiments, the MF is primary MF (PMF), post polycythemia vera MF (post-PV MF), post-essential thrombocythemia MF (post-ET MF), primary MF in blast phase (PMF-BP), post-PV MF in blast phase, or post-ET MF in blast phase.

[00138] In some embodiments, the MPN is in blast phase.

[00139] In some embodiments, the MPN is PMF-BP, post-PV MF in blast phase, or post-ET MF in blast phase. In some embodiments, the MPN is identified by physical examination, blood tests, bone marrow aspirate and biopsy, cytogenetic analysis, testing for mutations in the JAK2, MPL, ASXL1, TET2, or CALR gene, arterial oxygen saturation and carboxyhaemoglobin 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.

[00140] Regarding the cytogenetic analysis, cytogenetic abnormalities can serve as prognostic factors and may closely predict survival of the subjects. In some embodiments, cytogenetic testing is performed on bone marrow aspirates at initial diagnosis. Chromosomal abnormalities are considered clonal if the same structural abnormality appears in at least 3 metaphases. Subjects are determined to have a favorable or unfavorable cytogenetics on the basis of survival outcomes. Unfavorable cytogenetics refers to a cytogenetic risk profile associated with an unfavorable outcome. Unfavorable cytogenetics in a subject with MF may include, for example, complex karyotype (> 3 rearrangement abnormalities) or one or two abnormalities that include trisomy 8, deletion 7/7q, inversion 17q, inversion 3, deletion 5/5q, deletion 12p, or llq23 rearrangement, and favorable cytogenetics include all other scenarios, including normal karyotype.

C. Methods of measuring clinical efficacy for MPN

[00141] In some embodiments, inhibiting or treating MPN results in a reduction in the proliferation of MPN cells, a stabilization in the amount of MPN cells, and/ or a reduction in the amount of MPN cells.“MPN cells,” as used herein refers to any abnormal cell of the bone marrow that gives rise to an MPN.

[00142] In some embodiments, inhibiting or treating MPN results in a reduction in spleen and/ or liver size. In some embodiments, reduction in spleen size is at least 25%, at least 29%, at least 33%, at least 35%, at least 40%, or at least 50%. In some embodiments, the spleen size is measured by palpation. In some

embodiments, spleen size is measured by volumetric analysis, such as magnetic resonance imaging (MRI) or computed tomography (CT), at different timepoints pre- and post-treatment. In some embodiments, the subject with MPN has an abnormally large spleen (i.e., splenomegaly) or abnormally large liver. In some embodiments, splenomegaly is defined as > 5 cm below costal margin (BCM) by physical examination.

[00143] In some embodiments, the subject with MPN does not have an abnormally large spleen or abnormally large liver. In some embodiments, a subject with MPN is without baseline splenomegaly (£ 5cm BCM).

[00144] In some embodiments, the 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 spleen size. In some embodiments, the reduction in spleen size is a reduction of >33% in spleen size in subjects with baseline splenomegaly. Subjects who meet parameters for reduction in spleen measures are treatment“spleen responders.”

[00145] In some embodiments, clinical efficacy is measured by improvements in splenomegaly. In some embodiments, improvements in

splenomegaly in a group of subjects is measured by the percentage of spleen responders.

[00146] In some embodiments, improvements in splenomegaly is measured by the percent change in splenomegaly as measured by the cm palpable below the left costal margin. [00147] In addition to the above response criteria, response criteria for myelofibrosis (MF) include, but are not limited to, the exemplary response criteria in Table 2 below from the International Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) and European LeukemiaNet (ELN) for MF.

