Soluble thrombopoietin receptor fragments and uses thereof

Abstract

The present invention relates to a polypeptide comprising the extracellular domain of the thrombopoietin receptor or a fragment thereof, and its use as a medicament. The present invention also relates to the treatment and / or prevention of thrombopoietin-related diseases, in particular myeloproliferative neoplasms.

Description

[Technical field]

[0001] The present invention relates to a polypeptide comprising the extracellular domain of the thrombopoietin receptor or a fragment thereof and its use as a medicament. The present invention also relates to the treatment and / or prevention of thrombopoietin-related diseases, in particular myeloproliferative neoplasms. [Background technology]

[0002] Myeloproliferative neoplasms (MPNs) are malignant hematological disorders of the elderly and represent a major burden to the healthcare system. MPNs can progress to a very serious condition called secondary acute myeloid leukemia (AML), which is nearly always fatal. Three major types of MPNs, namely polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF), can occur with an incidence that can reach 1 / 5,000 cases per year.

[0003] In all three diseases, acquired mutations in hematopoietic stem cells (HSCs) lead to pathological activation of TPOR / JAK2 signaling that drives the pathology. The JAK2 V617F mutation is involved in 70% of all MPNs, is present in over 96% of PV and 60% of ET and MF cases, and leads to hyperactivation of the JAK2-STAT5 pathway via the thrombopoietin receptor (TPOR) and, to a lesser extent, other cytokine receptors (EpoR, G-CSFR).

[0004] Several studies have demonstrated a requirement for thrombopoietin (THPO) for the development of MPN pathology independent of driver mutations, and a recent study in particular showed that THPO inhibition led to significant amelioration of disease in mouse models.

[0005] Non-specific chemotherapy, radiotherapy and bone marrow transplantation are common strategies for the treatment of malignant hematological diseases. However, these therapeutic approaches are invasive and burdensome to patients. Therefore, there is an increasing need in this field to bring new resources or tools to improve therapeutic approaches.

[0006] Previous studies have identified the binding site of THPO to TPOR (Chen Wei-Min et al., 2010). Methods to generate soluble fragments of TPOR have also been described (Zhang Qing et al., 2004).

[0007] Chachoua et al. 2016 disclose the use of the D1-D2 fragment region of TPOR to inhibit TPOR-dependent signaling pathways, but through the ablation of CALR mutant activity in cells ("cis" acting), rather than the ablation of THPO. These models are not suitable for clinical use, as they all act intracellularly and therefore require gene transfer approaches. The present invention aims to use the extracellular domain of the receptor TPOR coupled to the Fc fragment of IgG1 as a capture molecule for circulating thrombopoietin (THPO). This approach aims to compete or partially block the signaling induced by abnormally elevated TPOR, which is important for the development of all types of MPN. Novel potent and specific THPO inhibitors would therefore provide a much-needed improvement for the treatment and / or prevention of MPN.

[0008] In addition to MPN, several hereditary and sporadic diseases result from overactivation of TPOR signaling. In hereditary thrombocytosis, germline mutations in TPOR or JAK2 result in cytokine-independent signaling. Such conditions are not clonal, but they do result in complications and need to be addressed early. In another condition, mutations in the untranslated region result in overproduction of THPO ligand by the liver. This type of condition would also benefit greatly from THPO inhibitors. In primary AML, studies have also suggested that high levels of THPO signaling may promote oncogenic signaling. Finally, infection and inflammation may result in higher THPO production by some tissues, resulting in thrombocytosis. For all these conditions, THPO inhibitors would be very useful. Summary of the Invention

[0009] The present invention relates to a polypeptide comprising an amino acid sequence having at least 75% sequence identity with SEQ ID NO:2 or a variant thereof, which binds to thrombopoietin (THPO). In one embodiment, the polypeptide is for use in the treatment and / or prevention of disease.

[0010] In some embodiments, the polypeptides are capable of inhibiting, reducing, preventing, and / or disrupting the binding of THPO to its receptor, hi some embodiments, the polypeptides are capable of inhibiting, reducing, preventing, and / or disrupting the TPOR signaling pathway or the TPOR-dependent signaling pathway.

[0011] In some embodiments, the amino acid sequence of the polypeptide has at least 75% sequence identity to SEQ ID NO:3.

[0012] In some embodiments, the amino acid sequence of the polypeptide has at least 75% sequence identity to SEQ ID NO:4.

[0013] The present invention further relates to a fusion protein comprising a polypeptide of the invention, in one embodiment, the fusion protein is for use in the treatment and / or prevention of a disease.

[0014] In one embodiment, the fusion protein further comprises a second polypeptide, preferably an Fc region of an immunoglobulin or a functional equivalent thereof, that improves the stability and / or reduces the immunogenicity of the first polypeptide.

[0015] In one embodiment, the immunoglobulin is IgA, IgD, IgE, IgG or IgM, preferably IgG.

[0016] The present invention further relates to a nucleic acid comprising a sequence encoding a polypeptide of the invention or a fusion protein comprising said polypeptide. In one embodiment, this nucleic acid is for use in the treatment and / or prevention of a disease.

[0017] The present invention further relates to a vector comprising a nucleic acid encoding a polypeptide of the invention or a fusion protein comprising said polypeptide, in one embodiment, said vector for use in the treatment and / or prevention of a disease.

[0018] The present invention further relates to a composition comprising a polypeptide, a fusion protein, a nucleic acid or a vector according to the invention and an acceptable vehicle. In one embodiment, this composition is for use in the treatment and / or prevention of a disease.

[0019] The present invention further relates to a pharmaceutical composition comprising (i) a polypeptide, a fusion protein, a nucleic acid or a vector according to the present invention and (ii) at least one pharma- ceutically acceptable vehicle. In one embodiment, this pharmaceutical composition is for use in the treatment and / or prevention of a disease.

[0020] The present invention further relates to a polypeptide, a fusion protein or a pharmaceutical composition according to the invention for use as a medicament.

[0021] The present invention further relates to a polypeptide, a fusion protein or a pharmaceutical composition according to the invention for use in the treatment and / or prevention of thrombopoietin-related diseases.

[0022] In some embodiments, the thrombopoietin-related disease is a disease, preferably an inflammatory and / or infectious disease, in which thrombopoietin receptor signaling is increased. Increased signaling can occur due to elevated levels of thrombopoietin or, even in the absence of elevated thrombopoietin levels, due to thrombopoietin enhancement of pathological signaling.

[0023] In some embodiments, the thrombopoietin-associated disease is selected from the group consisting of hematological diseases, chronic liver diseases, thrombopoietin-associated infections, thrombopoietin-associated inflammation, and hereditary thrombocytosis, preferably selected from the group consisting of myeloproliferative neoplasms (MPNs), hereditary thrombocytosis, and de novo AML (acute myeloid leukemia).

[0024] In some embodiments, the thrombopoietin-associated disease is a myeloproliferative neoplasm (MPN).

