Lymphotoxin alpha for use in therapy of myeloid leukemia

Abstract

To provide substances for use in the treatment of myeloid diseases and myeloid neoplasms, which enable longer and complete remission of the disease, cause little or no side effects, and are expected to lead to prolonged overall survival of these patients.SOLUTION: The present invention provides a polypeptide for use in the treatment of myeloid diseases or myeloid neoplasms, the polypeptide comprising the amino acid sequence of full length human lymphotoxin-α or of mature human lymphotoxin-α, or comprising an amino acid sequence having at least 80% identity to the amino acid sequences, and a pharmaceutical composition and kit comprising the polypeptide.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to polypeptides comprising the amino acid sequence of full-length human lymphotoxin alpha or mature human lymphotoxin alpha, or a sequence having at least 80% identity to one of these amino acid sequences, for use in the treatment of bone marrow diseases or myeloid neoplasms, pharmaceutical compositions comprising such polypeptides for use in the treatment of bone marrow diseases or myeloid neoplasms, and kits comprising such polypeptides for use in the treatment of bone marrow diseases or myeloid neoplasms. [Background technology]

[0002] Acute myeloid leukemia (AML) is a heterogeneous disorder characterized by the clonal proliferation of primitive myeloid lineage cells (blasts) in the bone marrow and peripheral blood, resulting in bone marrow failure. Recent studies have revealed that AML, chronic myeloid leukemia (CML), and other bone marrow disorders arise from the sequential acquisition of recurrent genetic alterations within hematopoietic stem cells (HSCs). Many somatic mutations in AML patients result in the inhibition of myeloid differentiation or induce self-renewal in primitive hematopoietic stem and progenitor cells (HSPCs). In CML patients, genetic alterations in the same cell type, i.e., HSPCs, lead to their increased proliferation. Similar mechanisms have been suggested in the development of other bone marrow disorders. The acquisition of additional genetic abnormalities within the pool of preleukemic HSPCs ultimately gives rise to leukemic stem cells (LSCs), which play a role in a wide range of bone marrow disorders, including AML, CML, myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), and others.

[0003] Despite rapid advances in the field, including new drug targets and an increased understanding of the biology of CML alongside AML, the general approach to current treatment strategies has not changed substantially for myeloid diseases. For example, the standard of care for AML consists of an induction phase involving the combination of cytarabine and an anthracycline in a standard intensive regimen called "7+3" chemotherapy, followed by consolidation chemotherapy.

[0004] This traditional approach achieves complete remission (CR) in 60%-80% of young adults and 40%-60% of elderly patients (generally defined as over 60 years of age) (Non-Patent Document 1). However, most are not cured, and long-term overall survival (OS) has changed little over the past few decades, with 5- and 3-year OS rates ranging from 23% to 35%. For elderly patients, who represent the majority of patients diagnosed with AML, outcomes have improved but remain particularly grim, with current expectations persisting for a median OS of less than one year from diagnosis.

[0005] Allogeneic stem cell transplantation is commonly considered as a strategy to extend remission, however, such transplantation is subject to other limitations and complications, including the accessibility of suitable donor cells, comorbidities, and individual variability in response to stem cell therapy.

[0006] One major obstacle to long-term remission in patients with bone marrow diseases or neoplasms, particularly AML and CML, is the persistence of LSCs even after rigorous treatment. While standard chemotherapy can effectively eliminate the majority of tumor cells (proliferating leukemic blasts), LSCs are relatively resistant to these treatments and can reactivate and maintain the disease (Non-Patent Document 2). Nevertheless, multiagent chemotherapy remains the standard general therapy for patients with bone marrow diseases, despite its various drawbacks, including high toxicity and low success rates. Therefore, it is essential to find alternative approaches to induce long-term remission and prevent relapse that target AML and LSCs, which may be the cause of relapse in various bone marrow stem cell diseases.

[0007] Various bone marrow stem cell disorders originate from the hematopoietic stem cell pool, including HSPCs. Although the metabolic abnormalities differ among the various types of disease at later stages, the abnormal growth behavior is largely the same among these bone marrow stem cell disorders. Therefore, approaches directed at the early stages of the disease and involving the hematopoietic stem cell pool appear promising in this setting.

[0008] Currently, only a few targeted therapies are available, each useful only for a small subgroup of patients who exhibit specific biomarkers. Furthermore, such targeted therapies can cause significant side effects that impair the quality of life of treated patients, potentially making such treatments unsuitable for application to elderly patients overall.

[0009] To date, research into cell death in AML has focused on apoptosis, but in recent years there has been great interest in activating alternative forms of cell death, such as necroptosis, as a novel therapeutic strategy (Non-Patent Document 3).

[0010] Necroptosis is a form of programmed necrosis mediated by the interaction of RIPK1 (receptor-interacting protein kinase 1) and RIPK3 in the absence of caspase-8 activation. Necroptosis is triggered by death receptor activation, the same stimulus that normally activates apoptosis. However, necroptosis is distinct from apoptosis because it does not involve key apoptotic regulators, such as caspases and Bcl-2 family members, or mitochondrial release of cytochrome c. Furthermore, necroptosis is morphologically distinct from apoptosis, involving membrane rupture and the release of cytoplasmic contents from dying cells, including cytokines, chemokines, and danger signals (damage-associated molecular patterns; DAMPs), which trigger inflammation.

[0011] We previously demonstrated that HSPCs undergoing FLT3-ITD or AML-ETO-driven transformation initiate RIPK3-mediated necroptosis and inflammasome activation as a tumor-suppressive mechanism. In this context, necroptosis demonstrated dual functionality by triggering LSC death and, in addition, propagating myeloid differentiation through the release of significant amounts of IL-1β, further limiting leukemogenesis. Consequently, AML arises from LSCs that successfully suppress the necroptotic pathway. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Dohner, H. et al. (2017). Blood 129, 424-447 [Non-patent document 2] Shlush, LI et al. (2014). Nature 506, 328-333 [Non-patent document 3] Hoeckendorf, U. et al. (2016). Cancer Cell 30, 75-91. Summary of the Invention [Problem to be solved by the invention]

[0013] Therefore, starting from and building upon previous research and the state of the art in the field of treatment of bone marrow diseases and myeloid neoplasms, it is an object of the present invention to provide useful and advantageous agents for use in the treatment of such bone marrow diseases and neoplasms, which are expected to enable longer and more complete remission of the disease, with little or no side effects, and to lead to increased long-term overall survival of these patients. [Means for solving the problem]

[0014] These objects have been achieved by the aspects of the present invention set out below.

[0015] According to a first aspect of the present invention, there is provided a method for treating a myeloid disease or myeloid neoplasm, comprising the amino acid sequence of SEQ ID NO: 1 (full-length human LT-α) or SEQ ID NO: 2 (mature human LT-α), or having at least 80% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. A polypeptide comprising an amino acid sequence of

[0016] According to a preferred embodiment of the first aspect of the present invention, the bone marrow disorder is a myeloid stem cell disorder, preferably one of myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), blastic plasmacytoid dendritic cell neoplasm (BPDCN), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), eosinophilia, and myeloid neoplasms associated with PDGFRA, PDGFRB or FGFR1 rearrangements, or PCM1-JAK2; more preferably, the bone marrow disorder is one of acute myeloid leukemia (AML) or chronic myelogenous leukemia (CML); even more preferably, the bone marrow disorder is acute myeloid leukemia (AML).

[0017] According to another preferred embodiment of the first aspect of the present invention, the polypeptide is capable of affecting a TNF receptor superfamily (TNFRSF)-dependent signaling cascade, preferably a TNF receptor (TNFR) 1 (TNFRSF1A), TNFR2 (TNFRSF1B), lymphotoxin beta receptor (TNFRSF3) or HVEM (TNFRSF14)-dependent signaling cascade, more preferably the signaling cascade is TNFR1 and / or TNFR2-dependent.

