Thrombopoietin receptor fragments and uses thereof - Patents.com

Abstract

The present invention relates to polypeptides comprising fragments of the extracellular domain of the thrombopoietin receptor, fusion proteins and pharmaceutical compositions comprising said polypeptides, and uses thereof.

Description

[Technical field]

[0001] The present invention relates to a polypeptide comprising at least one fragment of the thrombopoietin receptor and its use as a medicament. The present invention also relates to the treatment and / or prevention of myeloproliferative neoplasms (MPNs). [Background technology]

[0002] Malignant hematological diseases impose a serious burden on the healthcare system. Some such conditions, such as BCR-ABL-negative myeloproliferative neoplasms (MPNs), are diseases of the elderly, are associated with many complications including thrombosis and bleeding, and require many healthcare resources to treat. MPNs can develop into a very serious condition called secondary acute myeloid leukemia (AML), which is almost always fatal. The three main MPN types, namely polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF), can develop in children and adolescents, but are most common in the elderly, with incidence rates that can reach 1 in 5000 per year. In all three diseases, acquired mutations in hematopoietic stem cells (HSCs) lead to pathological activation of TPOR / JAK2 signaling, which drives the pathology.

[0003] JAK2 V617F mutations are responsible for 70% of all MPNs and are present in over 96% of PV and 60% of ET and MF cases, with 70% of cases involving BCR-ABL-negative MPNs. The majority of the remaining cases of ET and MF are due to acquired mutations in calreticulin (CALR) (20-30%), a chaperone that is retained in the endoplasmic reticulum for quality control of N-glycosylated proteins and calcium storage, and / or thrombopoietin receptor (TPOR) (3-5%).

[0004] It was shown that the JAK2 V617F mutation acted by inducing receptor dimerization through its intracellular domain in the absence of cytokines, through interactions between the pseudokinase domains of the mutant JAK2 molecules associated with the receptor.

[0005] It was also elucidated that CALR mutants bind to TPOR and induce its activation in the absence of its ligand thrombopoietin (THPO or TPO), resulting in sustained activation of the JAK2-STAT5 pathway in HSCs and megakaryocytes (Chachoua et al., 2016; Pecquet et al., 2019). Chachoua et al., 2016 also disclose that a region of TPOR and its associated N-glycosylation site (N117) are required for the activity of CALR mutants. Pecquet et al., 2019 describe that the FFPLHWLV motif of TPOR is important for activation but not binding.

[0006] Different mutations have been identified in exon 9, the last exon of CALR. In MPNs, deletions or insertions of more than 60 nucleotides in CALR have been described, all of which result in the same +1 frameshift, which contributes to the synthesis of a new C-terminus of CALR (also designated mutant C-tail or mutant tail) that is methionine-rich, positively charged, and lacks the retained KDEL motif. The new tail of CALR confers tight binding to TPOR. The TPOR-CALR mutant complex translocates to the cell surface and induces constitutive activation of the JAK2 / STAT5 pathway in megakaryocytic precursors and stem cells where TPOR is expressed.

[0007] Regardless of the driver mutation, the outcome is always hyperactivation of the JAK2-STAT5 pathway through TPOR and, to a lesser extent, through other cytokine receptors (EpoR in JAK2 mutants, G-CSFR in JAK2 mutants and CALR mutants). To date, therapeutic interventions have focused on non-specific JAK2 inhibitors, which did not provide adequate clinical responses in terms of reducing allele burden, blasts, and fibrosis. The absolute necessity and central role of TPOR for the development of all three diseases makes it a prime target for therapeutic intervention, but it remains understudied.

[0008] The inventors show that specific domains or subdomains of TPOR are involved in the interaction with mutant CALR and compete or partially block signaling induced by abnormally high TPOR, which is crucial for the development of all types of MPN. Summary of the Invention

[0009] Thus, the present invention relates to a polypeptide comprising an amino acid sequence having at least 75% sequence identity with SEQ ID NO:66, wherein said amino acid sequence of said polypeptide does not include SEQ ID NO:5.

[0010] Thus, the present invention relates to a polypeptide comprising an amino acid sequence having at least 75% sequence identity with SEQ ID NO:2, wherein said amino acid sequence of said polypeptide does not include SEQ ID NO:5.

[0011] Thus, the present invention relates to a polypeptide comprising an amino acid sequence having at least 75% sequence identity with SEQ ID NO:2, wherein said amino acid sequence of said polypeptide does not include SEQ ID NO:6.

[0012] In some embodiments, the amino acid sequence of the polypeptide has at least 75% sequence identity to SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0013] In some embodiments, the amino acid sequence of the polypeptide has at least 75% sequence identity to SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

[0014] In some embodiments, the amino acid sequence of the polypeptide does not include SEQ ID NO:5.

[0015] In some embodiments, the amino acid sequence of the polypeptide has at least 75% sequence identity to SEQ ID NO:3 or SEQ ID NO:4.

[0016] In some embodiments, the amino acid sequence of the polypeptide comprises at least SEQ ID NO:7.