[00148] 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 coastal margin, and UNL = upper limit of normal. * Baseline and posttreatment bone marrow slides are to be interpreted at one sitting by a central review process. Cytogenetic and molecular responses are not required for CR assignment.† Grading of MF is according to the European classification (Thiele et al., Haematologica 90:1128 (2005)). f Immature myeloid cells constitute blasts + promyelocytes + myelocytes + metamyelocytes + nucleated red blood cells. In splenectomized patients, <5% immature myeloid cells is allowed. § See above for definitions of anemia response, spleen response, and progressive disease. Increase in severity of anemia constitutes the occurrence of new transfusion dependency or a >20 g/L decrease in hemoglobin level from

pretreatment baseline that lasts for at least 12 weeks. Increase in severity of thrombocytopenia or neutropenia is defined as a 2-grade decline, from pretreatment baseline, in platelet count or absolute neutrophil count, according to the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. In addition, assignment to Cl requires a minimum platelet count of >25 000 x 10(9)/L and absolute neutrophil count of >0.5 x 10(9)/L. D Applicable only to patients with baseline hemoglobin of <100 g/L. In patients not meeting the strict criteria for transfusion dependency at the time of study enrollment, but have received transfusions within the previous month, the pretransfusion hemoglobin level should be used as the baseline. Transfusion dependency before study enrollment is defined as transfusions of at least 6 units of packed red blood cells (PRBC), in the 12 weeks prior to study enrollment, for a hemoglobin level of <85 g/L, in the absence of bleeding or treatment-induced anemia. In addition, the most recent transfusion episode must have occurred in the 28 days prior to study enrollment. Response in transfusion-dependent patients requires absence of any PRBC transfusions during any consecutive“rolling” 12-week interval during the treatment phase, capped by a hemoglobin level of >85 g/L. # In splenectomi2ed patients, palpable hepatomegaly is substituted with the same measurement strategy. ** Spleen or liver responses must be confirmed by imaging studies where a >35% reduction in spleen volume, as assessed by MRI or CT, is required. Furthermore, a >35% volume reduction in the spleen or liver, by MRI or CT, constitutes a response regardless of what is reported with physical examination.†† Symptoms are evaluated by the MPN-SAF TSS.17 The MPN-SAF TSS is assessed by the patients themselves and this includes fatigue, concentration, early satiety, inactivity, night sweats, itching, bone pain, abdominal discomfort, weight loss, and fevers. Scoring is from 0 (absent/ as good as it can be) to 10 (worst imaginable/ as bad as it can be) for each item. The MPN-SAF TSS is the summation of all the individual scores (0-100 scale). Symptoms response requires >50% reduction in the MPN-SAF TSS. ff Progressive disease assignment for splenomegaly requires confirmation my MRI or computed tomography showing a >25% increase in spleen volume from baseline. Baseline values for both physical examination and imaging studies refer to pretreatment baseline and not to

posttreatment measurements.

III. Administration of agents

[00149] These methods comprise a variety of administration schedules of DT-IL3. [00150] In some embodiments, the DT-IL3 is administered at least once a week. In some embodiments, the DT-IL3 is administered at least two times a week. In some embodiments, the DT-IL3 is administered at least three times a week.

[00151] In some embodiments, the one or more additional agents are administered at least once a week, at least two times a week, at least three times a week, every other day, or every day. In some embodiments, the one or more additional agents are administered more than one time a day, twice a day, or three times per day. In some embodiments, the DT-IL3 and the one or more additional agents are administered over a period of one week or more, or a period of two weeks or more. In some embodiments, the DT-IL3 and the one or more additional agents are administered for at least 3 consecutive days. In some embodiments, the one or more additional agents are one or more Jak inhibitors and/ or one or more

hypomethylating agents.

A. Treatment cycles

[00152] In some embodiments, the DT-IL3 is administered in multiple treatment cycles. As used herein,“treatment cycle” refers to a period of

administration of the DT-IL3. In some embodiments, there is a single treatment cycle. In some embodiments, there are multiple treatment cycles.

[00153] In some embodiments, there is a period of time between multiple treatment cycles when the DT-IL3 is not administered. In some

embodiments, 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.

[00154] In some embodiments, the DT-IL3 is administered for at least 3 consecutive days every 21 days for four cycles, followed by every 28 days for 3 cycles, and then every 42 days. In some embodiments, one or more additional agents are administered concurrentiy with the DT-IL3. [00155] In some embodiments, the DT-IL3 is administered until disease progression and/ or unacceptable toxicity is obtained as determined, for example, by a treating physician.