[0018] According to a preferred embodiment of the first aspect of the invention, the relevant signalling pathway affected by the polypeptide is fully or partially functional in the individual to be treated.

[0019] According to a preferred embodiment of the first aspect of the present invention, the polypeptide is capable of inducing programmed cell death, preferably the polypeptide is capable of inducing programmed cell death only in one or more of leukemia cells, leukemia progenitor cells, and leukemia stem cells.

[0020] According to a preferred embodiment of the first aspect of the present invention, the polypeptide is a recombinant polypeptide or a purified endogenous polypeptide, preferably a recombinant polypeptide.

[0021] According to another preferred embodiment of the first aspect of the invention, the polypeptide is used in the treatment of mammals, more preferably the polypeptide is used in the treatment of humans.

[0022] According to yet another preferred embodiment of the first aspect of the present invention, the polypeptide consists of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, preferably the polypeptide consists of the amino acid sequence of SEQ ID NO:2.

[0023] According to a second aspect of the present invention there is provided a pharmaceutical composition for the treatment of a bone marrow disease or myeloid neoplasm, the pharmaceutical composition comprising a polypeptide according to the first aspect of the invention and at least one pharmaceutically acceptable excipient.

[0024] According to a preferred embodiment of the second aspect of the present invention, the pharmaceutical composition further comprises one or more substances selected from the group comprising one or more SMAC mimetics such as birinapant, one or more chemotherapeutic agents such as cytarabine (cytosine arabinoside) or cerbidine (daunorubicin), and one or more TNF inhibitors such as Humira (adalimumab), Remicade (infliximab), Simponi (golimumab), Cimzia (certolizumab pegol).

[0025] According to another preferred embodiment of the second aspect of the present invention, the pharmaceutical composition is administered to the patient by systemic administration, preferably by intravenous or subcutaneous administration, More preferably, it is administered to the patient by intravenous administration.

[0026] According to yet another preferred embodiment of the second aspect of the present invention, the pharmaceutical composition does not comprise any type of TNF receptor molecule, including antibodies to TNFR1 (SEQ ID NO: 3), TNFR2 (SEQ ID NO: 4), lymphotoxin beta receptor (SEQ ID NO: 5), HVEM (SEQ ID NO: 6), or lymphotoxin, or antibodies to any type of TNF receptor, including TNFR1, TNFR2, HVEM, and lymphotoxin beta receptor.

[0027] According to a third aspect of the present invention there is provided a kit for treating a bone marrow disease or myeloid neoplasm comprising a polypeptide according to the first aspect of the invention and a container. [Brief explanation of the drawings]

[0028] [Figure 1]This figure shows that LT-α deficiency promotes FLT3-ITD-induced myeloproliferation by accumulating leukemic stem and progenitor cells, whereas TNF deficiency attenuates FLT3-ITD-induced myeloproliferation by failing to produce leukemic cells in the bone marrow. (a) Experimental design; (b) Survival of mice transplanted with BM from FLT3-ITD-transduced WT, Tnf- / -, LTa- / -, or LtaΔ / Δ mice. Median survival: WT FLT3-ITD → WT 42 days vs. Tnf- / - FLT3-ITD → Tnf- / - 47 days vs. LTa- / - FLT3-ITD → LTa- / - 34 days vs. LTaΔ / Δ FLT3-ITD → LTa- / - 28.5 days. Data are representative of two independent experiments. The number of mice is indicated in the figure; (c) Frequency of GFP+ cells in the bone marrow (BM), peripheral blood (PB), spleen (SPL), and liver (LIV); (d) Survival of mice serially transplanted with GFP+ splenocytes from the primary FLT3-ITD-transplanted mice in (b). LTa- / - FLT3-ITD → LTa- / - median survival time was 257 days, and LTaΔ / Δ FLT3-ITD → LTa- / - median survival time was 260 days. Data are representative of two independent experiments. The number of mice is indicated in the figure; (e) Frequency of GFP+ cells in the BM, PB, SPL, and LIV from the mice in (d); each dot represents a mouse, and error bars represent the mean ± SEM. p values ​​are based on the Mantel-Cox test (b, d); otherwise, by Student's t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 2]Figure 1 shows that TNF signaling promotes self-renewal of FLT3-ITD mutant LSCs, while LT-α restricts it. (a) Experimental design and colony counts of 5-FU-enriched HSPCs. Numbers of colony-forming units (CFUs) are shown—granulocytes, erythrocytes, macrophages, and megakaryocytes (GEMMs) (n = 5 biological replicates for WT and LtaΔ / Δ, n = 4 for Ripk3− / −, and n = 3 for Lta− / − and Tnf− / −). The p-values ​​shown were determined by comparison with WT. Data are representative of at least two independent experiments; (b) Experimental design; (c) GEMM colony counts of FLT3-ITD-transduced BM (n = 5 biological replicates for WT, n = 4 for Ripk3− / − and LTaΔ / Δ, and n = 3 for LTa− / − and Tnf− / −). Data are representative of at least two independent experiments. (d) GEMM colony counts (n=5 biological replicates) of FLT3-ITD-transduced BM treated as indicated. Data are representative of at least two independent experiments. p-values ​​shown were determined by comparison with control (-). Error bars represent mean ± SEM. p-values ​​are Student's t-test. **p<0.01, ***p<0.001, ****p<0.0001. [Figure 3] This figure shows that treatment of FLT3-ITD-transplanted mice with LT-α or TNF-neutralizing antibody (α-TNF) eradicates LSCs and leads to complete and long-term remission. Experimental design and survival of WT mice transplanted with FLT3-ITD-transduced WT BM and treated twice weekly as indicated. Median survival for isotype control was 76.5 days vs. 65 days for etanercept. Number of mice is indicated in the figure. Error bars represent mean ± SEM. p-values ​​are from the Mantel-Cox test. ***p<0.001, ****p<0.0001. [Figure 4]Figure 1 shows that LT-α treatment, in combination with anti-TNF, cytarabine, or IAP inhibition, kills LSCs and blasts but supports healthy HSPCs in multiple human primary AML samples. (a) Relative numbers of cells in AML cell lines treated as indicated (shown as the average of at least three technical replicates). Results are expressed as fold changes compared to untreated cells (ctr; set to 1, indicated by the dashed line); (b) Experimental design and relative numbers of hematopoietic cell subsets in healthy BM treated with 100 ng / ml LT-α. Results are expressed as fold changes compared to untreated cells (control; set to 1, indicated by the dashed line). The p-values ​​shown were determined by comparison with the control; (c) Relative numbers of Lin− cells, leukemic stem cells (LSCs), and non-leukemic (CD99−) HSPCs in AML BM samples treated as indicated (number of biological replicates per group shown in the figure). Results are expressed as fold change compared to untreated cells (control; set to 1, indicated by dashed line). The p-values ​​shown were determined by comparison with the control; error bars represent the mean ± SEM. p-values ​​are Student's t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 5] Figure 1 shows that LT-α treatment, either individually or in combination with imatinib treatment, kills blasts of a representative chronic myeloid leukemia (CML) cell line, K562. Relative numbers of cells treated as indicated (shown as the mean of three replicates) are shown. Results are expressed as fold changes compared to untreated cells (ctr; set to 1, indicated by the dashed line); error bars represent the mean ± SEM. p values ​​are paired Student's t-test. **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0029] The inventors have dedicated themselves to solving the problem of the present invention and have succeeded in finding that the polypeptides defined herein, used in the treatment of bone marrow diseases or myeloid neoplasms, result in programmed cell death and reliable destruction of leukemic cells and leukemic stem cells (LSCs), promising to bring about complete and long-term remission in patients suffering from these conditions.