[0017] In some embodiments, the polypeptide binds to a variant of calreticulin (CALR) that has a positively charged amino acid sequence in its C-terminal tail.

[0018] In some embodiments, the amino acid sequence of the polypeptide comprises SEQ ID NO:7, and / or at least one amino acid Asn at position 117, said positions being defined with respect to the amino acid sequence of SEQ ID NO:1.

[0019] In some embodiments, the amino acid Asn at position 117 is glycosylated.

[0020] In some embodiments, the amino acid Asn at position 117 is not glycosylated.

[0021] The present invention further relates to fusion proteins comprising a polypeptide of the invention.

[0022] In one embodiment, the fusion protein further comprises a second polypeptide that increases the stability and / or decreases the immunogenicity of the first polypeptide.

[0023] In one embodiment, the second polypeptide of the fusion protein is an immunoglobulin Fc region or a functional equivalent thereof, preferably selected from the group including or consisting of IgG, IgA, IgD, IgE, or IgM.

[0024] 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.

[0025] The present invention further relates to a vector comprising a nucleic acid encoding a polypeptide of the present invention or a fusion protein comprising said polypeptide.

[0026] 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 invention, and (ii) at least one pharma- ceutically acceptable vehicle.

[0027] The present invention further relates to a polypeptide, a fusion protein, a nucleic acid, a vector or a pharmaceutical composition according to the invention for use as a medicament.

[0028] In some embodiments, the polypeptide, fusion protein, nucleic acid, vector, or pharmaceutical composition of the invention is for use in the treatment and / or prevention of myeloproliferative neoplasms (MPNs).

[0029] In some embodiments, MPNs are derived by one or more mutations in calreticulin (CALR), preferably resulting in the production of a positively charged amino acid sequence at the C-terminus of CALR.

[0030] The present invention further relates to a kit for treating and / or preventing myeloproliferative neoplasms (MPNs), comprising (i) a polypeptide, fusion protein, nucleic acid, vector, or pharmaceutical composition for use according to the present invention, (ii) a means for administering said polypeptide, fusion protein, or pharmaceutical composition, and optionally (iii) a further anti-cancer agent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] definition In the present invention, the following terms have the following meanings.

[0032] The term "about" when preceding a number means ±10% of said numerical value.

[0033] The term "amino acid substitution" refers to the replacement of one amino acid with another amino acid in a polypeptide. In one embodiment, an amino acid is replaced with another amino acid having similar structural and / or chemical properties, e.g., conservative amino acid replacement. "Conservative amino acid substitution" may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, tyrosine, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, 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 substitution involves exchanging a member of one of these classes with a member of another class. For example, amino acid substitutions may 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 a different group (e.g., basic). Amino acid substitutions may be made using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. It is contemplated that methods other than genetic engineering, such as methods that alter the side chain of an amino acid by chemical modification, may also be useful.

[0034] The term "identity" refers to the measurement of the identity of nucleotide or amino acid sequences. Generally, sequences are aligned to obtain the top match. "Identity" itself 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. There are many methods for measuring the identity between two polynucleotide or polypeptide sequences, and 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), the GAP program. By way of illustration, a polynucleotide having a nucleotide sequence with at least, for example, 95% "identity" to a reference nucleotide sequence is intended to be identical to the reference sequence, except that the polynucleotide sequence may contain, on average, up to 5 point mutations per 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 of the reference sequence may be deleted or replaced with another nucleotide, or up to 5% of the number of nucleotides of the total nucleotides of the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5'-end or 3'-end position of the reference nucleotide sequence, or may occur anywhere between these end positions, either individually between the nucleotides of the reference sequence or scattered in one or more consecutive groups within the reference sequence.

[0035] The term "peptide linker" or "linker", also known as a "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.

[0036] The term "pharmaceutically acceptable excipient" refers to an excipient that does not cause 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, etc. Pharmaceutically acceptable carriers or excipients refer to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid. For human administration, they must meet the standards of sterility, pyrogenicity, general safety, and purity required by the FDA Agency's standards for biologics.

[0037] 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, more typically 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 contains one or more modified bases, and DNA or RNA with backbones modified for stability or 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 the chemically, enzymatically, or metabolically modified forms of polynucleotides commonly found in nature; as well as the chimeric forms of DNA and RNA that are characteristic of viruses and cells. "Polynucleotide" also encompasses shorter polynucleotides, often referred to as oligonucleotides.

[0038] The term "polypeptide" refers to any peptide or protein containing two or more amino acids joined to each other 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.

[0039] The term "protein" refers to a sequence of more than 100 amino acids and / or a multimeric entity. 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 other modifications known in the art, including naturally occurring and non-naturally occurring. Such modifications are well described in basic texts and more detailed monographs, as well as a large 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 is understood that the same type of modification can be present in the same or varying degrees at several sites in a given polypeptide or protein. A given polypeptide or protein can also contain many types of modifications. Polypeptides or proteins may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides or proteins may result from post-translational natural processes or may 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, formation of GPI anchors, 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, e.g., "Proteins-structure and molecular properties", 2nd Ed., TE Creighton, 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.