[00156] In some embodiments, the one or more additional agents are administered for at least the first 3 days, at least the first 4 days, at least the first 5 days, at least 6 days, or at least 7 days of at least one cycle.

[00157] In some embodiments, the one or more Jak inhibitors and/ or 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 3 cycles. In some embodiments, DT-IL3 is administered concurrentiy with the one or more Jak inhibitors and/ or the one or more hypomethylating agents. In some embodiments, the one or more Jak inhibitors and / or the one or more hypomethylating agents are administered following administration of DT-IL3 for four 21 -day cycles.

IV. Pharmaceutical compositions

[00158] In some embodiments, a pharmaceutical composition for treating or inhibiting MPN in a subject with monocytosis in need thereof comprises an effective amount of a DT-IL3. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[00159] In some embodiments, the invention provides a pharmaceutical composition comprising an effective amount of a conjugate of the invention and a pharmaceutically acceptable carrier or vehicle. In some embodiments, a

pharmaceutical composition comprises an effective amount of a conjugate of the invention and a pharmaceutically acceptable carrier or vehicle. The pharmaceutical compositions are suitable for veterinary and/ or human administration.

[00160] The pharmaceutical compositions of the present invention can be in any form that allows for the composition to be administered to a subject.

[00161] Materials used in preparing the pharmaceutical compositions can be non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of subject (e.g., human), the overall health of the subject, the type of cancer the subject has, the use of the composition as part of a multi-drug regimen, the particular form of the composition of the invention, and the manner of administration.

[00162] The term“carrier” refers to a diluent, adjuvant or excipient, with which a composition of the invention is administered. Any auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to a subject, the compositions of the invention and pharmaceutically acceptable carriers are sterile. Water may be a carrier when the composition of the invention is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The present compositions, if desired, can also contain minor amounts of pH buffering agents.

[00163] The liquid compositions of the invention, whether they are solutions, suspensions, or other like form, can also include one or more of the following: sterile diluents such as water for injection, saline solution, physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. A parenteral composition can be enclosed in an ampoule, a disposable syringe, or a multiple-dose vial made of glass, plastic or other material. In some embodiments, physiological saline is an adjuvant. An injectable composition may be sterile. [00164] The pharmaceutical compositions comprise an effective amount of a composition of the invention such that a suitable dosage will be obtained.

Typically, this amount is at least 0.01% of the composition of the invention by weight of the pharmaceutical composition. When intended for oral administration, this amount can be varied to be between 0.1% and 80% by weight of the pharmaceutical composition. Oral pharmaceutical compositions may comprise from between 4% and 50% of the composition of the invention by weight of the pharmaceutical

composition. Pharmaceutical compositions may be prepared so that a parenteral dosage unit contains from between 0.01% and 2% by weight of the composition of the invention.

[00165] The pharmaceutical compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa, etc.). Administration can be systemic or local. Various delivery systems are known, e.g., microparticles, microcapsules, capsules, etc., and may be useful for administering a composition of the invention. Methods of administration may include, but are not limited to, oral administration and parenteral administration; parenteral

administration including, but not limited to, intradermal, intramuscular,

intrap eritoneal, intravenous, subcutaneous; intranasal, epidural, sublingual, intranasal, intracerebral, intraventricular, intrathecal, intravaginal, transdermal, rectally, by inhalation, or topically to the ears, nose, eyes, or skin. The mode of administration is left to the discretion of the practitioner, and will depend, in-part, upon the site of the medical condition (such as the site of cancer, a cancerous tumor, or a pre-cancerous condition).

[00166] In some embodiments, the compositions of the invention are administered parenterally. In some embodiments, the compositions of the invention are administered intravenously. In another embodiment, the compositions of the invention are administered by continuous infusion. In a particular embodiment, the compositions of the invention are administered by an infusion that lasts for 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, or 2 hours.

[00167] In some embodiments, it can be desirable to administer one or more compositions of the invention locally to the area in need of treatment. This can be achieved, for example, and not by way of limitation, by local infusion during surgery; topical application, e.g., in conjunction with a wound dressing after surgery; by injection; by means of a catheter; by means of a suppository; or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In certain embodiments, one or more compositions of the invention can be injected intraperitoneally.