[0025] The present invention further relates to a kit for treating and / or preventing a thrombopoietin-related disease comprising (i) a polypeptide, a fusion protein or a pharmaceutical composition and (ii) a means for administering said means for administering said polypeptide, fusion protein or pharmaceutical composition, optionally further comprising an anti-cancer agent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] definition The term "amino acid substitution" refers to the replacement of one amino acid in a polypeptide with another amino acid. In one embodiment, an amino acid is replaced with another amino acid having similar structure and / or chemical properties, e.g., a conservative amino acid substitution. "Conservative amino acid substitutions" can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Non-conservative substitutions entail exchanging a member of one of these classes for a member of another class. For example, amino acid substitutions can also result in the replacement of one amino acid with another amino acid having different structural and / or chemical properties, e.g., replacing an amino acid from one group (e.g., polar) with an amino acid from another, different group (e.g., basic). Amino acid substitutions can be made using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is contemplated that methods of altering the side chain groups of amino acids by methods other than genetic engineering, such as chemical modification, can also be useful.

[0027] The term "identity" refers to a measure of the identity of nucleotide or amino acid sequences. Generally, sequences are aligned to obtain the largest order of correspondence. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. See, for example, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics And Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis Of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds, Humana Press, New Jersey, 1994; Sequence Analysis In Molecular Biology, von Heijne, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds, M Stockton Press, New York, 1991. While there are many methods for measuring the identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to those skilled in the art (Carillo and Lipton, SIAM J Applied Math, 1998, 48:1073). Commonly used methods for determining identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994; and Carillo and Lipton, SIAM J Applied Math, 1998, 48:1073. Methods for determining identity and similarity are codified in computer programs.Computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux et al., J Molec Biol, 1990, 215: 403), GAP program. By way of illustration, a polynucleotide having a nucleotide sequence with at least, for example, 95% "identity" with a reference nucleotide sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain an average of up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or up to 5% of the number of nucleotides in the reference sequence may be inserted into the reference sequence. These variations in the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere in between the terminal positions, and can be interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0028] The term "peptide linker" or "linker", also called "spacer peptide", refers to a peptide used to link two peptides or polypeptides together. In one embodiment, a peptide linker of the invention comprises 3-50 amino acids. Peptide linkers are known in the art or described herein.

[0029] The term "pharmaceutically acceptable excipient" refers to an excipient that does not produce adverse allergic or other undesirable reactions when administered to animals, preferably humans. This includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Pharmaceutically acceptable carriers or excipients refer to non-toxic solid, semisolid or liquid fillers, diluents, encapsulating materials or formulation auxiliary of any type. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.

[0030] The term "nucleic acid" or "polynucleotide" refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Nucleic acid" or "polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA that may be single-stranded or, more commonly, double-stranded, or a mixture of single-stranded and double-stranded regions. Furthermore, "nucleic acid" or "polynucleotide" refers to triple-stranded regions that include RNA or DNA or both RNA and DNA. The term "nucleic acid" or "polynucleotide" also includes DNA or RNA that contain one or more modified bases and DNA or RNA with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases, such as inosine. A variety of modifications have been made to DNA and RNA; thus, "nucleic acid" or "polynucleotide" encompasses chemically, enzymatically or metabolically modified forms of polynucleotides as found in nature generally, as well as chemical forms characteristic of viral and cellular DNA and RNA. "Polynucleotide" also encompasses shorter polynucleotides, often referred to as oligonucleotides.

[0031] The term "polypeptide" refers to any peptide or protein containing two or more amino acids linked together by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, commonly referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.

[0032] The term "protein" refers to sequences and / or multimeric entities of more than 100 amino acids. The proteins of the present invention are not limited to a particular length product. The terms "polypeptide" or "protein" do not refer to or exclude post-expression modifications of proteins, such as glycosylation, acetylation, phosphorylation, and the like, as well as other modifications, both natural and non-natural, known in the art. Such modifications are well described in basic textbooks and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide or protein, including the peptide backbone, the amino acid side chains, and the amino or carboxyl termini. It will be appreciated that the same type of modification can be present in the same or varying degrees at several sites in a given polypeptide or protein. Also, a given polypeptide or protein can contain many types of modifications. Polypeptides or proteins can be branched as a result of ubiquitination, and they can be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides or proteins can be the result of post-translational natural processes or can be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA mediated addition of amino acids to proteins, such as arginylation and ubiquitination.See, for example, "Proteins-structure and molecular properties", 2nd Ed., TECreighton, WH Freeman and Comany, New York, 1993; Wolt, F., "Posttranslational Protein Modifications: Perspectives and Prospects", Posttranslational covalent modification of proteins, BC Johnson, Ed., Academic Press, New York, 1983, pgs. 1-12; Seifter et al., "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol, 1990, 182:626-646; Rattan et al, "Protein Synthesis: Posttranslational Modifications and Aging", Ann NY Acad Sci, 1992, 663:48-62. The protein may be an entire protein or a subsequence thereof.

[0033] The term "fusion protein" refers to a protein having at least two heterologous polypeptides covalently linked either directly or via an amino acid linker. The polypeptides forming the fusion protein are generally linked C-terminus to N-terminus, but they can also be linked C-terminus to C-terminus, N-terminus to N-terminus or N-terminus to C-terminus. The polypeptides of the fusion protein can be fused in any order. The term also refers to conservatively modified variants, polymorphic variants, alleles, variants, subsequences and interspecies homologs of antigens that produce the fusion protein.

[0034] The term "fused" refers to components that are linked by a peptide bond, either directly or via one or more peptide linkers.

[0035] The term "immunoglobulin" includes any protein having a combination of two heavy chains and two light chains, whether or not it retains any relevant specific immunological reactivity.

[0036] The terms "treating" or "treatment" or "alleviation" refer to both therapeutic treatment and prophylactic or preventative measures; where the object is to prevent or delay (alleviate) a disease or condition, preferably a thrombopoietin-related disease, more preferably an MPN. Those in need of treatment include those already suffering from a disease or condition, preferably a thrombopoietin-related disease, more preferably an MPN, as well as those susceptible to a disease or condition, preferably a thrombopoietin-related disease, more preferably an MPN, or those in whom a disease or condition, preferably a thrombopoietin-related disease, more preferably an MPN, is to be prevented. A subject or mammal has been successfully "treated" with respect to a disease or condition, preferably a thrombopoietin-related disease, more preferably an MPN, if, after receiving a therapeutic amount of a polypeptide or fusion protein according to the methods of the invention, the patient exhibits an observable and / or measurable reduction or absence of one or more of the following: a reduction in the number of pathogenic cells; a reduction in the percentage of total cells that are pathogenic; and / or an alleviation to some extent of one or more of the symptoms associated with the disease or condition, preferably a thrombopoietin-related disease, more preferably an MPN; a reduction in morbidity and mortality and an improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in a disease or condition, preferably a thrombopoietin-related disease, more preferably an MPN, are readily measurable by routine procedures familiar to physicians.

[0037] The term "variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another reference polynucleotide. Changes in the nucleotide sequence of a variant may or may not change the amino acid sequence of a polypeptide encoded by a reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in a polypeptide encoded by a reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, the differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions (preferably conservative), additions, deletions, in any combination. The substituted or inserted amino acid residues may or may not be those encoded by the genetic code. A polynucleotide or polypeptide variant may be a naturally occurring one, such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis. The variant should retain one or more of the biological activities of the reference polypeptide.