[0040] The term "fusion protein" refers to a protein having at least two heterologous polypeptides covalently linked directly or via an amino acid linker. The polypeptides forming the fusion protein are typically 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, multimeric variants, alleles, mutants, subsequences, and interspecies homologs of the antigens that make up the fusion protein.

[0041] The term "fused" refers to components that are linked by a peptide bond, either directly or through one or more peptide linkers.

[0042] The term "immunoglobulin" refers to a protein having a combination of two heavy chains and two light chains, regardless of whether it has any associated specific immunoreactivity.

[0043] The terms "treating" or "treatment" or "alleviation" refer to both therapeutic treatment and prophylactic or preventative measures, the object of which is to prevent or slow down (attenuate) the onset of a disease or condition, preferably a hematological cancer, more preferably an MPN. Those in need of treatment include those already suffering from the disease or condition, preferably a hematological cancer, more preferably an MPN, as well as those prone to having the disease or condition, preferably a hematological cancer, more preferably an MPN, or those in which the disease or condition, preferably a hematological cancer, more preferably an MPN, is to be prevented. "Treatment" of the disease or condition, preferably blood cancer, more preferably MPN, is successful if, after administration of a therapeutic amount of the fusion protein, the patient exhibits an observable and / or measurable reduction or absence of one or more of the following: reduction in the number of pathogenic cells; reduction in the percentage of the total number of cells that are pathogenic; and / or alleviation to some extent of one or more symptoms associated with the disease or condition, preferably blood cancer, more preferably MPN; reduction in morbidity and mortality; and improvement in quality of life issues. Parameters for assessing the success and improvement of treatment of the disease or condition, preferably blood cancer, more preferably MPN, are readily measurable by routine procedures well known to physicians.

[0044] 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. The change in the nucleotide sequence of the variant may or may not change the amino acid sequence of the polypeptide encoded by the reference polynucleotide. The change in the nucleotide sequence may result in amino acid substitution, addition, deletion, fusion, and truncation in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. In general, the differences are limited, such that the sequences of the reference polypeptide and the variant are closely similar overall, and identical in many regions. The variant and the reference polypeptide may differ in amino acid sequence by one or more substitutions (preferably conservative substitutions), additions, deletions, in any combination. The substituted or inserted amino acid residues may or may not be encoded by the genetic code. A variant of a polynucleotide or polypeptide, such as an allelic variant, may be naturally occurring, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis techniques or by direct synthesis. The variant should retain one or more of the biological activities of the reference polypeptide.

[0045] Detailed Description The thrombopoietin receptor, referred to herein as TPOR, is a transmembrane protein expressed predominantly in the cell membrane of megakaryocytes, platelets, hemangioblasts, and hematopoietic stem cells, which upon binding to its ligand thrombopoietin induces the JAK / STAT signaling pathway. In this disclosure, the complete amino acid sequence of TPOR is referred to as SEQ ID NO:1 (corresponding to UniProt ID P40238).

[0046] 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.

[0047] The present invention relates to a polypeptide comprising an amino acid sequence having at least 75% sequence identity with SEQ ID NO:66, wherein X1 is D (Asp, aspartic acid) or E (Glu, glutamic acid) and X2 is R (Arg, arginine) or K (Lys, lysine), and said amino acid sequence of the polypeptide does not include SEQ ID NO:5.

[0048] SEQ ID NO:66: PLKCFSX2TFX1X1LTCFWX1X1X1X1AAPSGTYQLLYAYPREKPRACPLSSQSMPHFGTRYVCQFPX1QX1X1VX2

[0049] In one embodiment, the polypeptide of the present invention relates to a polypeptide comprising an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 66, the amino acid sequence comprising at least the motifs TFX1X1 (SEQ ID NO: 72), WX1X1X1X1 (SEQ ID NO: 74), and X1QX1X1 (SEQ ID NO: 76), where X1 is D (Asp, aspartic acid) or E (Glu, glutamic acid). In one embodiment, the polypeptide of the present invention comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 66, the amino acid sequence comprising at least the motifs TFED (SEQ ID NO: 60), WDEEE (SEQ ID NO: 73), and DQEE (SEQ ID NO: 75).

[0050] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO:66, the amino acid sequence comprising at least the motifs X2TFX1X1 (SEQ ID NO:78), WX1X1X1X1 (SEQ ID NO:74), and X1QX1X1VX2 (SEQ ID NO:80), where X1 is D (Asp, aspartic acid) or E (Glu, glutamic acid) and X2 is R (Arg, arginine) or K (Lys, lysine). In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO:66, the amino acid sequence comprising at least the motifs RTFED (SEQ ID NO:77), WDEEE (SEQ ID NO:73), and DQEEVR (SEQ ID NO:79).

[0051] 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: 66. In one embodiment, the amino acid sequence of a polypeptide of the invention comprises or consists of SEQ ID NO:66.

[0052] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:67, wherein said amino acid sequence of the polypeptide does not include SEQ ID NO:5.