[00168] In yet another embodiment, the compositions of the invention can be delivered in a controlled release system.

[00169] In some embodiments, a pump can be used to deliver the compositions of the invention (see, e.g., Sefton, CRC Crit. Ref. Biomed. Eng. 1987, 14, 201; Buchwald et al., Surgery 1980, 88: 507; Saudek et al., N. Engl. J. Med. 1989, 321: 574). In some embodiments, the pump may be, but is not limited to, an insulin-like pump.

[00170] The present compositions can take the form of solutions, suspensions, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays,

suspensions, or any other form suitable for use. Examples of suitable pharmaceutical carriers are described in emington's Pharmaceutical Sciences by E.W. Martin.

[00171] In some embodiments, the compositions of the invention are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to animals, particularly human beings.

Typically, the carriers or vehicles for intravenous administration are sterile isotonic aqueous buffer solutions. Where necessary, the compositions can also include a solubilizing agent. Compositions for intravenous administration can optionally comprise a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where a composition of the invention is to be administered by infusion, it can be dispensed, for example, with an infusion botde containing sterile

pharmaceutical grade water or saline. Where the composition of the invention is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[00172] The compositions of the present invention can comprise an additional active agent selected from among those including, but not limited to, an additional prophylactic agent, an additional therapeutic agent, an antiemetic agent, a hematopoietic colony stimulating factor, an adjuvant therapy, a vaccine or other immune stimulating agent, an antibody/ antibody fragment-based agent, an anti depressant and an analgesic agent. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier or vehicle.

[00173] The pharmaceutical compositions can be prepared using methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining a

composition of the invention with water so as to form a solution. A surfactant can be added to facilitate the formation of a homogeneous solution or suspension.

Surfactants are complexes that can non-covalentiy interact with a composition of the invention so as to facilitate dissolution or homogeneous suspension of the composition of the invention in the aqueous delivery system.

EXAMPLES

Example 1. Strong Association of Positive Clinical Outcomes with

Monocytosis

[00174] A non-randomized, open label, dose escalation multicenter study was conducted to determine the optimal dosage of DT-IL3 in advanced, high- risk MPN subjects. Subjects > 18 years of age (a median age of 69) with chronic myelomonocytic leukemia (CMML), MF, advanced systemic mastocytosis (SM), or advanced symptomatic primary eosinophilic disorder (PED) were administered 7, 9, or 12 pg/kg DT-IL3 via IV infusion. No intra-patient dose escalation was allowed. DT-IL3 was administered on days 1-3 of a 21 -day cycle for four cycles (cycles 1-4), followed by a 28-day cycle for three cycles (cycles 5-7), and a 42-day cycle thereafter. Extension of cycles 1-4 to 28 days as permitted in the setting of specific adverse events (AEs) and/ or other difficulties during Cycle 1 and in subsequent cycles. Subjects with evidence of response (or disease control) at the end of Cycle 7 (after approximately 24 weeks of treatment) could opt to continue treatment beyond Cycle 7 or to remain on-study without treatment but with ongoing observation.

[00175] Physical examination and clinical assessments were performed at day 1, 8, 15, and 21 of cycles 1-4, day 1, 15, and 28 during cycles 5-7, and then every 12 weeks for cycle 8 and over. Clinical assessments included, for example, an assessment of hematology, serum albumin, serum chemistry and electrolytes, coagulation parameters, and urinalysis. The hematology assessment included, for example, white blood cell (WBC) count, differential white cell count (lymphocytes, monocytes, basophils, eosinophils, and neutrophils), red blood cell (RBC) count, hematocrit, hemoglobin (Hb), and platelet count.

[00176] Twenty three subjects had MF, of which 13 (57%) had primary MF (PMF), 4 (17%) had post-polycythemia MF, and 2 (9%) subjects had post essential thrombocythemia MF (3 patients did not have these data available at the time of the data cutoff). Of the 23 subjects with MF, 16 subjects had baseline splenomegaly (> 5 cm below coastal margin (BCM) by physical exam). See Table 3 below.