[0030] The main advantages of the present invention are its highly specific effect on leukemic cells with no or negligible toxicity to healthy cells, its applicability to most patients suffering from bone marrow diseases or neoplasms caused by hematopoietic stem and progenitor cells (HSPCs), such as AML or CML, and its significant effect on LSCs, resulting in significant remission to the point of cure of patients.

[0031] As already mentioned above, RIPK3-mediated necroptosis and inflammasome activation act as tumor suppressor mechanisms. Importantly, consistent with their role in RIPK3 activation, LSC survival and differentiation are controlled by TNF receptor (TNFR) 1 and 2 signaling. TNFR1 is ubiquitously expressed, whereas TNFR2 expression is highly regulated, induced by inflammatory stimuli, and restricted to hematopoietic lineage cells (typically lymphocytes) and endothelial cells. TNFR1 and TNFR2 transduce signals from their two cognate ligands, TNF trimers and lymphotoxin α (LTα) trimers.

[0032] TNF is a pleiotropic cytokine produced by a wide range of cell types, exerting beneficial activities in immunoregulation and host defense, as well as dangerous pro-inflammatory and cytotoxic functions during inflammation. TNF exists in both transmembrane and soluble forms. Both forms of TNF are biologically active, but soluble TNF exhibits a higher affinity for TNFR1, while TNFR2 can only be properly activated by membrane-bound TNF.

[0033] Compared to TNF, the role of LT-α appears to be much more restricted. LT-α is produced primarily by CD4 T cells, B cells, and natural killer (NK) cells and is involved in the development and function of the immune system, primarily in lymphoid organ development, organization and maintenance of the lymphoid microenvironment, host defense, and immune system function. LT-α plays a specific role in the regulation of inflammatory processes. LT-α binds to TNFR1 and TNFR2, as well as to HVEM (herpes virus entry mediator), but this binding is relatively weak. In contrast to TNF, LT-α is converted to a soluble form with high efficiency. LT-α is anchored to the cell membrane only as a heterotrimer associated with membrane-bound LT-β, ​​i.e., the predominant LTα1β2 form and the minor LTα2β1 form, both of which interact with the LT-β receptor (LTβR), but not with TNFR1 or TNFR2.

[0034] Like TNF, LT-α binds with high affinity to TNFR1 and TNFR2. However, depending on the specific cellular context, TNFR activation can result in survival, death, or differentiation. The multifaceted nature of TNFR signaling is due to the sequential formation of different signaling complexes / cascades upon activation of TNFR1 and TNFR2. TNFR1 contains a death domain (DD), whereas TNFR2 does not.

[0035] Activation of TNFR1 can lead to inflammation through induction of NF-κB and mitogen-activated protein kinase (MAPK) JNK and p38 signaling, as well as cell death, which can be either apoptosis or necrosis. Although TNFR2 can also activate canonical NF-κB and JNK signaling, activation of TNFR2 is thought to primarily trigger non-canonical NF-κB signaling via the E3 ligases TRAF2 and TRAF3, resulting in numerous changes in gene expression that drive cell survival, proliferation, inflammation, immune regulation, and tissue homeostasis. Furthermore, TNFR2 can directly bind to TRAF2, along with TRAF1, thereby inducing intracellular crosstalk between both receptors and influencing the outcome of signaling initiated by TNF binding.

[0036] Both TNFR1 and TNFR2 limit the self-renewal capacity of healthy HSPCs in vivo. Interestingly, current data suggest that TNFR1 and TNFR2 differentially block HSPCs, with TNF regulating committed progenitors primarily by binding to TNFR1, whereas primitive hematopoietic progenitors express TNFR2. Furthermore, the TNF / TNFR2 axis is involved in the proper development of embryonic HSCs.

[0037] TNFR1 / 2 signaling is skewed in myeloid diseases or neoplasms, such as some AML subtypes, by upregulating TNFR2 surface expression and directing TNF-dependent signaling toward promoting, rather than suppressing, HSPC self-renewal. In this oncogenic setting, LT-α was surprisingly found to be a ligand capable of binding to TNFR1 / 2 to induce cell death.

[0038] The present inventors have successfully developed clinically relevant models of AML and CML as exemplary myeloid stem cell diseases to demonstrate, based on in vitro and in vivo data, that targeting LSCs with LT-α is effective in treating myeloid stem cell diseases such as AML or CML and how it can be most effectively used to increase the chances of cure for AML, CML, and other diseases. Using these models and primary AML patient samples, we can show that LT-α can indeed kill LSCs in AML and blasts in CML, and induce complete and long-term remissions in myeloid neoplasms and myeloid diseases.

[0039] Thus, the present invention relates to a polypeptide comprising the amino acid sequence of SEQ ID NO:1 (full-length human LT-α) or SEQ ID NO:2 (mature human LT-α), or comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, for use in the treatment of bone marrow diseases or myeloid neoplasms.

[0040] In the context of the present invention, SEQ ID NO:1 represents full-length human LT-α, including the signal peptide of amino acids 1 to 34. According to a preferred embodiment, a polypeptide for use according to the present invention comprises SEQ ID NO:1, and preferably, a polypeptide for use according to the present invention consists solely of SEQ ID NO:1.

[0041] The amino acid sequence of SEQ ID NO:1 is as follows: MTPPERLFLPRVCGTTLHLLLLGLLLVLLPGAQGLPGVGLTPSAAQTARQHPKMHLAHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVP TSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL

[0042] In the context of the present invention, SEQ ID NO:2 represents the mature form of human LT-α in which the signal peptide has been replaced by an N-terminal methionine. According to a preferred embodiment, a polypeptide for use according to the present invention comprises SEQ ID NO:2, and preferably, a polypeptide for use according to the present invention consists solely of SEQ ID NO:2.

[0043] The amino acid sequence of SEQ ID NO:2 is as follows: MLPGVGLTPSAAQTARQHPKMHLAHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL

[0044] The full-length amino acid sequence of human LT-α (SEQ ID NO: 1) is available under UniProt accession number P01374. The mature sequence of SEQ ID NO: 2 can be obtained as amino acids 35-205 of the entry under UniProt accession number P01374, with the addition of an N-terminal methionine.

[0045] In the present invention, polypeptides comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 1 or SEQ ID NO: 2 are also envisaged as polypeptides for use according to the invention. According to a preferred embodiment, a polypeptide for use according to the invention comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 1, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99% identity to SEQ ID NO: 1. Even more preferably, a polypeptide for use according to the invention consists solely of an amino acid sequence having such degrees of identity.

[0046] According to another preferred embodiment, the polypeptide for use according to the invention comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 2, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% identity to SEQ ID NO: 2. Even more preferably, the polypeptide for use according to the invention consists solely of an amino acid sequence having such degree of identity.

[0047] The determination of percent identity between two sequences is accomplished, in accordance with the present invention, by employing the mathematical algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90:5873-5877). Such an algorithm is the basis for the BLASTN and BLASTP programs of Altschul et al. (J. Mol. Biol. (1990) 215:403-410). Nucleotide searches are performed with the BLASTN program. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described by Altschul et al. (Nucleic Acids Res. (1997) 25:3389-3402). BLAST When using the Gapped BLAST program, Use the default parameter.

[0048] The mechanism of action of LT-α on abnormal myeloid cells such as leukemia cells and LSCs also makes it reasonable to apply this molecule to patients suffering from various myeloid diseases, particularly diseases derived from abnormal myeloid stem cells or myeloid progenitor cells. Therefore, according to a preferred embodiment, the myeloid disease is a myeloid stem cell disease, preferably one of myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), blastic plasmacytoid dendritic cell neoplasms (BPDCN), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), eosinophilia, and myeloid neoplasms associated with PDGFRA, PDGFRB, or FGFR1 rearrangements, or PCM1-JAK2; more preferably, the myeloid disease is one of acute myeloid leukemia (AML) or chronic myeloid leukemia (CML); even more preferably, the myeloid disease is acute myeloid leukemia (AML). In the present invention, bone marrow stem cell disease is a bone marrow disease caused by hematopoietic stem and progenitor cells (HSPCs) in the bone marrow.