[0053] SEQ ID NO:67: PLKCFSRTFEDLTCFWDEEEAAPSGTYQLLYAYPREKPRACPLSSQSMPHFGTRYVCQFPDQEEVR

[0054] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 67, preferably at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 67, the amino acid sequence comprising at least the motifs RTFED, WDEEE, and DQEEVR. In one embodiment, the amino acid sequence of a polypeptide of the invention comprises or consists of SEQ ID NO: 67.

[0055] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:68, wherein X1 is D (Asp, aspartic acid) or E (Glu, glutamic acid), and X2 is R (Arg, arginine) or K (Lys, lysine), and said amino acid sequence of the polypeptide does not include SEQ ID NO:5.

[0056] SEQ ID NO:68: QX1VSLLASX1SX1PLKCFSX2TFX1X1LTCFWX1X1X1X1AAPSGTYQLLYAYPREKPRACPLSSQSMPHFGTRYVCQFPX1QX1X1VX2

[0057] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 68, the amino acid sequence comprising at least the motifs QX1V, SX1SX1 (SEQ ID NO: 82), TFX1X1, WX1X1X1X1, and X1QX1X1, where X1 is D (Asp, aspartic acid) or E (Glu, glutamic acid). In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 68, the amino acid sequence comprising at least the motifs QDV, SDSE (SEQ ID NO: 81), TFED, WDEEE, and DQEE.

[0058] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 68, the amino acid sequence comprising at least the motifs QX1V, SX1SX1, X2TFX1X1, WX1X1X1X1, and X1QX1X1VX2, where X1 is D (Asp, aspartic acid) or E (Glu, glutamic acid) and X2 is R (Arg, arginine) or K (Lys, lysine). In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 68, the amino acid sequence comprising at least the motifs QDV, SDSE, RTFED, WDEEE, and DQEEVR.

[0059] 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: 68. In one embodiment, the amino acid sequence of a polypeptide of the invention comprises or consists of SEQ ID NO: 68.

[0060] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:69, wherein said amino acid sequence of the polypeptide does not include SEQ ID NO:5.

[0061] SEQ ID NO:69: QDVSLLASDSEPLKCFSRTFEDLTCFWDEEEAAPSGTYQLLYAYPREKPRACPLSSQSMPHFGTRYVCQFPDQEEVR

[0062] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:69, wherein the amino acid sequence comprises at least the motifs QDV, SDSE, TFED, WDEEE, and DQEE.

[0063] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:69, wherein the amino acid sequence comprises at least the motifs QDV, SDSE, RTFED, WDEEE, and DQEEVR.

[0064] In one embodiment, a polypeptide of the invention comprises an amino acid sequence that has at least 80% sequence identity, preferably at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 69. In one embodiment, the amino acid sequence of a polypeptide of the invention comprises or consists of SEQ ID NO: 69.

[0065] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:70, wherein X1 is D (Asp, aspartic acid) or E (Glu, glutamic acid), and X2 is R (Arg, arginine) or K (Lys, lysine), and said amino acid sequence of the polypeptide does not include SEQ ID NO:5.

[0066] SEQ ID NO:70:

[0067] QX1VSLLASX1SX1PLKCFSX2TFX1X1LTCFWX1X1X1X1AAPSGTYQLLYAYPREKPRACPLSSQSMPHFGTRYVCQFPX1QX1X1VX2LFFPLHLWV

[0068] In one embodiment, a polypeptide of the invention comprises an amino acid sequence that has at least 80% sequence identity, preferably at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 70. In one embodiment, the amino acid sequence of a polypeptide of the invention comprises or consists of SEQ ID NO: 70.

[0069] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 70, the amino acid sequence comprising at least the motifs QX1V, SX1SX1, TFX1X1, WX1X1X1X1, and X1QX1X1, where X1 is D (Asp, aspartic acid) or E (Glu, glutamic acid). In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 70, the amino acid sequence comprising at least the motifs QDV, SDSE, TFED, WDEEE, and DQEE.

[0070] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 70, the amino acid sequence comprising at least the motifs QX1V, SX1SX1, X2TFX1X1, WX1X1X1X1, and X1QX1X1VX2, where X1 is D (Asp, aspartic acid) or E (Glu, glutamic acid) and X2 is R (Arg, arginine) or K (Lys, lysine). In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 70, the amino acid sequence comprising at least the motifs QDV, SDSE, RTFED, WDEEE, and DQEEVR.

[0071] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:71, wherein said amino acid sequence of the polypeptide does not include SEQ ID NO:5.

[0072] SEQ ID NO:71: QDVSLLASDSEPLKCFSRTFEDLTCFWDEEEAAPSGTYQLLYAYPREKPRACPLSSQSMPHFGTRYVCQFPDQEEVRLFFPLHLWV

[0073] 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: 71. In one embodiment, the amino acid sequence of a polypeptide of the invention comprises or consists of SEQ ID NO: 71.

[0074] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 71, wherein the amino acid sequence comprises at least the motifs QDV, SDSE, TFED, WDEEE, and DQEE.