[00177] In Table 3, a = Measured by physical exam (cm below coastal margin); b = Monocyte count > 1 x 10⁹/L; c = Platelet count £50 x 10⁹/L; d = Platelet count <100 x 10⁹/L; JAKi = JAK inhibitor; HMA = hypomethylating agent; Benda = bendamustine; PST = prior systemic therapy; SCT=stem cell transplant; Invest. Agent = patient had previous treatment with a different investigative agent; N/A = not applicable /no measurement currentiy available; and N/E = not evaluable.

[00178] Nine of the 16 (56%) MF subjects with baseline splenomegaly had a spleen response. See Table 3 above, and Figure 1. Splenomegaly reduction is accepted by the FDA as a primary endpoint in PMF. The study demonstrated that DT-IF3 treated patients have splenomegaly reductions.

[00179] It was unexpectedly found that splenomegaly reductions (any

%), were significantly more prevalent in patients with monocytosis (i.e., had a monocyte count > 1 x 10⁹/F). See Table 3 above and Table 4 below.

[00180] In Table 4, a = patients with palpable spleen at baseline; and BCM = below coastal margin (by physical exam).

[00181] Moreover, while for evaluable patients with monocytosis and splenomegaly, the response rate was 100% (5/ 5), see Tables 3 and 4 and Figure 2, all patients that showed an increase in splenomegaly did not have monocytosis (Table 3). Furthermore, this enrichment effect of sensitivity to DT-IL3 in patients with MF and monocytosis is maintained when splenomegaly responses were sub-grouped by all size reductions, reductions equal or greater than 29%, and equal or greater than 45% See Tables 3 and 4.

[00182] This data shows a strong association of positive clinical outcomes with DT-IL3 treatment in the presence of monocytosis.

Example 2. Clinical trials of MPN patients with monocytosis

[00183] A clinical trial may be used to further assess the efficacy of a DT-IL3 in MPN patients with monocytosis.

[00184] The patients in the trial may have an MPN with or without monocytosis.

[00185] Patients may be diagnosed with an MPN based on physical examination, blood tests, bone marrow aspirate and biopsy, cytogenetic analysis, testing for mutations in the JAK2, MPL, ASXL1, TET2, or CALR gene, arterial oxygen saturation and carboxyhaemoglobin levels, neutrophil alkaline phosphatase levels, vitamin B 12 or B 12 binding capacity, or serum urate levels.

[00186] In some embodiments, the patient has mutations in the JAK2, MPL, ASXL1, TET2, or CALR gene. In some embodiments, the patient has the JAK2V617F mutation.

[00187] In some embodiments, the MPN is myelofibrosis (MF) or polycythemia vera (PV). Within the study, patients may have the same or different forms of MPN.

[00188] Patients may be determined to have monocytosis based on standard monocytes counts (> 1 x 10⁹/L). [00189] Any of the doses of agents and treatment cycles described in this application may be used. In some embodiments, the dose is 12 pg/kg via IV infusion. The DT-IL3 may be DT₃₈sIL-3.

[00190] The trial may have an adaptive design. In other words, the trial may allow modifications to the trial and/ or procedures during the trial. Alternatively, the trial may have set treatments groups that are maintained throughout the trial.

[00191] Treatment groups may include comparison of DT-IL3 administration in MPN patients with and without monocytosis. For example, treatment groups may include comparison of DT-IL3 in MF or PV patients with and without monocytosis.

[00192] Treatment success or failure in MPN patients may be assessed by standard measures used in MPN trials, such as reduction in spleen and/ or liver size. Changes in blood composition, disease recurrence, and patient survival would also be assessed over treatment. Toxicities and adverse events would be measured during treatment. Response criteria for MF patients may include, for example, the criteria listed in Table 2 above.

[00193] Applicant expects that the treated MPN patients with monocytosis will see improved clinical improvement over MPN patients without monocytosis.

EQIJ IVALENTS

[00194] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[00195] As used herein, all numeric values are presumed to“about.” As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., +/ -5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.