[0049] According to a particularly preferred embodiment, the polypeptide for use according to the invention is capable of affecting a TNF receptor superfamily-dependent signaling cascade, more preferably a TNFR1 (TNFRSF1A), TNFR2 (TNFRSF1B), lymphotoxin beta receptor (TNFRSF3) or HVEM (TNFRSF14)-dependent signaling cascade. Therefore, it is preferred that the polypeptide for use according to the invention is capable of binding specifically and with high affinity to TNFR1, TNFR2, lymphotoxin beta receptor or HVEM, more preferably TNFR1 and / or TNFR2.

[0050] Since the polypeptides of the invention perform their function based on TNF receptor superfamily member-dependent signaling cascades, it is preferred that said pathways are fully or at least partially functional in the subject to be treated. Preferably, it may be tested whether the relevant pathways are fully or at least partially functional in the subject to be treated prior to administration of the polypeptides of the invention.

[0051] Preferably, the polypeptides for use according to the present invention are isolated diseased cells or in The polypeptides are capable of inducing programmed cell death in abnormal cells in vivo, and preferably, the polypeptides are capable of inducing programmed cell death only in one or more cells defined as leukemia cells, leukemia progenitor cells, and leukemia stem cells, more preferably, leukemia stem cells. The polypeptides for use according to the present invention are also preferably capable of acting as ligands capable of binding to TNF receptor superfamily members TNFR1, TNFR2, lymphotoxin beta receptor, or HVEM, more preferably, TNFR1 and / or TNFR2. Such binding preferably induces cell death of the specific abnormal cells.

[0052] Polypeptides for use according to the present invention may preferably be prepared and obtained by recombinant protein production, as is generally known in the art, or preferably, polypeptides for use according to the present invention may be purified from endogenous sources.

[0053] In the context of the present invention, the polypeptide for use in the present invention is preferably for use in the treatment of a mammal, such as a cat or a dog. According to a particularly preferred embodiment, the polypeptide is for use in the treatment of a human patient.

[0054] The present invention also relates to a method for treating a bone marrow disease or myeloid neoplasm, wherein an effective amount of a polypeptide according to the invention or a pharmaceutical composition according to the invention is administered to an individual in need of such treatment.

[0055] The present invention also relates to a method for treating a bone marrow disease or myeloid neoplasm using the method of the present invention in the manufacture of a medicament for the treatment of a bone marrow disease or myeloid neoplasm. The present invention relates to the use of a polypeptide according to the present invention or a pharmaceutical composition according to the present invention.

[0056] Furthermore, the present invention also relates to a pharmaceutical composition for the treatment of a bone marrow disease or myeloid neoplasm, which comprises a polypeptide according to the present invention and at least one pharmaceutically acceptable excipient, such as a suitable carrier or diluent.

[0057] Preferably, the polypeptide for use according to the invention constitutes the active ingredient of a pharmaceutical composition and / or is present in an effective amount, the term "effective amount" meaning the amount of polypeptide for use according to the invention that has a prophylactically, diagnostically or therapeutically relevant effect against a disease or pathological condition.

[0058] A prophylactic effect prevents the onset of a disease. A therapeutic effect alleviates to some extent one or more symptoms of a disease or partially or completely restores to normal one or more physiological or biochemical parameters associated with or contributing to a disease or pathological condition. The respective amounts for administering polypeptides for use according to the present invention are sufficiently large to achieve the desired prophylactic, diagnostic, or therapeutic effect. Those skilled in the art will understand that the specific dose level, frequency, and duration for any particular mammal will depend on a variety of factors, including the activity of the specific components used, age, body weight, general health, sex, diet, time of administration, route of administration, drug combinations, and the severity of the particular treatment. Using known means and methods, the exact amount can be determined by those skilled in the art as a matter of routine experimentation.

[0059] In pharmaceutical compositions for use according to the present invention, the polypeptide for use according to the present invention preferably contains a polypeptide suitable for administration to a patient at a dose of 250 ng / kg to 250 μg / kg body weight, more preferably 100 ng / kg to 100 μg / kg body weight, even more preferably 500 ng / kg to 50 μg / kg body weight, even more preferably 1 μg / kg to 10 μg / kg body weight, even more preferably 3 μg / kg to 8 μg / kg body weight, and particularly about 5 μg / kg body weight. Accordingly, the polypeptide of the present invention is preferably administered to a patient at a dose of 250 ng / kg to 250 μg / kg body weight, more preferably 100 ng / kg to 100 μg / kg body weight, even more preferably 500 ng / kg to 50 μg / kg body weight, even more preferably 1 μg / kg to 10 μg / kg body weight, even more preferably 3 μg / kg to 8 μg / kg body weight, and particularly about 5 μg / kg body weight.

[0060] The pharmaceutical compositions of the present invention are generally administered as formulations in association with one or more pharmaceutically acceptable excipients. The term "excipient" is used herein to describe any component other than the polypeptide for use according to the present invention. The choice of excipient will largely depend on the particular mode of administration. The excipient may be a suitable carrier and / or diluent.

[0061] The pharmaceutical composition for use according to the present invention may preferably be administered to a patient to provide systemic effectiveness. To this end, the pharmaceutical composition is preferably administered by systemic administration, more preferably by intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular or subcutaneous administration, even more preferably by subcutaneous or intravenous administration, especially by intravenous administration.

[0062] Suitable devices for administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques. Parenteral formulations useful herein are typically aqueous solutions which may contain additives such as salts, carbohydrates, and buffers (preferably pH 3-9), although for some applications they may be more suitably formulated as sterile non-aqueous solutions or as a dry form used in combination with an appropriate vehicle such as sterile, pyrogen-free water.

[0063] The preparation of parenteral formulations under sterile conditions, for example by lyophilization, may be readily accomplished using standard pharmaceutical techniques known to those skilled in the art. The solubility of pharmaceutical compositions for use according to the present invention used in the preparation of parenteral solutions may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.

[0064] According to a preferred embodiment of the present invention, the pharmaceutical composition further comprises one or more substances selected from the group comprising one or more SMAC mimetics such as birinapant, one or more chemotherapeutic agents such as cytarabine (cytosine arabinoside) or cerbidine (daunorubicin), and one or more TNF inhibitors such as Humira (adalimumab), Remicade (infliximab), Simponi (golimumab), Cimzia (certolizumab pegol).

[0065] Preferably, the pharmaceutical composition does not contain any type of TNF receptor superfamily molecule. More preferably, the pharmaceutical composition does not contain SEQ ID NO: 3. SEQ ID NO: 3 represents the amino acid sequence of TNFR1, which has UniProt accession number P19438.

[0066] The amino acid sequence of SEQ ID NO:3 is as follows: MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPPGGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVE ISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTVLLPLVIFFGLCLLSL LFIGLMYRYQRWKSKLYSIVCGKSTPEKEGELEGTTTKPLAPNPSFSPTPGFTPTLGFSPVPSSTFTSSSTYTPGDCPNFAAPRREVAPPYQGADPILATALASDPIPNPLQKW EDSAHKPQSLTDTDDPATLYAVVENVPPLRWKEFVRRLGLSDHEIDRLELQNGRCLREAQYSMLATWRRRTPRREATLELLGRVLRDMDLLGCLEDIEEALCGPAALPPAPSLLR

[0067] Also preferably, the pharmaceutical composition does not comprise SEQ ID NO: 4. SEQ ID NO: 4 represents the amino acid sequence of TNFR2 having UniProt accession number P20333.