[0075] In one embodiment, a polypeptide of the invention comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 71, wherein the amino acid sequence comprises at least the motifs QDV, SDSE, RTFED, WDEEE, and DQEEVR.

[0076] The present invention also relates to a polypeptide comprising an amino acid sequence having at least 75% sequence identity with SEQ ID NO:2, wherein said amino acid sequence of the polypeptide does not include SEQ ID NO:5.

[0077] The present invention also relates to a polypeptide comprising an amino acid sequence having at least 75% sequence identity with SEQ ID NO:2, wherein said amino acid sequence of the polypeptide does not include SEQ ID NO:6.

[0078] SEQ ID NO: 2 relates to the sequence of subdomain D1 of the ECD domain (hereinafter referred to as D1).

[0079] SEQ ID NO:2: PLKCFSRTFEDLTCFWDEEEAAPSGTYQLLYAYPREKPRACPLSSQSMPHFGTRYVCQFPDQEEVRLFFPLHLWVKNVFLN

[0080] SEQ ID NO:6 relates to the sequence of the complete extracellular domain (ECD) of wild-type TPOR, including subdomains D1, D2, D3, and D4 of the ECD (hereinafter referred to as D1D2D3D4).

[0081] In one embodiment, a polypeptide of the present invention is an isolated polypeptide.

[0082] In one embodiment, the amino acid sequence of a polypeptide of the invention does not include SEQ ID NO:1.

[0083] In some embodiments, a polypeptide of the invention comprises at least 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids.

[0084] In some embodiments, a polypeptide of the invention comprises between 66 and 464 amino acids, between 66 and 258 amino acids, between 66 and 96 amino acids, or between 66 and 85 amino acids.

[0085] In some embodiments, the polypeptide of the present invention comprises between 66 and 400 amino acids, preferably between 66 and 300 amino acids, more preferably between 66 and 200 amino acids, and even more preferably between 66 and 150 amino acids. In one embodiment, the polypeptide of the present invention comprises between 66 and 100 amino acids, 66 and 95, 66 and 90, or 66 and 85 amino acids.

[0086] In some embodiments, a polypeptide of the invention comprises between 77 and 464 amino acids, between 77 and 258 amino acids, between 77 and 96 amino acids, or between 77 and 85 amino acids.

[0087] In some embodiments, the polypeptide of the present invention comprises between 77 and 400 amino acids, preferably between 77 and 300 amino acids, more preferably between 77 and 200 amino acids, and even more preferably between 77 and 150 amino acids. In one embodiment, the polypeptide of the present invention comprises between 77 and 100 amino acids, 77 and 95, 77 and 90, or 77 and 85 amino acids.

[0088] In some embodiments, a polypeptide of the invention comprises between 81 and 464 amino acids, between 81 and 258 amino acids, between 81 and 96 amino acids, or between 81 and 85 amino acids.

[0089] In some embodiments, the polypeptide of the present invention comprises between 81 and 400 amino acids, preferably between 81 and 300 amino acids, more preferably between 81 and 200 amino acids, and even more preferably between 81 and 150 amino acids. In one embodiment, the polypeptide of the present invention comprises between 81 and 100 amino acids, 81 and 95, 81 and 90, or 81 and 85 amino acids.

[0090] In some embodiments, the polypeptide of the present invention comprises between 85 and 400 amino acids, preferably between 85 and 300 amino acids, more preferably between 85 and 200 amino acids, and even more preferably between 85 and 150 amino acids. In one embodiment, the polypeptide of the present invention comprises between 85 and 100 amino acids, 85 and 95, or 85 and 90 amino acids.

[0091] In some embodiments, the polypeptide of the present invention comprises between 96 and 400 amino acids, preferably between 96 and 300 amino acids, more preferably between 96 and 200 amino acids, even more preferably between 96 and 150 amino acids, or between 96 and 100 amino acids.

[0092] In some embodiments, the polypeptides of the invention comprise up to 635 amino acids. In one embodiment, the polypeptides comprise up to 456 amino acids. In another embodiment, the polypeptides comprise up to 464 amino acids.

[0093] In one embodiment, a polypeptide of the invention comprises up to 600, 500, 400, 300, or 200 amino acids. In one embodiment, a polypeptide of the invention comprises up to 190, 180, 170, 160, 150, 140, 130, 125, 120, 115, 110, 105, or 100, 95, 90, or 85 amino acids. In one embodiment, a polypeptide of the invention comprises up to 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, or 67 amino acids.

[0094] In one embodiment, the amino acid sequence of a polypeptide of the invention is at least 80% identical, 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 consists of SEQ ID NO: 2.

[0095] In some embodiments, the FFPLHLWV (SEQ ID NO:83) motif in SEQ ID NO:2, 3, 4, 70, or 71 may be absent or mutated. As we show, mutation of this motif does not prevent competition between TPOR and D1 for binding of CALA mutants.

[0096] 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 SEQ ID NO: 3.

[0097] 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.