[0038] Detailed Description The thrombopoietin receptor, referred to herein as TPOR, is a transmembrane protein expressed primarily in the plasma membrane of megakaryocytes, platelets, hemangioblasts, and hematopoietic stem cells that initiates the JAK / STAT signaling pathway upon binding to its ligand, thrombopoietin. Aberrant TPOR activation is associated with pathological conditions, including myeloproliferative neoplasms. In this disclosure, the full amino acid sequence of TPOR is referred to as SEQ ID NO: 1 (corresponding to UniProt IDP40238).

[0039] TPOR is a 635 amino acid long protein with three functional domains: an extracellular domain (ECD) containing the thrombopoietin binding site, a transmembrane domain (TMD) and a cytoplasmic / intracellular domain (ICD). In particular, the ECD is divided into four subdomains, namely D1, D2, D3 and D4.

[0040] Thrombopoietin is referred to herein as THPO, however, it may also be referred to as TPO, MPL ligand, or megakaryocyte colony stimulating factor.

[0041] The present invention relates to polypeptides comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO:2 or variants thereof that bind to thrombopoietin (THPO).

[0042] SEQ ID NO: 2 relates to the sequence of subdomain D1 of the ECD (hereinafter referred to as D1).

[0043] In one embodiment, a polypeptide of the present invention is an isolated polypeptide.

[0044] In one embodiment, the polypeptides of the invention are capable of preventing, inhibiting, reducing or disrupting the binding of thrombopoietin (THPO) to its receptor (TPOR / MPL).

[0045] In one embodiment, the amino acid sequence of a polypeptide of the invention does not include SEQ ID NO:1.

[0046] In some embodiments, a polypeptide of the invention comprises at least 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 amino acids. In some embodiments, a polypeptide of the invention comprises at least 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, or 270 amino acids. In some embodiments, a polypeptide of the invention comprises at least 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, or 480 amino acids.

[0047] In some embodiments, the polypeptide of the present invention comprises between 96 and 465 amino acids or between 96 and 258 amino acids. In some embodiments, the polypeptide of the present invention comprises between 96 and 500 amino acids, between 96 and 400 amino acids, between 96 and 300 amino acids, or between 96 and 200 amino acids.

[0048] In some embodiments, the polypeptide of the present invention comprises 258 to 465 amino acids. In some embodiments, the polypeptide of the present invention comprises 258 to 500 amino acids, 258 to 400 amino acids, or 258 to 300 amino acids.

[0049] In some embodiments, a polypeptide of the invention comprises between 465 and 500 amino acids.

[0050] In some embodiments, the polypeptide of the invention comprises up to 635 amino acids. In one embodiment, the polypeptide comprises up to 634 amino acids.

[0051] In one embodiment, a polypeptide of the invention comprises at most 600, 500, 400, 300, or 200 amino acids.

[0052] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 80% sequence identity, preferably at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2. In one embodiment, the amino acid sequence of a polypeptide of the invention comprises or consists of SEQ ID NO:2.

[0053] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity, preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3. In one embodiment, the amino acid sequence of a polypeptide of the invention comprises or consists of SEQ ID NO: 3. In one embodiment, the amino acid sequence of a polypeptide of the invention does not consist of SEQ ID NO: 3.

[0054] SEQ ID NO: 3 relates to the sequence of subdomains D1 and D2 of the ECD (hereinafter referred to as D1D2).

[0055] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity, preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4. In one embodiment, the amino acid sequence of a polypeptide of the invention comprises or consists of SEQ ID NO: 4.

[0056] SEQ ID NO: 4 relates to the sequences of subdomains D1 to D4 of the ECD (hereinafter referred to as D1D2D3D4).

[0057] In some embodiments, the polypeptides of the invention may act intracellularly ("cis" acting) or extracellularly ("trans" acting).

[0058] In a preferred embodiment, the polypeptides of the invention are capable of acting extracellularly ("trans" acting).

[0059] In some embodiments, the polypeptide of the invention is soluble. Advantageously, the solubility of the polypeptide of the invention allows it to act outside the cell ("trans" acting).

[0060] In some embodiments, the amino acid sequence of the polypeptide of the invention comprises at least one asparagine residue (Asn) at a position selected from the group consisting of or including positions 117, 178, 298, and 358, which positions are defined relative to the amino acid sequence SEQ ID NO:1. As used herein, at least one Asn residue means 1, 2, 3, or 4 Asn residues. In some embodiments, the amino acid sequence of the polypeptide of the invention comprises Asn residues at positions 117, 178, 298, and 358, which positions are defined relative to the amino acid sequence SEQ ID NO:1.

[0061] In some embodiments, the Asn residue is modified. In some embodiments, the Asn residue is glycosylated. In one embodiment, the glycan is mature. Non-limiting examples of mature glycosylations include Hex(5)HexNAc(2)dHex(1) and HexNAc(2)Hex(3)Fuc(1)Sias(1). In another embodiment, the glycan is immature. Non-limiting examples of immature glycosylations include HexNAc(2)Hex(10), HexNAc(2)Hex(9) and HexNAc(2)Hex(8). In some embodiments, the Asn residue is phosphorylated, glycosylated, ubiquitinated, nitrosylated, methylated, acetylated or lipidated.

[0062] In some embodiments, at least one Asn residue is unmodified. In some embodiments, the Asn residue is unmodified. In some embodiments, at least one Asn residue is unglycosylated. In some embodiments, the Asn residue is unglycosylated.

[0063] In some embodiments, the polypeptides of the invention bind to thrombopoietin (THPO) of SEQ ID NO: 5 or variants thereof.

[0064] In some embodiments, the amino acid sequence of a polypeptide of the invention comprises at least one mutation compared to the amino acid sequence of SEQ ID NO:2, which mutation improves the affinity of the polypeptide for THPO or a variant thereof.

[0065] As used herein, the term "mutation" refers to the insertion, deletion, truncation or substitution of at least one amino acid with at least one naturally occurring and / or non-naturally occurring different amino acid. Non-limiting examples of substitutions of amino acids with different chemical properties include the substitution of positively charged arginine with negatively charged glutamic acid or the substitution of polar asparagine with non-polar tryptophan.

[0066] As used herein, the term "affinity" refers to the "binding affinity" between two molecules, preferably proteins or polypeptides herein. Binding affinity is generally assessed by determining the dissociation constant (Kd) or measuring the kinetics of the reaction (Michaelis constant, Km), for example, using methods known in the art. In particular, when measuring the effect of an inhibitor, such as a competitive inhibitor or a non-competitive inhibitor, the inhibition constant (Ki) can be calculated.

[0067] In some embodiments, the polypeptides of the invention, particularly the fragment region D1D2D3D4, have a better interaction with THPO than the fragment region D1D2. Thus, the polypeptides of the invention are more effective at competing for THPO than the fragment region D1D2.

[0068] In one embodiment, the polypeptide is for use as a medicament. In one embodiment, the polypeptide is for use to treat and / or prevent a disease.

[0069] The present invention further relates to a fusion protein comprising a polypeptide as described herein above and at least one other polypeptide. As used herein in the context of a fusion protein, the polypeptide of the present invention is referred to as the "first polypeptide" and the at least one other polypeptide is referred to as the "second polypeptide."