[0068] The amino acid sequence of SEQ ID NO:4 is as follows: MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQN RICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPT PEPSTAPSTSFLLPMGPSPPAEGSTGDFALPVGLIVGVTALGLLIIGVVNCVIMTQVKKKPLCLQREAKVPHLPADKARGTQGPEQQHLLITAPSSSSSSLESSASALDRRAPTR NQPQAPGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS

[0069] Also preferably, the pharmaceutical composition does not comprise SEQ ID NO: 5. SEQ ID NO: 5 represents the amino acid sequence of lymphotoxin beta receptor having UniProt accession number P36941.

[0070] The amino acid sequence of SEQ ID NO:5 is as follows: MLLPWATSAPGLAWGPLVLGLFGLLAASQPQAVPPYASENQTCRDQEKEYYEPQHRICCSRCPPGTYVSAKCSRIRDTVCATCAENSYNEHWNYLTICQLCRPCDPVM GLEEIAPCTSKRKTQCRCQPGMFCAAWALECTHCELLSDCPPGTEAELKDEVGKGNNHCVPCKAGHFQNTSSPSARCQPHTRCENQGLVEAAPGTAQSDTTCKNPLEPL PPEMSGTMLMLAVLLPLAFFLLLATVFSCIWKSHPSLCRKLGSLLKRRPQGEGPNPVAGSWEPPKAHPYFPDLVQPLLPISGDVSPVSTGLPAAPVLEAGVPQQQSPLD LTREPQLEPGEQSQVAHGTNGIHVTGGSMTITGNIYIYNGPVLGGPPGPGDLPATPEPPYPIPEEGDPGPPGLSTPHQEDGKAWHLAETEHCGATPSNRGPRNQFITHD

[0071] Also preferably, the pharmaceutical composition does not comprise SEQ ID NO: 6. SEQ ID NO: 6 is identified in the art as HVEM (Herpesvirus Entry Mediator) having UniProt accession number Q92956 (TNFRSF14 (Tumor Necrosis Factor Receptor Superfamily Member)). 1 shows the amino acid sequence of CD270 (also known as CD270 member 14).

[0072] The amino acid sequence of SEQ ID NO:6 is as follows: meppgdwgpppwrstpktdvlrlvlyltflgapcyapalpsckedeypvgseccpkcspgyrvkeacgeltgtvcepcppgtyiahlnglskclqcqmcdpamglrasrncsrtenavcgcspghfcivqdgdhcaacray atsspgqrvqkggtesqdtlcqncppgtfspngtleecqhqtkcswlvtkagagtssshwvwwflsgslvivivcstvgliicvkrrkprgdvvkvivsvqrkrqeaegeatviealqappdvttvaveetipsftgrspnh

[0073] According to one preferred embodiment, the pharmaceutical composition does not comprise a protein comprising any of the sequences SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, or any of the respective mature proteins lacking the signal peptide sequence.

[0074] Also preferably, pharmaceutical compositions for use according to the invention do not comprise antibodies to lymphotoxin or to any type of TNF receptor, including TNFR1, TNFR2, lymphotoxin beta receptor, and HVEM. Most preferably, pharmaceutical compositions for use according to the invention do not comprise any of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or antibodies to lymphotoxin or to any type of TNF receptor, including TNFR1, TNFR2, lymphotoxin beta receptor, and HVEM.

[0075] The present invention also relates to a kit for use according to the invention comprising a polypeptide for use according to the invention, a container and, optionally, written instructions for use and / or administration means.

[0076] It is contemplated that all embodiments of the present invention described herein can be combined in any combination unless a person skilled in the art would consider such combination to make no technical sense. [Example]

[0077] I. Materials and Methods I.1. Cell culture All cells were cultured at 37°C and 5% CO2 in a fully humidified atmosphere.

[0078] Human primary samples: Bone marrow mononuclear cells (BMMCs) were isolated from heparinized bone marrow samples or bone fragments by centrifugation through a 1.077 g / ml Ficoll-Hypaque layer (Biochrom). Cells were harvested and used directly for immunophenotyping or cultured on EL08-1D2 stromal cells in 20% fetal calf serum (FCS; PAN-Biotech), penicillin / streptomycin, and PBS. The cells were cultured in IMDM (Gibco) supplemented with 0.05 mM tomycin, 2 mM L-glutamine, and 0.05 mM 2-mercaptoethanol (Gibco).

[0079] The mouse embryonic liver stromal cell line EL08-1D2 was obtained from Prof. R. Oostendorp (TUM, Germany) and cultured in 10% fetal bovine serum, 10% horse serum (StemCell Technologies), 2-mercaptoethanol (DMSO), and 10% ethanol. These cells were cultured in MEM Alpha+GlutaMAX medium (Gibco) supplemented with 0.05 mM captoethanol and 0.05 mM penicillin / streptomycin. The cells were grown on gelatin-coated (0.1% in PBS; Sigma) cell culture plates. Prior to co-culture with primary human BMMCs, EL08-1D2 cells were irradiated (30 Gy) and incubated for at least 3 hours before use.

[0080] AML and CML cell lines were maintained as directed by the ATCC or DSMZ Bioresource Center. All cell lines were quantified using the MycoProbe Mycoplasma Detection Kit. Tested regularly for mycoplasma using a Short T The cell line was authenticated using STR (strand repeat) profiling (ATCC). PLB-985 is present in the database of commonly misidentified cell lines maintained by ICLAC, but was included in this study as a necessary model of oncogenic driver-independent AML. DNA fingerprinting of this cell line clearly demonstrated that PLB-985 is a subclone of the HL-60 cell line, but displays different characteristics, including cytogenetics (e.g., Myc negativity), DNA profile, differentiation, and treatment response. This cell line was authenticated by DNA fingerprinting and compared to a published STR profile (Cellosaurus).

[0081] Progenitor cell analysis (human) For cytokine measurement, BM supernatant was obtained from BM aspirate samples by centrifugation. The supernatant was concentrated using a Vivaspin 10 kDa filter (Sartorius), and cytokines were analyzed. was quantified using a Cytometric Bead Array (CBA; BD Biosciences) according to the manufacturer's instructions.

[0082] For immunophenotyping, BM cells or AML cell lines were rinsed three times with PBS, stained for viability using the Zombie NIR™ Fixable Viability Kit (Biolegend) according to the manufacturer's instructions, and preincubated with Fc-block. Afterwards, the cells were stained with fluorescently labeled antibodies as described below. All centrifugation steps were performed at 400 × g.

[0083] For post-treatment flow cytometry analysis, BM or AML cell lines were treated as described above for 7 days, and, where appropriate, with etanercept (Enbrel™; 50 ng / ml; Pfizer), recombinant human (rh)LT-α (100 ng / ml; R&D), or other anti-inflammatory drugs, unless otherwise specified. , rhTNF (100 ng / ml; R&D), emricasan (2.5 nM; SelleckChem), birinapant (10 nM; SelleckChem), cytarabine (100 nM), α-LTα-I g (10 μg / ml; 359-238-8; Biolegend), adalimumab (Humira (TM); 500 ng / ml; Abbvie), and the respective isotype controls mouse IgG1, κ (MOPC-21; Biolegend), and Ultra-LEAF™ purified human Cultured in the presence of cytokines and inhibitors of IgG1 (QA16A12; Biolegend). .

[0084] Progenitor cell analysis (mouse) For colony-forming cell assays, all duplicate cultures were performed in 35-mm Petri dishes in mouse methylcellulose medium (Methocult, Stem Cell Technologies). BM cells from unchallenged or 5-FU-challenged mice were plated in M3434. FLT3-ITD-infected BM cells were treated with recombinant mouse (rm) LT-α (100 ng / ml), rmTNF (100 ng / ml; R&D), and etanercept (50 ng / ml) cytokines and inhibitors, as needed, unless otherwise specified. Colonies were plated in M3234 medium in the presence of 0.8 μg / ml Sigma-Aldrich (Sigma-Aldrich) and counted by light microscopy after 10 days. Cells infected with empty vector were used as a negative control and did not result in colony formation. To analyze colony-forming unit (CFU) cell viability, colonies were grown for 10 days in the presence of propidium iodide (PI) (0.8 μg / ml; Sigma-Aldrich) and counted by light microscopy after 10 days. Representative CFU-GEM images were obtained using a Yence BZ-9000 (Biorevo) fluorescence microscope. I photographed M.