[0098] SEQ ID NO:3: PLKCFSRTFEDLTCFWDEEEAAPSGTYQLLYAYPREKPRACPLSSQSMPHFGTRYVCQFPDQEEVRLFFPLHLWVKNVFLNQTRT

[0099] 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.

[0100] SEQ ID NO:4: QDVSLASDSEPLKCFSRTFEDLTCFWDEEEAAPSGTYQLLYAYPREKPRACPLSSQSMPHFGTRYVCQFPDQEEVRLFFPLHLWVKNVFLNQTRT

[0101] In some embodiments, the amino acid sequence of the polypeptide has at least 75% sequence identity to SEQ ID NO:3 or SEQ ID NO:4.

[0102] 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: 5. In one embodiment, the amino acid sequence of a polypeptide of the invention comprises or consists of SEQ ID NO:5.

[0103] SEQ ID NO:5 relates to the sequence of subdomains D1 and D2 of the ECD (hereinafter referred to as D1D2).

[0104] SEQ ID NO:5: QDVSLLASDSEPLKCFSRTFEDLTCFWDEEEAAPSGTYQLLYAYPREKPRACPLSSQSMPHFGTRYVCQFPDQEEVRLFFPLHLWVKNVFLNQTRTQRVLFVDSVGLPAPPSIIKAMGGSQPGELQISW EEPAPEISDFLRYELRYGPRDPKNSTGPTVIQLIATETCCPALQRPHSASALDQSPCAQPTMPWQDGPKQTSPSREASALTAEGGGSCLISGLQPGNSYWLQLRSEPDGISLGGSWGSWSLPTVDLPGD

[0105] In some embodiments, the amino acid sequence of a polypeptide of the invention does not comprise SEQ ID NO: 5. In some embodiments, the amino acid sequence of a polypeptide of the invention does not consist of SEQ ID NO:5.

[0106] In one embodiment, the amino acid sequence of a polypeptide of the invention comprises the amino acid sequence SEQ ID NO:7.

[0107] SEQ ID NO:7: PLKCFSRTFEDLTCFWDEE

[0108] In some embodiments, the polypeptides of the invention may act intracellularly ("cis" acting) or extracellularly ("trans" acting).

[0109] In some embodiments, the polypeptides of the invention are soluble. Suitably, the solubility of the polypeptides of the invention enables them to act outside the cell ("trans" acting).

[0110] In some embodiments, the amino acid sequence of a polypeptide of the invention comprises an asparagine residue (Asn) at position 117, said position being defined with respect to the amino acid sequence of SEQ ID NO:1.

[0111] In some embodiments, one or more amino acids of the polypeptide of the present invention are modified. Examples of post-translational modifications include, but are not limited to, phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, and proteolysis. In one embodiment, one or more amino acids of the polypeptide of the present invention are glycosylated. As used herein, the term "glycosylation" defines the state of an amino acid with a glycan, i.e., a polysaccharide, reversibly attached to one of its moieties. In a particular embodiment, the residue Asn at position 117 above is glycosylated.

[0112] In one embodiment, the glycosylation at Asn at position 117 is immature. In one embodiment, the immature glycan attached to Asn at position 117 is HexNAc(2)Hex(10), preferably HexNAc(2)Man(9)Glc(1). In another embodiment, the immature glycan attached to Asn at position 117 is HexNAc(2)Hex(9), preferably HexNAc(2)Man(9), or HexNAc(2)Man(8)Glc(1). In another embodiment, the immature glycan attached to Asn at position 117 is HexNAc(2)Hex(8), preferably HexNAc(2)Man(8).

[0113] In another embodiment, the glycosylation at Asn 117 is mature. In one embodiment, the mature glycan attached to Asn 117 is HexNAc(2)Hex(3)Fuc(1)Sias(1). In another embodiment, the mature glycan attached to Asn 117 is Hex(5)HexNAc(2)dHex(1).

[0114] In some embodiments, the amino acid sequence of a polypeptide of the invention comprises an asparagine residue (Asn) at position 117 that is unmodified.

[0115] In some embodiments, the amino acid sequence of the polypeptide of the present invention comprises an asparagine residue (Asn) at position 117 that is not glycosylated. Without wishing to be bound by theory, the inventors have shown that the asparagine residue (Asn) at position 117 (N117) of the polypeptide of the present invention (said position is defined with respect to the amino acid sequence SEQ ID NO: 1) is not required for its "trans" action (i.e., extracellular action), i.e., is not required for interaction with CALR mutants. Chachoua et al., 2016 discloses that the D1D2 fragment region of TPOR corresponding to 258 amino acids and its associated N-glycosylation site at N117 is required for the activity of CALR mutants. In the present invention, the inventors define a fragment of TPOR as a fragment that binds to CALR mutants and can compete for the activation of TPOR. Importantly, this identified fragment binds to TPOR even when N117 lacks immature sugar. As a result, the polypeptides of the present invention can compete with endogenous TPOR for binding to CALR mutants even if they lack glycosylation at N117. As a result, the polypeptides of the present invention advantageously inhibit activation of TPOR by CALR mutants even if N117 is not glycosylated, can be soluble, and can act from the extracellular space.