Claims

What is Claimed is:

1. A method for inhibiting or treating monocytosis in a subject in need thereof, comprising administering to the subject a diphtheria toxin-human interleukin-3 conjugate (DT-IL3).

2. A method for treating or inhibiting myeloproliferative neoplasm (MPN) cells in a subject having an MPN and monocytosis, comprising administering an effective amount of a diphtheria toxin-human interleukin-3 conjugate (DT-IL3) to the subject.

3. A method for treating or inhibiting an MPN in a subject in need thereof,

comprising administering to the subject a diphtheria toxin-human interleukin-3 conjugate (DT-IL3), wherein the subject has monocytosis.

4. A method for treating an MPN in a subject in need thereof, comprising: a. determining whether a subject with the MPN has monocytosis, and b. administering an effective amount of a diphtheria toxin-human interleukin-3 conjugate (DT-IL3) to the subject if the subject is determined to have monocytosis.

5. The method of any one of claims 2 to 5, wherein the method results in a reduction in the proliferation of MPN cells, a stabilization in the amount of MPN cells, a reduction in the amount of MPN cells, and/ or a reduction in spleen and/ or liver size.

6. The method of claim 5, wherein said stabilization or reduction is measured by blood tests; blast count; blast percentage; physical examination; complete blood count; flow cytometric analyses; bone marrow analyses; hematopoietic function; marrow blast index; the amount of normal white blood cells, red blood cells, and/ or platelets; histology; immunohistochemistry; frequency of transfusion; and/ or bone marrow biopsy.

7. The method of any one of claims 2 to 6, wherein the method results in the inhibition and/ or treatment of the monocytosis.

8. The method of any one of claims 2 to 7, wherein the cells of the MPN express the IL-3 receptor.

9. The method of any one of claims 2 to 8, wherein the growth of the MPN cells is inhibited.

10. The method of any one of claims 1 to 9, wherein the DT-IL3 is administered at a dose of 0.1 pg/kg to 50 pg/kg.

11. The method of any one of claims 1 to 10, wherein the DT-IL3 is administered at a dose of 4 pg/kg to 50 pg/kg.

12. The method of any one of claims 1 to 11, wherein the DT-IL3 is administered at a dose of 4 pg/kg to 20 pg/kg.

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

14. The method of any one of claims 1 to 13, wherein the DT-IL3 is administered at a dose of 5 pg/kg, 7 pg/kg, 9 pg/kg, or 12 pg/kg.

15. The method of any one of claims 1-9, wherein the DT-IL3 is administered at a dose that is the maximum tolerated dose.

16. The method of any one of claims 1-15, wherein the DT-IL3 is administered at least once a week.

17. The method of any one of claims 1-16, wherein the DT-IL3 is administered at least two times a week.

18. The method of any one of claims 1-17, wherein the DT-IL3 is administered at least three times a week.

19. The method of any one of claims 1-18, wherein the DT-IL3 is administered over a period of one week or more.

20. The method of any one of claims 1-19, wherein the DT-IL3 is administered over a period of two weeks or more.

21. The method of any one of claims 1-20, wherein the conjugate is administered once every day for three days.

22. The method of any one of claims 1-20, wherein the conjugate is administered once every day for five days.

23. The method of any one of claims 1 to 22, wherein the DT-IL3 is administered in multiple treatment cycles.

24. The method of claim 23, 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.

25. The method of claim 23 or claim 24, wherein the DT-IL3 is administered for at least 3 consecutive days of a 21-day cycle for four cycles, followed by at least three consecutive days of a 28-day cycle for 3 cycles.

26. The method of claim 25, further comprising at least three consecutive days of one or more 42-day cycles following the three 28-day cycles.

27. The method of any one of claims 1 to 26, wherein the DT-IL3 is administered until disease progression and/ or unacceptable toxicity is obtained.

28. The method of any one of claims 1 to 27, wherein the subject is human.

29. The method of claim 28, wherein the human has unfavorable cytogenetics.

30. The method of any one of claims 1 to 29, wherein the DT-IL3 is a chemical conjugate.

31. The method of any one of claims 1 to 30, wherein the DT-IL3 is a

recombinantiy expressed protein.