[0070] In some embodiments, the second polypeptide enhances the stability of the first polypeptide. Enhanced stability refers to improved resistance of the polypeptide to modification or degradation, including but not limited to dissolution, truncation, irreversible post-translational modification or unfolding. In effect, enhanced stability results in an increased half-life of the polypeptide in an organism.

[0071] Methods for measuring protein or peptide stability are known in the art and include, but are not limited to, differential scanning calorimetry (DSC), bleach-chase, cycloheximide-chase, circular dichroism (CD) spectroscopy, and SDS-PAGE electrophoresis.

[0072] In some embodiments, the second polypeptide reduces the immunogenicity of the first polypeptide, which refers to the efficacy to elicit a detectable immune response in vivo, such as the production of antibodies, B cells, helper T cells and / or cytotoxic T cells, specifically directed against one or more antigens.

[0073] Methods for measuring T cell immune responses are well known to those of skill in the art and include, but are not limited to, monitoring the production of IFN-gamma.

[0074] In one embodiment, the second polypeptide of the fusion protein is an immunoglobulin Fc region or a functional equivalent thereof. The Fc fragment of an immunoglobulin refers to the carboxy-terminal portions of both H chains held together by disulfides.

[0075] In some embodiments, the second polypeptide of the fusion protein is an Fc region of an immunoglobulin selected from the group including or consisting of IgA, IgD, IgE, IgG, IgM and functional equivalents thereof.

[0076] In a preferred embodiment, the immunoglobulin is IgG, preferably IgG1 or IgG2. In another embodiment, the immunoglobulin is IgA. In another embodiment, the immunoglobulin is IgD. In another embodiment, the immunoglobulin is IgE. In another embodiment, the immunoglobulin is IgM.

[0077] In a preferred embodiment, the immunoglobulin is a human immunoglobulin. In another embodiment, the immunoglobulin is a non-human immunoglobulin.

[0078] In some embodiments, the immunoglobulin is modified, for example genetically and / or post-translationally modified, in order to indeed improve the stability of the fusion protein and / or its immunogenicity.

[0079] In one embodiment, the polypeptides of a fusion protein of the invention are arranged in a single, continuous polypeptide chain.

[0080] In one embodiment, the fusion protein of the invention further comprises at least one peptide linker. In one embodiment, the polypeptides of the fusion protein of the invention are linked to each other through one or more peptide linkers.

[0081] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the fusion protein (fragment) with appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, NY (1989). The expression vector can be part of a plasmid, a virus, or can be a nucleic acid fragment. The expression vector contains an expression cassette into which the polynucleotide (i.e., coding region) encoding the fusion protein (fragment) is cloned in operative association with a promoter and / or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of a nucleic acid consisting of codons that are translated into amino acids. Although a "stop codon" (TAG, TGA or TAA) is not translated into amino acids, it may be considered to be part of the coding region; however, any adjacent sequences, if present, such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, etc., are not part of the coding region. Two or more coding regions may be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector may contain a single coding region or may comprise two or more coding regions, e.g., a vector of the invention may encode one or more polypeptides that are post-translationally or co-translationally separated into a final protein via proteolytic cleavage.Additionally, the vectors, polynucleotides or nucleic acids of the invention may encode heterologous coding regions, either fused or unfused to the polynucleotide encoding the fusion protein (fragment) of the invention or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. Operable association is when a coding region for a gene product, such as a polypeptide, is linked to one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and an associated promoter) are "operably linked" if induction of promoter function results in transcription of an mRNA encoding the desired gene product, and if the nature of the link between the two DNA fragments does not interfere with the ability of the expression control sequences to direct expression of the gene product or the ability of the DNA template to be transcribed. Thus, a promoter region is operably linked to a nucleic acid encoding a polypeptide if the promoter is capable of affecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in a given cell. Other transcriptional control elements besides promoters, such as enhancers, operators, repressors, and transcription termination signals, can be operably linked to the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. A variety of transcriptional control regions are known to those of skill in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., immediate early promoter, linked to intron-A), Simian Virus 40 (e.g., early promoter), and retroviruses (e.g., Rous Sarcoma Virus, etc.). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone, and rabbit a-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells.Further suitable transcription control regions include tissue-specific promoters and enhancers as well as inducible promoters (e.g., tetracycline-inducible promoters). Similarly, various translation control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons and elements derived from viral systems (particularly internal ribosome entry sites or IRES, also called CITE sequences). The expression cassette may also include other features such as an origin of replication, and / or chromosomal integration elements such as retroviral long terminal repeats (LTRs) or adeno-associated virus (AAV) inverted terminal repeats (ITRs).

[0082] Fusion proteins prepared as described herein may be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to one of skill in the art. For affinity chromatography purification, an antibody, ligand, receptor, or antigen to which the fusion protein binds may be used. The purity of the fusion protein may be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, etc.

[0083] In one embodiment, the fusion protein as described herein above is soluble. In some embodiments, the second polypeptide improves the solubility of the first polypeptide.

[0084] In one embodiment, the fusion protein as described herein above is for use as a medicament.

[0085] The present invention also relates to a nucleic acid comprising a sequence encoding a polypeptide or a fusion protein as described herein above.

[0086] In one embodiment, nucleic acids encoding a fusion protein of the invention can be expressed as a single nucleic acid molecule encoding the entire fusion protein or as multiple (e.g., two or more) co-expressed nucleic acid molecules. The polypeptides encoded by the co-expressed nucleic acid molecules can associate, for example, through disulfide bonds or other means, to form a functional fusion protein.

[0087] In one embodiment, the nucleic acid molecule is DNA. In another embodiment, the nucleic acid molecule is in the form of RNA, such as messenger RNA (mRNA).

[0088] In one embodiment, the nucleic acid is linear, hi another embodiment, the nucleic acid is circular.

[0089] In one embodiment, the nucleic acid as described herein above is for use as a medicament.

[0090] Another object of the invention is a vector comprising a nucleic acid according to the invention. In some embodiments, at least one nucleic acid molecule is comprised in one vector.

[0091] Within the scope of the present invention, the expression "at least one nucleic acid" is intended to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleic acid molecules.

[0092] In some embodiments, the vector allows for the controlled expression of at least one polypeptide and / or fusion protein. As used herein, the expression "controlled expression" is intended to refer to expression that is controlled in time and / or space. In other words, the controlled expression of the polypeptide according to the present invention can occur at a specific location in the body, such as a specific organ, and / or for a specific time.

[0093] In certain embodiments, the vector is a viral vector. Non-limiting examples of viral vectors include adenoviruses, adeno-associated viruses (AAV), alphaviruses, herpes viruses, lentiviruses, non-integrating lentiviruses, retroviruses, vaccinia viruses, and baculoviruses.

[0094] In certain embodiments, the vector is a non-viral vector. Non-limiting examples of non-viral vectors include inorganic particles (e.g., gold, calcium phosphate), lipid emulsions, lipid nanoparticles (e.g., liposomes), DNA-binding proteins or peptides.

[0095] In one embodiment, the vector as described herein above is for use as a medicament.

[0096] The present invention further relates to a composition comprising a polypeptide, a fusion protein, a nucleic acid or a vector according to the invention and an acceptable vehicle.

[0097] The present invention further relates to a pharmaceutical composition comprising a polypeptide, a fusion protein, a nucleic acid or a vector according to the invention and a pharma- ceutically acceptable vehicle.