[0116] In some embodiments, the polypeptides of the invention bind to a variant of calreticulin (CALR) that has a positively charged amino acid sequence in its C-terminal tail.

[0117] 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 increases the affinity of the polypeptide for a variant of calreticulin.

[0118] 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.

[0119] As used herein, the term "affinity" refers to the "binding affinity" between two molecules, preferably proteins or polypeptides. Binding affinity is usually evaluated by determining the dissociation constant (Kd) or, for example, measuring the kinetics of the reaction (Michaelis constant, Km) using methods known in the art. In particular, when measuring the effect of an inhibitor, for example a competitive inhibitor or a non-competitive inhibitor, the inhibition constant (Ki) can be calculated.

[0120] In some embodiments, the polypeptides of the invention contain at least one mutation that prevents or reduces binding of the polypeptide to thrombopoietin (THPO).

[0121] In some embodiments, at least one mutation is located in at least one domain of the polypeptide involved in interaction with THPO. In some embodiments, at least one mutation alters the three-dimensional structure of the polypeptide, wherein the alteration of the three-dimensional structure prevents interaction with THPO.

[0122] In some embodiments, the polypeptides of the invention have better affinity for a CALR mutant than they have affinity for THPO.

[0123] In one embodiment, "good affinity" means an affinity difference of at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more. Affinity comparisons can be assessed by comparing Kd, Km, or Ki.

[0124] In some embodiments, the polypeptide of the present invention, particularly the fragment region D1, has a better interaction with CALR mutants than the fragment region D1D2D3D4. Thus, the polypeptide of the present invention competes more efficiently for CALR mutants than the fragment region D1D2D3D4.

[0125] In one embodiment, the polypeptide is for use as a pharmaceutical.

[0126] The present invention further relates to a fusion protein comprising the polypeptide described hereinabove and at least one other polypeptide. When used herein in the context of a fusion protein, the polypeptide of the present invention is referred to as a "first polypeptide" and the at least one other polypeptide is referred to as a "second polypeptide."

[0127] In some embodiments, the second polypeptide increases the stability of the first polypeptide.Increasing stability refers to improving the resistance of the polypeptide to modification or degradation, including but not limited to dissolution, truncation, irreversible post-translational modification, or unfolding.In fact, increasing stability leads to an increase in the half-life of the polypeptide in organism.

[0128] 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.

[0129] In some embodiments, the second polypeptide reduces the immunogenicity of the first polypeptide, by which is meant the ability to induce a detectable immune response in an organism, such as the production of antibodies, B cells, helper T cells, and / or cytotoxic T cells specifically against one or more antigens.

[0130] 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-γ.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] In a preferred embodiment, the immunoglobulin is a human immunoglobulin, hi another embodiment, the immunoglobulin is a non-human immunoglobulin.

[0135] In some embodiments, the immunoglobulin is modified, for example genetically and / or by post-translational modification, in order to increase the stability of the fusion protein and / or its immunogenicity.

[0136] In one embodiment, the polypeptides of the fusion protein of the invention are arranged in a single continuous polypeptide chain.

[0137] 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 via one or more peptide linkers.

[0138] 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) in conjunction 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 a nucleic acid fragment. The expression vector contains an expression cassette in which the polynucleotide (i.e., the coding region) encoding the fusion protein (fragment) is cloned in operable 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 part of the coding region when present, but any adjacent sequences, e.g., promoters, ribosome binding sites, transcription termination codons, 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 stranded polynucleotide construct, e.g., on a single vector or on separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector may contain a single coding region or may contain two or more coding regions, e.g., the vectors of the invention may encode one or more polypeptides that are post- or co-translationally separated into a final protein via proteolytic cleavage.Furthermore, the vectors, polynucleotides, or nucleic acids of the present invention may encode heterologous coding regions, or variants or derivatives thereof, either fused or not fused to the polynucleotide encoding the fusion protein (fragment) of the present invention. 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 associated with one or more control sequences such that expression of the gene product is under the influence or control of the regulatory sequences. Two DNA fragments (e.g., a polypeptide coding region and its associated promoter) are "operably associated" when induction of promoter function results in transcription of an mRNA encoding the desired gene product, and when the nature of the linkage 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 to be transcribed. Thus, a promoter region is operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of said nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in a given cell. In addition to the promoter, other transcription control elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as, but are not limited to, promoters and enhancer segments from cytomegalovirus (e.g., immediate early promoters linked to intron A), promoters and enhancer segments from simian virus 40 (e.g., early promoters), and promoters and enhancer segments from retroviruses (e.g., Rous sarcoma virus).Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit alpha globulin, 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 start and stop 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 the long terminal repeats (LTRs) of retroviruses, or the inverted terminal repeats (ITRs) of adeno-associated viruses (AAV).

[0139] The 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, molecular sieve 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 example, affinity chromatography purification may use an antibody, ligand, receptor, or antigen to which the fusion protein binds. 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.