32. The method of any one of claims 1 to 31, wherein the DT-IL3 is expressed as a single polypeptide comprising the catalytic and translocation domains of diphtheria toxin and human IL-3.

33. The method of any one of claims 1 to 32, wherein the DT-IL3 comprises amino acid residues 1 to 388 of diphtheria toxin linked via a peptide bond to human IL-3.

34. The method of any one of claims 2 to 33, wherein the MPN is polycythemia vera (PV), essential thrombocythemia (ET), myelofibrosis (MF), chronic

myelomonocytic leukemia (CMML), chronic neutrophilic leukemia, chronic eosinophilic leukemia, systemic mastocytosis (SM), symptomatic hypereosinophilic disorder, or other bone marrow disorder that causes the production of excess red blood cells, white blood cells, and/ or platelets, or a primary eosinophilic disorder (PED).

35. The method of claim 34, wherein the MPN is polycythemia vera (PV)

36. The method of claim 35, wherein the MPN is myelofibrosis (MF).

37. The method of claim 36, wherein the myelofibrosis is primary myelofibrosis, post-polycythemia vera myelofibrosis, post-essential thrombocythemia myelofibrosis, blast phase primary myelofibrosis, post-polycythemia vera myelofibrosis in blast phase, or post-ET myelofibrosis in blast phase.

38. The method of any one of claims 2 to 37, wherein the MPN is refractory to at least one prior MPN treatment.

39. The method of any one of claims 2 to 37, wherein the subject is in a state of remission from the MPN.

40. The method of any one of claims 1 to 39, wherein the subject has been previously treated with a therapeutic agent and/ or has undergone radiation therapy.

41. The method of any one of claims 1 to 40, wherein the subject is currently being administered a therapeutic agent other than a human IL-3-diphtheria toxin conjugate and/ or is undergoing radiation therapy.

42. The method of any one of claims 2 to 38, 40 and 41, wherein the subject has relapsed from prior MPN treatment.

43. The method of any one of claims 1 to 42, wherein the subject has failed prior MPN treatment.

44. The method of any one of claims 1 to 43, wherein the subject is susceptible to adverse reactions from other MPN therapies.

45. The method of any one of claims 1 to 44, wherein the subject is refractory to chemotherapy.

46. The method of any one of claims 1 to 39, wherein the subject has not been previously treated for the MPN.

47. The method of any one of claims 1 to 46, further comprising administering one or more Jak inhibitors and/ or one or more hypomethylating agents.

48. The method of claim 47, wherein at least one Jak inhibitor and at least one hypomethylating agent is administered.

49. The method of claim 47 or claim 48, wherein the one or more Jak inhibitors comprises ruxolitinib.

50. The method of any one of claims 47 to 49, wherein the one or more hypomethylating agents comprise a2acitidine, decitabine, and/or SGI-110.

51. The method of any one of claims 47 to 50, wherein the subject is administered a pharmaceutical composition comprising the DT-IL3 and the (a) one or more Jak inhibitors and/ or (b) one or more hypomethylating agents.

52. The method of any one of claims 1 to 51, wherein the subject with

monocytosis has a monocyte count equal to or greater than 1 x 10⁹/L.

53. A pharmaceutical composition for treating or inhibiting an MPN in a subject in need thereof comprising an effective amount of a diphtheria toxin-human interleukin-3 conjugate (DT-IL3), wherein the subject has monocytosis.

54. The pharmaceutical composition of claim 53, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

55. The pharmaceutical composition of claim 53 or 54, further comprising one or more Jak inhibitors and/ or one or more hypomethylating agents.

56. The pharmaceutical composition of any one of claims 53 to 55, wherein the MPN is myelofibrosis.

57. The pharmaceutical composition of any one of claims 53 to 55, wherein the MPN is polycythemia vera.

58. The method of any one of claims 53 to 57, wherein the subject with monocytosis has a monocyte count equal to or greater than 1 x 10⁹/L.