[0098] In some embodiments, the pharma- ceutically acceptable vehicle is selected from the group consisting of or includes solvents, diluents, carriers, excipients, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, and any combination thereof. The carriers, diluents, solvents, or excipients must be "acceptable" in the sense of being compatible with the polypeptide or derivative thereof and not harmful when administered to an individual. Generally, the vehicle does not cause harmful allergic or other untoward reactions when administered to an individual, preferably a human individual.

[0099] For particular purposes for human administration, the pharmaceutical compositions should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory agencies, such as the Food and Drugs Administration (FDA) Office or the European Medicines Agency (EMA).

[0100] In some embodiments, the carrier can be water or saline (e.g., physiological saline), which is sterile and pyrogen-free. Suitable excipients include mannitol, dextrose, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.

[0101] Acceptable carriers, solvents, diluents and excipients for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro ed. 1985). The selection of a suitable pharmaceutical carrier, solvent, excipient or diluent can be made with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition can contain any suitable binder, lubricant, suspending agent, coating agent or solubilizing agent as, or in addition to, the carrier, excipient, solvent or diluent. Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition.

[0102] In some embodiments, the polypeptides, fusion proteins, nucleic acids or vectors may be included in delivery particles, particularly in combination with other natural or synthetic compounds, such as lipids, proteins, peptides or polymers.

[0103] Within the scope of the present invention, said delivery particles are intended to provide or "deliver" a polypeptide, a nucleic acid or a nucleic acid vector according to the invention to a target cell, tissue or organ.

[0104] In some embodiments, the delivery particles can be in the form of lipoplexes containing cationic lipids; lipid nanoemulsions; solid lipid nanoparticles; peptide-based particles; polymer-based particles, especially including natural and / or synthetic polymers; and mixtures thereof.

[0105] In some embodiments, polymer-based particles may include synthetic polymers, particularly polyethyleneimine (PEI), dendrimers, poly(DL-lactide) (PLA), poly(DL-lactide-co-glycoside) (PLGA), polymethacrylates, and polyphosphoesters.

[0106] In some embodiments, the delivery particle further comprises one or more ligands on its surface suitable for targeting the polypeptide, nucleic acid or nucleic acid vector to a target cell, tissue or organ.

[0107] In some embodiments, the pharmaceutical composition as described hereinabove is for use as a medicament.

[0108] In one embodiment the polypeptide, fusion protein, pharmaceutical composition or medicament of the invention is for the treatment and / or prevention of a disease.

[0109] In one embodiment, the polypeptide, fusion protein, pharmaceutical composition or medicament of the invention is for the treatment and / or prevention of a thrombopoietin-related disease.

[0110] As used herein, the term "thrombopoietin-related disease" refers to any disease or condition in which THPO contributes, is involved at any level, or promotes its development or progression.

[0111] In some embodiments, the thrombopoietin-related disorders of the invention include diseases or conditions associated with elevated blood levels of THPO. Elevated blood levels of THPO can result from abnormally high production (or overproduction) of THPO and / or abnormally low degradation of THPO.

[0112] In some other embodiments, the thrombopoietin-related disease of the present invention includes diseases or conditions associated with hypersensitivity of TPOR to its ligand THPO. Hypersensitivity of TPOR can be due to abnormally high activation (or overactivation) of TPOR. Overactivation of TPOR can be due to elevated blood levels of THPO. Overactivation of TPOR can be due to one or more mutations in the genes encoding TPOR and / or JAK2. In one embodiment, the thrombopoietin-related disease is associated with or due to constitutive activation of TPOR.

[0113] In some embodiments, thrombopoietin-related disorders include diseases or conditions that highlight pathological mechanisms in which THPO-induced signaling affects other signaling pathways.

[0114] In some embodiments, the polypeptides, fusion proteins, pharmaceutical compositions or medicaments of the invention are for the treatment and / or prevention of a condition or disease associated with elevated blood levels of THPO in a patient. In some embodiments, elevated blood levels of THPO means greater than 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310 or 320 pg / mL. In some embodiments, elevated blood levels of THPO means greater than 350, 400, 450, 500, 750 or 1000 pg / mL.

[0115] In some embodiments, the thrombopoietin-associated disorder is associated with an elevated platelet count, and the elevated platelet count is greater than or equal to 150x10 platelets. 3 , 200x10 3 , 250x10 3 , 300x10 3 , 350x10 3 , 400x103 , 450x10 3 Or 500x10 3 This means more than cells / μL of blood.

[0116] In some embodiments, a thrombopoietin-associated disease includes a disease or condition associated with elevated blood levels of THPO, such that THPO-induced signaling is increased. In some embodiments, the polypeptides, fusion proteins, pharmaceutical compositions or medicaments of the invention are for the treatment and / or prevention of inflammatory conditions and infectious diseases associated with increased THPO-induced signaling.

[0117] In some embodiments, the polypeptides, fusion proteins, pharmaceutical compositions or medicaments of the invention are for the treatment and / or prevention of a thrombopoietin-related disease selected from the group consisting of hematological diseases, chronic liver diseases, thrombopoietin-associated infections, thrombopoietin-associated inflammation and hereditary thrombocytosis in a subject in need thereof.

[0118] In some embodiments, the polypeptides, fusion proteins, pharmaceutical compositions or medicaments of the present invention are for the treatment and / or prevention of diseases or conditions in which TPOR is overactivated.

[0119] In one embodiment the polypeptide, fusion protein, pharmaceutical composition or medicament is for the treatment and / or prevention of a hematological disorder in a subject in need thereof.

[0120] In some embodiments, the polypeptide, fusion protein, pharmaceutical composition or medicament of the invention is for the treatment and / or prevention of a hematological disorder selected from the group comprising or consisting of myeloproliferative neoplasm (MPN), de novo acute myeloid leukemia (de novo AML), secondary acute myeloid leukemia (secondary AML) and hereditary thrombocytosis, preferably myeloproliferative neoplasm (MPN) or de novo acute myeloid leukemia (AML), in a subject in need thereof.

[0121] In a preferred embodiment, the polypeptide, fusion protein, pharmaceutical composition or medicament of the invention is for the treatment and / or prevention of myeloproliferative neoplasms (MPNs) in a subject in need thereof.

[0122] As used herein, MPN refers to a type of blood cancer caused by pathological constitutive activation of the JAK / STAT pathway, which primarily affects hematopoietic stem cells and induces the abnormal expansion of cells of the myeloid lineage.

[0123] In one embodiment, the MPN comprises or is selected from the group consisting of essential thrombocythemia (ET), primary myelofibrosis (PMF) and polycythemia vera (PV).

[0124] In one embodiment, the polypeptides according to the invention alleviate, reduce and / or inhibit THPO-induced signaling and / or constitutive activation of JAK / STAT that induces MPN.

[0125] In one embodiment, the polypeptides according to the invention alleviate, reduce and / or inhibit THPO-induced signaling and / or constitutive activation of JAK / STAT, which induces MPNs through transactivation.

[0126] In one embodiment, a polypeptide according to the present invention comprises an amino acid sequence having at least 75% identity to SEQ ID NO:4 and ameliorates, reduces and / or inhibits THPO-induced signal transduction and / or constitutive activation of JAK / STAT that induces MPN.