[0085] I.2. Other mouse Tnf tm1Gkl(Tnf - / -), Tnfrsf1 atm1Mak / J(Tn fr1 - / - ), Tnfrsf1 btm1Mwm / J(Tnfr2 - / - ), and Lta tm1Dch / J(Lta - / - ) mice were purchased from Jackson Laboratories. - / - Mice were kindly provided by Prof. M. Heikenwaelder (DKFZ, Heidelberg, Germany), and Lta Δ / Δ Mice were obtained from Dr. A. Kruglov (DRFZ, Berlin, Germany). - / - Mice were obtained from Genentech under a material transfer agreement. All animal experiments were performed in accordance with protocols approved by the guidelines of the local animal ethics committee.

[0086] Human primary samples Primary and secondary AML samples were collected from the clinical trial AMLCG-2008 (http: / / clinicaltrial.gov, identifier NCT01382147), the AML Register and Biomaterial Database of the AML Registry Protocol of the German Leukemia Study Alliance, from January 6, 2011 AML Registry Protocol of the German AML Cooperative Group Version 2.0, German A Patients were enrolled in the AML Registry Protocol of the ML Study Group or treated at the Faculty of Medicine III of the Technical University of Munich after approval by the local ethics committee (approval number 62 / 16S from February 10, 2016, and approval number 2790 / 10 from April 30, 2010). Informed consent was obtained from patients at the start of the study.

[0087] Non-leukemic control samples were collected from individuals who underwent diagnostic bone marrow aspiration after which hematopoietic disorders had been excluded. Healthy controls were isolated from the femoral heads of patients undergoing surgical hip replacement.

[0088] In vivo treatment in the FLT3-ITD mouse model Retrovirus preparation, transduction, and mouse bone marrow transplantation were performed as previously described (Non-Patent Document 3; supra).

[0089] For in vivo therapeutic experiments, FLT3-ITD-transduced C57BL / 6 WT cells were resuspended in PBS (Sigma) and preconditioned with busulfan (20 mg / kg busulfan (Sigma)) before transplantation. Ent mice were injected iv for 5 consecutive days as previously described (Peake, K., et al., (2015). J Vis Exp, e52553.).

[0090] For treatment, animals were randomly divided into groups of eight at 2 and 8 weeks after transplantation. Mice received either isotype control (5 mg / kg; RTK2071; Biolegend), etanercept (E Mice were given either rmbrel™ (50 ng / ml; Pfizer), rmLT-α (250 μg / kg; Cusabio), α-TNF (5 mg / kg; MP6-XT22; Biolegend), or rmLT-α plus α-TNF by iv injection twice weekly.

[0091] Diseased mice were sacrificed. Peripheral blood white blood cell counts (WBC) were measured by scil Vet abc (scil animal care company). Single-cell suspensions of designated tissue samples were prepared, and peripheral blood red blood cells were lysed before analysis. Cells were preincubated with Fc-block and then stained with fluorescently labeled antibodies. Dead cells were identified by PI (Sigma) or Zombie. ie Aqua (trademark) Fixable Viability Kit (Biolegend) dyeing Color was excluded according to the manufacturer's instructions. Flow cytometry immunophenotyping of transplanted mice was performed as previously described (Non-Patent Document 3; supra).

[0092] statistical analysis For statistical analysis, p values ​​were determined by applying a two-tailed t-test for independent samples. Survival curves were analyzed using the Mantel-Cox test implemented in GraphPad Prism software. Throughout this paper, all values ​​are presented as mean ± SEM. and statistical significance is expressed as p<0.0001 (****), p<0.001 (***), p<0.01 (**), p<0.05 (*), or ns (not statistically significant).

[0093] Unless otherwise stated, all experiments represent at least 8 mice per group.

[0094] I.3. Reagents and Antibodies Antibodies used to isolate mouse hematopoietic cell subsets: Fc-block, as well as B220 (RA3_6B2), CD19 (eBio1D3), Thy1.2 (53-2.1), CD3 (17A2), TCR-b (H57-597), CD4 (Gk1.5), CD8a (53-6.7), CD11b (M1 / 70), F4 / 80 (BM8), Gr-1 (RB6-8C5), Ly6B.2 (AbD Serotec), CD34 (700011; R&D Fluorescently conjugated antibodies against IL-7Ra (A7R34), Sca-1 (D7), c-Kit (2B8), CD16 / 32 (93), CD244.2 (eBio244F4), CD150 (mShad150), CD48 (HM48-1), Ly6C (HK1.4), Ter119 (TER-119), and IL-7Ra (A7R34) were obtained from eBioscience, unless otherwise stated. The gating strategy used to identify subsets of murine hematopoietic stem and progenitor cells has been previously described (Non-Patent Document 3; supra).

[0095] Antibodies used for isolation of human hematopoietic cell subsets for immunophenotyping and intracellular protein expression analysis: Lineage Cocktail (catalog no. 348801; 348703), fluorescently labeled antibodies against CD45RA (HI100), CD34 (581), CD38 (HB-7), CD99 (3B2 / TA8), and CD123 (5B11) were obtained from Biolegend. Flow analysis was performed using a BD FACS Canto II (BD Biosciences). ) and data were analyzed using FlowJo software (Tree Star).

[0096] II. Results LTα restricts malignant myeloproliferation To determine the role of TNF and LT-α in AML, we utilized a mouse bone marrow transplantation model of FLT3-ITD-driven myeloproliferative disorder in mice (Non-Patent Document 3; supra). As previously reported, transplantation of FLT3-ITD-transduced wild-type (WT) bone marrow into lethally irradiated syngeneic WT recipient mice (abbreviated as WT FLT3-ITD → WT) resulted in a rapidly fatal myeloproliferative neoplasm (MPN) characterized by peripheral leukocytosis, hepatosplenomegaly, and infiltration of the bone marrow (BM), spleen, and liver (Figures 1a-c).

[0097] To investigate the role of LT-α in AML development, we utilized two different strains of LT-α-deficient mice: conventional Lta deficiency, which is defective in TNF production, and LT-α-deficient mice, which are defective in TNF production. - / - Unlike mice, neo-free Lta Δ / Δ Animals are able to produce normal amounts of TNF both in vivo and in vitro.

[0098] FLT3-ITD transduced Lta - / - or Lta Δ / Δ LT-α-deficient recipient mice (Lta - / - FLT3-ITD → Lta - / - and Lta Δ / Δ FLT3-ITD → Lta - / -Lta (abbreviated as Lta) died significantly earlier from MPN compared with WT FLT3-ITD (Fig. 1b), which was associated with significantly worsening clinical features, including elevated white blood cell (WBC) counts and increased hepatosplenomegaly (data not shown). - / - FLT3-ITD → Lta - / - and Lta Δ / Δ FLT3-ITD → Lta - / - The increased leukemia burden in + This was also observed by flow cytometry of the cells (Fig. 1c). Δ / Δ FLT3-ITD → Lta - / - Lta - / - FLT3-ITD → Lta - / - Died of disease that was significantly worse than Figure 1b).

[0099] In clear contrast, FLT3-ITD transduced Tnf - / - TNF-deficient recipient mice (Tnf - / - FLT3-ITD → Tnf - / - (abbreviated as TNF) demonstrated a significant delay in disease progression compared to WT FLT3-ITD. - / - FLT3-ITD → Tnf - / - Histological examination of the BM showed that hematopoietic tissue was replaced by connective tissue, similar to primary myelofibrosis (data not shown). - / - FLT3-ITD → Tnf - / - The reduction in leukemic burden was also observed by flow cytometry of GFP+ cells (Fig. 1c).