[0127] In some embodiments, the polypeptide of the invention comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 4 has better inhibition of THPO-induced signal transduction and / or constitutive activation of JAK / STAT inducing MPN than the fragment region D1D2. In one embodiment, the polypeptide, fusion protein, pharmaceutical composition or medicament of the invention is for the treatment and / or prevention of newly developed AML in a subject in need thereof.

[0128] In another embodiment, the polypeptide, fusion protein, pharmaceutical composition or medicament is for the treatment and / or prevention of hereditary thrombocytosis in a subject in need thereof. As used herein, hereditary thrombocytosis relates to a familial myeloproliferative disorder with clinical characteristics resembling sporadic essential thrombocythemia, a disorder characterized by excessive platelet production, which may be caused by mutations in the genes encoding THPO and / or TPOR.

[0129] In another embodiment, the polypeptide, fusion protein, pharmaceutical composition or medicament is for the treatment and / or prevention of chronic liver disease in a subject in need thereof. In another embodiment, the polypeptide, fusion protein, pharmaceutical composition or medicament is for the treatment and / or prevention of thrombopoietin-associated infection in a subject in need thereof. In another embodiment, the polypeptide, fusion protein, pharmaceutical composition or medicament is for the treatment and / or prevention of thrombopoietin-associated inflammation in a subject in need thereof.

[0130] In some embodiments, the polypeptide, fusion protein, nucleic acid, vector or pharmaceutical composition of the invention is administered in combination with an additional anti-cancer agent or with an anti-cancer vaccine.

[0131] In a particular embodiment, the further anti-cancer agent or vaccine is administered in combination, simultaneously or sequentially, with the polypeptide, fusion protein, nucleic acid, vector, pharmaceutical composition or agent according to the invention.

[0132] In one embodiment, the further anti-cancer agent or vaccine is administered simultaneously (i.e., co-administered, optionally in a co-formulation) with the polypeptide, fusion protein, nucleic acid, vector, pharmaceutical composition or agent according to the invention of the present invention. In one embodiment, the further anti-cancer agent or vaccine is administered at a different time than the polypeptide, fusion protein, nucleic acid, vector, pharmaceutical composition or agent according to the present invention (i.e., sequential administration, where the further anti-cancer agent or vaccine is administered before or after the polypeptide is administered). In some embodiments, the further anti-cancer agent or vaccine may be administered in the same manner as the polypeptide, fusion protein, nucleic acid, vector, pharmaceutical composition or agent according to the present invention, or by using the usual route of administration for the further anti-cancer agent or vaccine.

[0133] As used herein, the term "subject" refers to an animal, preferably a mammal, more preferably a human. In one embodiment, the subject is a male. In another embodiment, the subject is a female. In one embodiment, the subject is a "patient", i.e., the subject is awaiting or receiving medical care, or has been / is / will be subject to a medical procedure or treatment aimed at curing or treating a thrombopoietin-related disease, preferably MPN, and / or alleviating the symptoms of a blood cancer, preferably MPN. In some embodiments, the subject is monitored for the progression of a thrombopoietin-related disease, preferably MPN. In one embodiment, the subject is given a prophylactic treatment against a thrombopoietin-related disease, preferably MPN. In one embodiment, the subject is an adult (e.g., a subject over 18 years of age). In another embodiment, the subject is a child (e.g., a subject under 18 years of age).

[0134] In some embodiments, a polypeptide, nucleic acid, vector or pharmaceutical composition according to the invention is administered orally, parenterally, topically, by inhalation spray, rectally, nasally, buccally, vaginally, or via an implanted reservoir. As used herein, the term administration includes subcutaneous, intravenous, intramuscular, intraocular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0135] In a preferred embodiment, a polypeptide, nucleic acid, vector or pharmaceutical composition of the invention is administered parenterally, subcutaneously, intravenously or via an implanted reservoir.

[0136] In some embodiments, a polypeptide, nucleic acid, vector or pharmaceutical composition of the invention is in a form suitable for injection, e.g., for intraocular, intramuscular, subcutaneous, intradermal, transdermal or intravenous injection or infusion.

[0137] Examples of forms suitable for injection include, but are not limited to, liquid solutions, e.g., sterile aqueous solutions, dispersions, emulsions, suspensions, solid forms suitable for use in preparing solutions or suspensions upon addition of liquid prior to use, e.g., powders, liposomal forms, etc.

[0138] Treatment may consist of a single dose or multiple doses over a period of time. The polypeptide, nucleic acid, vector or pharmaceutical composition according to the invention may be formulated in a sustained release formulation to provide sustained release over an extended period of time, such as over at least 2 or 4 or 6 or 8 weeks. Preferably, sustained release is provided for at least 4 weeks.

[0139] In certain embodiments, the effective amount of a polypeptide, nucleic acid, vector or pharmaceutical composition administered may depend on a variety of parameters, including the material selected for administration, whether the administration is a single dose or multiple doses, and subject parameters including age, health, size, weight, sex and the severity of the thrombopoietin-related disorder being treated.

[0140] In some embodiments, a polypeptide, nucleic acid, vector or pharmaceutical composition according to the invention is administered to a subject in need thereof in a therapeutically effective amount.

[0141] A "therapeutically effective amount" means a level or amount that is necessary and sufficient to slow or stop the progression, worsening, or aggravation of one or more symptoms of a thrombopoietin-related disorder, or to alleviate the symptoms of cancer, or to cure a thrombopoietin-related disorder, without causing significant negative or harmful side effects to the individual. In certain embodiments, an effective amount of a polypeptide according to the present invention may range from about 0.001 mg to about 3,000 mg per dosage unit, preferably from about 0.05 mg to about 1,000 mg per dosage unit.

[0142] Within the scope of the present invention, about 0.001 mg to about 3,000 mg is about 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg , 0.3mg, 0.4mg, 0.5mg, 0.6mg, 0.7mg, 0.8mg, 0.9mg, 1mg, 2mg, 3mg, 4mg, 5mg, 6mg, 7mg, 8mg, 9mg, 10mg, 20mg, 30 mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg , 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1,000mg, 1,100mg, 1,150mg, 1,200mg, 1,250mg, 1, 300mg, 1,350mg, 1,400mg, 1,450mg, 1,500mg, 1,550mg, 1,600mg, 1,650mg, 1,700mg, 1,750mg, 1,800mg, 1,850 mg, 1,900mg, 1,950mg, 2,000mg, 2,100mg, 2,150mg, 2,200mg, 2,250mg, 2,300mg, 2,350mg, 2,400mg, 2,450mg, 2,500mg, 2,550mg, 2,600mg, 2,650mg, 2,700mg, 2,750mg, 2,800mg, 2,850mg, 2,900mg, 2,950mg and 3,000mg.

[0143] In certain embodiments, the polypeptides according to the present invention are administered at a dosage level sufficient to deliver from about 0.001 mg / kg to about 100 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, preferably from about 0.1 mg / kg to about 40 mg / kg, preferably from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, and more preferably from about 1 mg / kg to about 25 mg / kg of subject body weight per day.

[0144] In some embodiments, the effective amount of the nucleic acid or vector administered is about 1x10 5 ~ approx. 1x10 15 Copies / dosage unit may range.