[0100] Taken together, these data suggest that LT-α delayed AML progression, and TNF enhanced the clonogenic potential of LSCs, increasing the number of leukemic cells and promoting the development of AML.

[0101] To determine the factors responsible for the differential disease characteristics of LT-α and TNF-deficient animals, we characterized the composition of the HSPC compartment in FLT3-ITD transplanted mice. Similar to human AML, which is characterized by the accumulation of primitive HSPCs, we found that Lta Δ / Δ FLT3-ITD → Lta - / - and, to a lesser extent, Lta - / - FLT3-ITD → Lta - / - Similarly, the FLT3-ITD-expressing line (Lin - ) We found significant expansion of cells (data not shown).

[0102] Myeloid progenitor population (Lin - Sca1 - c-Kit + ) (including common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), and megakaryocyte-erythroid progenitors (MEPs)) compared with Lin - Sca1 + c-Kit + Characterization of the (LSK) compartment (containing long-term and short-term HSCs (LT- and ST-HSCs) and multipotent progenitor cells (MPPs)) revealed that expansion was primarily a CMP population (BM:Lta) only in LTα-deficient mice, as previously reported (Non-Patent Document 3; supra). - / - vs. WT, p=0.0115; Lta Δ / Δ vs. WT, p<0.0001) and ST-HSC population (BM:Lta Δ / Δ We found that WT FLT3-ITD expanded the GMP compartment and depleted the HSC compartment, whereas TNF-α was responsible for a significant increase in the number of FLT3-ITD-expressing cells (vs. WT, p = 0.0287) (data not shown). Furthermore, despite a significant decrease in the absolute number of FLT3-ITD-expressing cells in all organs, TNF-α was not significantly affected. - / - FLT3-ITD → Tnf - / - (BM:Tnf - / - A clear increase in the GMP population was observed in the Lta (vs. WT, p=0.0151) (data not shown). - / - FLT3-ITD and Lta Δ / ΔThis was supported by the presence of leukemic blasts exclusively in the BM of mice transplanted with FLT3-ITD (data not shown).

[0103] Notably, serial transplantation of splenocytes from diseased mice resulted in Lta - / - FLT3-ITD cells and Lta Δ / Δ Only FLT3-ITD cells could be reconstituted, and neither WT control nor Tnf - / - FLT3-ITD was unable to reconstitute and resulted in transplantable leukemia in secondary recipients (Fig. 1d). + Leukocytosis and bone marrow organ infiltration were confirmed by Lta - / - FLT3-ITD → Lta - / - and Lta Δ / Δ FLT3-ITD → Lta - / - was only detected in secondary transplants (Fig. 1e).

[0104] Because FLT3-ITD-driven MPNs in WT mice were not serially engraftable (Fig. 1d), this finding indicated a strongly enhanced ability of transformed HSPCs to survive and propagate myeloid neoplasms when Lta was deleted.

[0105] FLT3-ITD mediates TNF-dependent TNFR1 / 2 signaling to promote HSPC self-renewal. Distorts signal transmission Although both TNFR1 and TNFR2 have been shown to play a role in limiting HSPC self-renewal, the role of LT-α in this process is unclear. Therefore, we investigated the functional consequences of TNFR signaling on the survival and differentiation capacity of HSPCs before and after FLT3-ITD expression by assaying their colony-forming ability.

[0106] At steady state, 5-FU challenged (HSPC-enriched) WT, Ripk3 - / - , TNF - / - , Lta - / - , and Lta Δ / ΔBM from mice showed normal differentiation and distribution to all myeloid lineages (data not shown). However, the number of multipotent granulocyte-erythroid-macrophage-megakaryocyte (GEMM) colonies differed between genotypes. BM Lta cells enriched for HSPCs - / - and Lta △ / △ showed a GEMM colony number comparable to that of WT, whereas Ripk3 - / - and TNF - / - showed a higher number of GEMM colonies (Fig. 2a).

[0107] When GEMM colonies were analyzed after FLT3-ITD expression, the opposite effects of TNF and LT-α were observed. - / - , Lta - / - , and Lta △ / △ has a high number of GEMM colonies, whereas Tnf - / - showed comparable numbers to WT (Fig. 2b and Fig. 2c). Furthermore, fluorescence microscopy analysis of the same colonies revealed that WT and Tnf colonies were significantly more abundant than Lta-deficient colonies. - / - showed higher cell death (detected as propidium iodide uptake, data not shown).

[0108] In contrast to nontransformed cells, in WT FLT3-ITD-transduced cultures, exogenous TNF specifically and significantly increased the number of GEMM colonies (Figure 2d), but restricted differentiated and mature progeny in a dose-dependent manner (data not shown). This is consistent with previous studies in which TNF was observed to promote the survival and proliferation of patient-derived AML blasts. Surprisingly, in WT FLT3-ITD-transduced cultures, exogenous LT-α significantly reduced the number of GEMM colonies (Figure 2d).

[0109] As expected, TNF / LT-α blockade with TNFR2-Ig fusion protein etanercept in FLT3-ITD-transduced cells induced an increase in GEMM colonies in WT cultures compared with controls (Figure 2d), supporting the finding that LT-α is the ligand that causes a decrease in GEMM colonies.

[0110] Taken together, these data suggest that the FLT3-ITD oncogene skews TNFR signaling toward promoting rather than suppressing HSPC self-renewal. In this oncogenic context, LT-α is a ligand capable of inducing cell death.

[0111] Administration of LT-α eliminates AML in vivo We further tested whether exogenous LT-α, an inhibitory antibody against TNF (α-TNF), or a combination thereof could be useful for the treatment of leukemia. Because mouse AML models have previously been used to predict the behavior of chemotherapy in the clinic, we generated WT FLT3-ITD mice and initiated dosing at 2 or 8 weeks after transplantation, respectively (Figure 3).

[0112] Treatment with LT-α and α-TNF was well tolerated in vivo, reduced the FLT3-ITD disease burden to less than 1% in all organs analyzed, and induced long-term remission in treated animals, prolonging survival from 285 to 300 days after treatment initiation (Figure 3). Only two animals failed to respond to α-TNF alone, while mice treated with an isotype control and mice receiving etanercept rapidly succumbed to disease (Figure 3). Using HSPC-enriched BM, Consistent with previous studies, we found that TNF / LT-α blockade with etanercept dramatically increased the number of primitive leukemic cells compared with controls, which was associated with a shorter latency period, a significantly higher WBC count, increased hepatosplenomegaly, and a higher leukemic burden (data not shown).

[0113] The combination treatment, LT-α + α-TNF, was as effective as LT-α monotherapy in reducing FLT3-ITD disease burden (<1%) and inducing long-term remission in WT FLT3-ITD mice, but the addition of α-TNF did not accelerate leukemic cell death compared to LT-α monotherapy. On the other hand, mice receiving the combination therapy gained weight more rapidly compared to LT-α monotreated animals (data not shown). This may indicate that treated mice experienced fatigue accompanied by increased LT-α concentrations, for example, due to the onset of an inflammatory process, whereas the simultaneous reduction of TNF improved fatigue over time.

[0114] Although we believe that the depth and durability of the responses seen here is primarily due to selective LSC targeting, the ability of these regimens to eradicate the majority of leukemic cells (even those in the periphery) is impressive.

[0115] In conclusion, LT-α (±α-TNF) was highly active in FLT3-ITD-AML, effectively eradicating LSCs and achieving complete and long-lasting remissions.