[0145] Within the scope of the present invention, about 1x10 5 ~ approx. 1x10 15 Copies are 1x10 per dosage unit 5 , 2x10 5 , 3x10 5 , 4x10 5 , 5x10 5 , 6x10 5 , 7x10 5 , 8x10 5 , 9x10 5 , 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , 9x10 9 , 1x10 10 , 2x10 10 , 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x10 10 , 8x10 10 , 9x10 10 , 1x10 11 , 2x10 11 , 3x10 11 , 4x10 11 , 5x10 11 , 6x10 11 , 7x10 11 , 8x10 11 , 9x10 11 , 1x10 12 , 2x10 12 , 3x10 12 , 4x10 12 , 5x10 12 , 6x10 12 , 7x10 12 , 8x10 12 , 9x10 12 , 1x10 13 , 2x10 13 , 3x10 13 , 4x10 13 , 5x10 13 , 6x10 13 , 7x10 13 , 8x10 13 , 9x10 13 , 1x10 14 , 2x10 14 , 3x10 14 , 4x10 14 , 5x10 14 , 6x10 14 , 7x10 14 , 8x10 14 , 9x10 14 and 1x10 15 Includes copy.

[0146] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a lyophilized (freeze-dried) condition requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations for use in the present invention may further contain other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may contain flavoring agents.

[0147] Another object of the invention is a kit comprising (i) at least one polypeptide, at least one fusion protein, at least one nucleic acid, at least one vector, at least one pharmaceutical composition or at least one vaccine according to the invention, and (ii) means for administering said polypeptide, fusion protein, nucleic acid, vector, pharmaceutical composition or medicament. In one embodiment, the kit is for treating and / or preventing a disease, preferably a thrombopoietin-associated disease or a thrombopoietin receptor-associated disease, more preferably an MPN.

[0148] In certain embodiments, the means for administering a polypeptide, a fusion protein, a nucleic acid, a vector, a pharmaceutical composition or a medicament according to the invention may include a syringe, a trocar, a catheter, a cup, a spatula, and the like.

[0149] In one embodiment, the kit further comprises an anti-cancer agent or vaccine, preferably for treating MPNs.

[0150] In some further aspects, the present invention also relates to the use of a polypeptide, a fusion protein, a nucleic acid, a vector, a pharmaceutical composition or a medicament according to the invention for the manufacture or preparation of a medicament.

[0151] The present invention also relates to a polypeptide, a fusion protein, a nucleic acid, a vector, a pharmaceutical composition or a medicament according to the invention for the manufacture of a medicament for the treatment and / or prevention of thrombopoietin-related diseases, preferably selected from the group consisting of myeloproliferative neoplasms (MPN), blood diseases, hereditary thrombocytosis, first-onset AML (acute myeloid leukemia), chronic liver diseases, thrombopoietin-associated infections and thrombopoietin-associated inflammation, preferably MPN.

[0152] The present invention also relates to a method for treating and / or preventing a thrombopoietin-related disease selected from the group consisting of myeloproliferative neoplasms (MPN), blood diseases, hereditary thrombocytosis, de novo AML (de novo acute myeloid leukemia), secondary acute myeloid leukemia (secondary AML), chronic liver disease, thrombopoietin-related infections and thrombopoietin-related inflammation, preferably MPN, in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a polypeptide, fusion protein, nucleic acid, vector, pharmaceutical composition or medicament according to the invention.

[0153] The present invention also relates to a polypeptide, a fusion protein, a nucleic acid, a vector, a pharmaceutical composition or a medicament according to the invention for use in inhibiting the proliferation and spread of cancer cells.

[0154] The present invention also relates to a method for inhibiting the proliferation and growth of cancer cells in an individual in need thereof, the method comprising at least the step of administering to the individual a therapeutically effective amount of a polypeptide, fusion protein, nucleic acid, vector, pharmaceutical composition or agent according to the present invention.

[0155] The present invention also relates to a polypeptide, a fusion protein, a nucleic acid, a vector, a pharmaceutical composition or a medicament according to the invention for use in improving the overall survival of an individual with cancer.

[0156] The present invention also relates to a method for improving the overall survival of an individual having cancer, the method comprising at least the step of administering to the individual a therapeutically effective amount of a polypeptide, a fusion protein, a nucleic acid, a vector or a pharmaceutical composition according to the present invention.

[0157] The present invention also relates to a polypeptide, a fusion protein, a nucleic acid, a vector, a pharmaceutical composition or a medicament according to the invention for use in improving the prognosis of an individual with cancer.

[0158] The present invention also relates to a method for improving the prognosis of an individual having cancer, the method comprising at least the step of administering to the individual a therapeutically effective amount of a polypeptide, a fusion protein, a nucleic acid, a vector, a pharmaceutical composition or a medicament according to the present invention.

[0159] Another object of the present invention is a method for inhibiting or reducing or preventing or disrupting the binding of THPO to TPOR in an individual, the method comprising at least administering to the individual an effective amount of a polypeptide, fusion protein, nucleic acid, vector, pharmaceutical composition or agent according to the present invention. In one embodiment, inhibiting or reducing the binding of THPO to TPOR inhibits or reduces or prevents or disrupts TPOR signaling in said subject. In one embodiment, inhibiting or reducing or preventing or disrupting the binding of THPO to TPOR inhibits the activation of TPOR in said subject. In one embodiment, inhibiting or reducing or preventing or disrupting the binding of THPO to TPOR treats or prevents a thrombopoietin-related disease.

[0160] The present invention also relates to a method of competing endogenous THPO in an individual with a polypeptide or fusion protein of the present invention, the method comprising at least administering to the individual an effective amount of a polypeptide, fusion protein, nucleic acid, vector, pharmaceutical composition or agent according to the present invention. In one embodiment, competing with endogenous THPO inhibits or reduces or prevents or interferes with TPOR signaling in the subject. In one embodiment, competing with endogenous THPO inhibits activation of TPOR in the subject. In one embodiment, competing endogenous THPO treats or prevents a thrombopoietin-related disease.

[0161] A further object of the present invention is a method for inhibiting, reducing, preventing or disrupting TPOR signaling or TPOR-dependent signaling in an individual, the method comprising administering to the individual at least an effective amount of a polypeptide, fusion protein, nucleic acid, vector, pharmaceutical composition or agent according to the present invention. Another object of the present invention is a method for inhibiting, reducing, preventing or disrupting activation of TPOR in an individual, the method comprising administering to the individual at least an effective amount of a polypeptide, fusion protein, nucleic acid, vector, pharmaceutical composition or agent according to the present invention. In one embodiment, inhibiting, reducing, preventing or disrupting TPOR signaling or TPOR-dependent signaling treats or prevents a thrombopoietin-related disease. [Brief description of the drawings]