[0116] LT-α induces cell death in AML in combination with cytarabine and birinapant. / The tolerability and efficacy of LT-α therapy were evaluated in human primary cells. / LT-α is effective in CML alone and in combination with imatinib. To confirm that LT-α / α-TNF targeting TNFR signaling is effective in treating human AML, we tested a panel of nine different AML cell line models for their sensitivity to LT-α / α-TNF (Fig. 4a). Indeed, exogenous LT-α specifically and significantly reduced the number of AML cells in a dose-dependent manner.

[0117] Accordingly, we also observed a dramatic decrease in cell number upon TNF blockade with the TNF-specific antibody adalimumab. In contrast, blockade of exogenous TNF or LT-α with an LT-α-neutralizing antibody significantly increased the number of AML cells. As expected, this effect was not observed with TNF / LT-α blockade with etanercept (Fig. 4a). This indicates that, consistent with our mouse model, the trophic activity of TNF and the suppression of LT-α are equally important for the survival and proliferation of AML cells. Importantly, the ability of LT-α and TNF blockade to promote cell death was independent of oncogenic mutations present in the cell lines.

[0118] To define the effects of exogenous LT-α on healthy hematopoiesis, we processed bone marrow samples from healthy control patients and evaluated HSC and myeloid progenitor cell subsets. LT-α not only did not induce any detectable toxic effects, but LT-α treatment maintained healthy hematopoiesis (Fig. 4b). Collectively, these data suggest that LT-α represents an interesting approach for AML therapy.

[0119] To investigate how targeting TNF / LT-α signaling can be most effectively used to treat AML, we treated primary human AML samples in vitro. LT-α and α-TNF (adalimumab) were evaluated alone or in combination. Furthermore, LT-α was tested in combination with the standard AML chemotherapy drug cytarabine (CYT), the clinical SMAC mimetic birinapant, and the clinical pan-caspase inhibitor emricasan. Drug concentrations were determined after titration in healthy bone marrow samples and AML cell lines (data not shown). After 7 days of treatment, CD99 was used to identify non-leukemic HSPCs (CD99 - ) and LSCs and cell viability was assessed (Fig. 4c).

[0120] LTα is Lin -LT-α killed leukemia cells (including leukemia cells (AML blasts) and the majority of healthy HSPCs) and LSCs, whereas α-TNF killed only LSCs. Both LT-α and α-TNF promoted the expansion of non-leukemogenic HSPCs. The combination of cytarabine or birinapant with LT-α increased blast cell death compared with cytarabine or birinapant alone. Birinapant was better than cytarabine at promoting both blast and LSC cell death. However, leukemia cells pretreated with emricasan were resistant to LT-α and birinapant-induced cell death (Figure 4c), demonstrating that LT-α / bilinapant also induced caspase-dependent cell death in some AML subtypes.

[0121] LT-α killed AML cells more effectively than the standard AML chemotherapy drug cytarabine (ara-C), and further increased cell death in combination with cytarabine without increasing toxicity to healthy controls. Furthermore, while LT-α can kill leukemia cells and leukemia stem cells in a more efficient manner than α-TNF, LT-α can also support healthy hematopoiesis without any significant cytotoxicity to healthy cells.

[0122] Furthermore, experiments were performed using K562, a representative cell line of chronic myeloid leukemia. Based on the above considerations, it is believed that the effects of LT-α are not limited to AML, but may extend to other bone marrow diseases or neoplasms originating from hematopoietic progenitor cells and hematopoietic stem cells.

[0123] As a model of CML, LT-α was tested for its potential effect on the cell line K562, separately and in combination with the standard therapy imatinib (at concentrations of 1 μM or 10 μM) (Fig. 5). Based on the results obtained, it was possible to demonstrate that LT-α has significant effects both alone and in combination with the standard therapy imatinib. Therefore, LT-α may be further used as a monotherapy or in combination to complement the effects of imatinib for treating CML.

[0124] The results observed in CML, a different type of bone marrow disease that has a different clinical picture from AML but also originates from HSPCs and may be related to the role of LSCs in the disease, suggest the general applicability of the claimed treatments to a variety of bone marrow diseases and neoplasms.

[0125] Based on these properties, LT-α is a promising candidate for a highly advantageous method of treating patients suffering from bone marrow diseases or myeloid neoplasms, such as AML, CML, etc., compared with currently available therapies.

Claims

1. A polypeptide for use in treating a bone marrow disease or myeloid neoplasm, comprising the amino acid sequence of SEQ ID NO: 1 (full-length human LT-α) or SEQ ID NO: 2 (mature human LT-α), or comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

2. 2. The polypeptide of claim 1, wherein the myeloid disease is a myeloid stem cell disease, preferably one of myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), blastic plasmacytoid dendritic cell neoplasms (BPDCN), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), eosinophilia, and myeloid neoplasms associated with PDGFRA, PDGFRB or FGFR1 rearrangements, or PCM1-JAK2, more preferably the myeloid disease is one of acute myeloid leukemia (AML) or chronic myeloid leukemia (CML), and even more preferably the myeloid disease is acute myeloid leukemia (AML).

3. 3. The polypeptide of claim 1 or 2, wherein the polypeptide is capable of affecting a TNF receptor superfamily (TNFRSF)-dependent signal cascade, preferably a TNFR1 (TNFRSF1A), TNFR2 (TNFRSF1B), lymphotoxin beta receptor (TNFRSF3) or HVEM (TNFRSF14)-dependent signal cascade, more preferably wherein the signal cascade is TNFR1 and / or TNFR2-dependent.

4. The polypeptide of any one of claims 1 to 3, wherein the relevant signaling pathway affected by said polypeptide is fully or partially functional in the individual to be treated.

5. 5. The polypeptide according to any one of claims 1 to 4, wherein the polypeptide is capable of inducing programmed cell death, preferably wherein the polypeptide is capable of inducing programmed cell death only in one or more of leukemia cells, leukemia progenitor cells, and leukemia stem cells.

6. The polypeptide according to any one of claims 1 to 5, wherein said polypeptide is a recombinant polypeptide or a purified endogenous polypeptide, preferably a recombinant polypeptide.

7. The polypeptide of any one of claims 1 to 6, wherein said polypeptide is used in the treatment of mammals, more preferably said polypeptide is used in the treatment of humans.

8. 8. The polypeptide according to any one of claims 1 to 7, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, preferably wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:

2.

9. A pharmaceutical composition for use in treating a bone marrow disease or myeloid neoplasm, comprising a polypeptide according to any one of claims 1 to 8 and at least one pharmaceutically acceptable excipient.

10. The pharmaceutical composition further comprises one or more substances selected from the group comprising an SMAC mimetic such as birinapant, a chemotherapeutic agent such as cytarabine (cytosine arabinoside) or cerbidine (daunorubicin), and a TNF inhibitor such as Humira (adalimumab), Remicade (infliximab), Simponi (golimumab), or Cimzia (certolizumab pegol). Item 10. The pharmaceutical composition according to Item 9.

11. 11. The pharmaceutical composition according to claim 9 or 10, wherein the pharmaceutical composition is adapted to be administered to a patient by systemic administration, preferably the pharmaceutical composition is adapted to be administered to the patient by intravenous or subcutaneous administration, more preferably by intravenous administration.

12. 12. The pharmaceutical composition of any one of claims 9 to 11, wherein the pharmaceutical composition does not contain any type of TNF receptor molecule, including TNFR1 (SEQ ID NO: 3), TNFR2 (SEQ ID NO: 4), lymphotoxin beta receptor (SEQ ID NO: 5), HVEM (SEQ ID NO: 6), or antibodies to lymphotoxin, or antibodies to any type of TNF receptor, including TNFR1, TNFR2, lymphotoxin beta receptor, and HVEM.

13. A kit for treating a bone marrow disease or myeloid neoplasm, comprising a polypeptide according to any one of claims 1 to 8 and a container.

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