[0162] [Figure 1]1A-1B are graphs showing THPO capture by soluble extracellular domain (ECD)-TPOR and Fc-bound soluble ECD-TPOR in vitro. Soluble ECD-TPOR (A) and soluble ECD-TPOR-Fc fusion protein (B) were collected after their secretion by HEK293T in culture. They were incubated with human recombinant THPO and then added to the medium of Baf3 expressing TPOR on their cell surface. The proliferation of cells incubated with soluble ECD-TPOR species (A) or with soluble ECD-TPOR-Fc fusion protein (B) in the presence (dotted line) or absence (solid line) of THPO was measured by daily cell counting for 5 days. Cell proliferation was measured in the presence of soluble D1+ / -Fc (dark grey, triangles), soluble D1D2+ / -Fc (light grey, circles), and D1D2D3D4+ / -Fc (black, squares) and compared to cell proliferation in the absence of soluble ECD-TPOR (black, circles). [Diagram 2] Figures 2A-2B are histograms showing the dose of circulating THPO by ELISA from MPL knockout mice before and after presentation to soluble TPOR in vivo. MPL- / - mice were subcutaneously injected with MEF cells transduced with retroviral particles (pMEGIX vector) expressing mouse soluble D1D2D3D4 fused to Fc (soluble ECD-TPOR-Fc). Two weeks after transplantation of MEF cells, vascularized cell masses under the skin were developed and two consecutive blood sampling experiments were performed at a one-week interval: the first blood sampling (Bleed 1) 3 weeks after transplantation (A) and the second blood sampling (Bleed 2) 4 weeks after transplantation (B). The graph shows the circulating THPO concentration in blood for each mouse group (5-6 mice / group). * indicates significant difference p<0.05; ** indicates significant difference p<0.01; *** indicates significant difference p<0.001 (one-way ANOVA, Holm-Sidak multiple comparison test), ns: no significant difference.

[0163] Working Example The present invention is further illustrated by the following examples.

[0164] Example 1: In vitro THPO capture by soluble TPOR extracellular domain fused or not to Fc Materials and Methods Soluble extracellular domain (ECD) species of TPOR, namely D1, D1D2 and D1D2D3D4, and -Fc fusion proteins of these ECD species were collected after their secretion by cultured HEK293T cells. They were incubated with human recombinant THPO to allow the interaction between soluble ECD-TPOR (or soluble ECD-TPOR-Fc fusion protein) and THPO, and were added to the culture medium of hematopoietic cell line Baf3 cells stably expressing human TPOR on their surface, whose proliferation is strictly dependent on cytokines. Cell proliferation was measured by daily cell counting for 5 days to demonstrate the ability of soluble ECD-TPOR and ECD-TPOR-Fc to sequester THPO in the culture medium.

[0165] result The results revealed significant inhibition of Baf3 cell proliferation in the presence of any one of the soluble ECDs of TPOR (D1, D1D2, and D1D2D3D4) (Figure 1A) as well as by the soluble species of the Fc-linked receptor (Figure 1B).

[0166] Notably, THPO inhibition was most efficient with the full-length ECD of TPOR (D1D2D3D4) rather than the truncated forms (D1 and D1D2) (Figure 1A). This difference in inhibitory potency was significantly increased by coupling soluble D1D2D3D4 to the Fc fragment compared to D1-Fc and D2-Fc (Figure 1B).

[0167] Example 2: In vivo validation of THPO signaling inhibition by soluble D1D2D3D4 of TPOR fused to Fc Materials and Methods MPL- / - mice, i.e. mice constitutively deficient in TPOR (because it is not degraded by its receptor) that express very high plasma concentrations of thrombopoietin, were subcutaneously injected (5-6 mice per group) with mouse embryonic fibroblast (MEF) cells transduced with retroviral particles (pMEGIX vector) containing DNA encoding the murine soluble D1D2D3D4 of TPOR fused to Fc, allowing their production by muscle cells and release into the blood circulation. Two weeks after transplantation of MEF cells, vascularized cell masses developed under the skin, and two consecutive blood sampling experiments were performed at 1-week intervals: the first blood sampling 3 weeks after transplantation and the second blood sampling 4 weeks after transplantation.

[0168] result The first bleed 3 weeks after MEF cell transplantation did not yield any significant difference between transplanted and non-transplanted mice (Figure 2A). However, in the second bleed 1 week later (4 weeks after transplantation), measurements of plasma THPO showed that soluble D1D2D3D4 linked to Fc was able to significantly reduce circulating levels of THPO (Figure 2B). This result therefore revealed that D1D2D3D4-Fc acts as a capture molecule for circulating THPO in vivo, thereby validating the in vitro results.

Claims

1. 2, comprising an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 2 or a variant thereof, which binds to thrombopoietin (THPO) for use in the treatment and / or prevention of diseases; Polypeptides.

2. 2. The polypeptide of claim 1, which is capable of inhibiting, reducing, preventing and / or disrupting the binding of thrombopoietin to its receptor (TPOR / MPL).

3. 3. The polypeptide of claim 1 or 2, which is capable of inhibiting, reducing, preventing and / or disrupting the TPOR signaling pathway or the TPOR-dependent signaling pathway.

4. The polypeptide according to claims 1 to 3, wherein the amino acid sequence has at least 75% sequence identity with SEQ ID NO:

3.

5. The polypeptide according to claims 1 to 3, wherein the amino acid sequence has at least 75% sequence identity with SEQ ID NO:

4.

6. A fusion protein comprising the polypeptide according to any one of claims 1 to 5.

7. The fusion protein of claim 6, further comprising a second polypeptide that improves the stability and / or immunogenicity of the polypeptide, preferably the Fc region of an immunoglobulin or a functional equivalent thereof.

8. The fusion protein according to claim 7, wherein the immunoglobulin is IgA, IgD, IgE, IgG or IgM, preferably IgG.

9. A nucleic acid comprising a sequence encoding a polypeptide according to any one of claims 1 to 5 or a fusion protein according to any one of claims 6 to 8.

10. A vector comprising the nucleic acid of claim 9.

11. A composition comprising a polypeptide according to any one of claims 1 to 5, a fusion protein according to any one of claims 6 to 8, a nucleic acid according to claim 9, or a vector according to claim 10, and an acceptable vehicle.

12. A pharmaceutical composition comprising (i) a polypeptide according to any one of claims 1 to 5, a fusion protein according to any one of claims 6 to 8, a nucleic acid according to claim 9, or a vector according to claim 10, and (ii) at least one pharma- ceutical acceptable vehicle.

13. The polypeptide according to any one of claims 1 to 5, the fusion protein according to any one of claims 6 to 8, or the pharmaceutical composition according to claim 10, wherein the disease is a thrombopoietin-associated disease.

14. 14. The polypeptide, fusion protein or pharmaceutical composition of claim 13, wherein the thrombopoietin-related disease comprises or is selected from the group consisting of blood diseases, chronic liver diseases, thrombopoietin-related infections, thrombopoietin-related inflammation and hereditary thrombocytosis, and preferably comprises or is selected from the group consisting of myeloproliferative neoplasms (MPN), newly diagnosed AML (acute myeloid leukemia).

15. 15. The polypeptide, fusion protein or pharmaceutical composition of claim 13 or 14, wherein the thrombopoietin-associated disease is a myeloproliferative neoplasm (MPN).

16. A kit for treating and / or preventing a thrombopoietin-related disease comprising (i) a polypeptide according to any one of claims 1 to 5, a fusion protein according to any one of claims 6 to 8, or a pharmaceutical composition according to claim 12, and (ii) a means for administering said polypeptide, fusion protein or pharmaceutical composition, optionally further comprising an anti-cancer agent or a vaccine.

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