[0140] In one embodiment, the fusion protein described herein above is for use as a medicament.

[0141] The present invention also relates to a nucleic acid comprising a sequence encoding a polypeptide or a fusion protein as described hereinabove.

[0142] In one embodiment, a nucleic acid encoding a fusion protein of the invention can be represented 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, via disulfide bonds or other means, to form a functional fusion protein.

[0143] In one embodiment, the nucleic acid molecule is DNA, hi another embodiment, the nucleic acid molecule is RNA, for example in the form of messenger RNA (mRNA).

[0144] In one embodiment, the nucleic acid is linear, hi another embodiment, the nucleic acid is circular.

[0145] In one embodiment, the nucleic acid described herein above is for use as a medicament.

[0146] Another subject of the present invention is a vector comprising a nucleic acid according to the invention. In some embodiments, at least one nucleic acid molecule is comprised in a vector.

[0147] 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.

[0148] In some embodiments, the vector allows for controlled expression of at least one polypeptide and / or fusion protein. As used herein, the expression "controlled expression" is intended to refer to time- and / or space-controlled expression. In other words, the controlled expression of the polypeptide of the present invention may occur at a specific location in the body, such as a specific organ, and / or at a specific time.

[0149] In certain embodiments, the vector is a viral vector. Non-limiting examples of viral vectors include adenovirus, adeno-associated virus (AAV), alphavirus, herpes virus, lentivirus, non-integrating lentivirus, retrovirus, vaccinia virus, and baculovirus.

[0150] 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.

[0151] In one embodiment, the vector described herein above is for use as a medicament.

[0152] 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.

[0153] In some embodiments, the pharma- ceutically acceptable vehicle is selected from the group consisting of solvents, diluents, carriers, excipients, dispersion media, coating agents, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, and any combination thereof. The carrier, diluent, solvent, or excipient must be "acceptable" in the sense of being compatible with the polypeptide or its derivative and not harmful after administration to an individual. Typically, the vehicle does not cause adverse, allergic, or other undesirable reactions when administered to an individual, preferably a human individual.

[0154] For certain purposes of administration to humans, the pharmaceutical compositions must meet sterility, pyrogenicity, general safety, and purity requirements as required by regulatory agencies, such as the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA).

[0155] In some embodiments, the carrier can be water or saline (e.g., physiological saline), which are sterile and pyrogen-free. Suitable excipients include mannitol, dextrose, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.

[0156] 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 suitable pharmaceutical carriers, solvents, excipients, or diluents can be made with reference 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 excipient. Preservatives, stabilizers, dyes, and even flavorings can be provided in the pharmaceutical composition.

[0157] In some embodiments, the polypeptide, fusion protein, nucleic acid, or vector may be included in a delivery particle, particularly a delivery particle in combination with other natural or synthetic compounds such as, for example, lipids, proteins, polypeptides, or polymers.

[0158] 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.

[0159] In some embodiments, the delivery particles may be in the form of lipoplexes comprising cationic lipids; lipid nanoemulsions; solid lipid nanoparticles; peptide-based particles; polymer-based particles, particularly polymer-based particles comprising natural and / or synthetic polymers; and mixtures thereof.

[0160] In some embodiments, the polymer-based particles may include synthetic polymers, particularly polyethyleneimine (PEI), dendrimers, poly(DL-lactide) (PLA), poly(DL-lactide-co-glycoside) (PLGA), polymethylmethacrylate, and polyphosphate esters.

[0161] In some embodiments, the delivery particle further comprises one or more ligands on its surface suitable for directing the polypeptide, nucleic acid, or nucleic acid vector to a target cell, tissue, or organ.

[0162] In one embodiment, the medicament is for the treatment and / or prevention of hematological cancer in a subject in need thereof. In a preferred embodiment, the medicament is for the treatment and / or prevention of myeloproliferative neoplasms (MPNs) in a subject in need thereof.

[0163] As used herein, the term "subject" refers to an animal, preferably a mammal, more preferably a human. In one embodiment, the subject is male. In another embodiment, the subject is female. In one embodiment, the subject is a "patient", i.e., the subject is waiting for medical treatment or is receiving medical treatment, or has / is / may be a past / present / future subject of medical treatment or treatment aimed at curing or treating hematological cancer, preferably MPN, and / or alleviating symptoms of hematological cancer, preferably MPN. In some embodiments, the subject is being monitored for the development of hematological cancer, preferably MPN. In one embodiment, the subject is undergoing preventative treatment for hematological cancer, 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).

[0164] The present invention also relates to a polypeptide, a fusion protein or a pharmaceutical composition according to the invention for use in the treatment and / or prevention of hematological cancers, preferably myeloproliferative neoplasms (MPNs).

[0165] 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 proliferation of cells of the myeloid lineage.

[0166] In one embodiment, the MPN is essential thrombocythemia (ET) or primary fibrosis myelofibrosis (PMF).

[0167] In one embodiment, the polypeptides of the present invention reduce, decrease and / or inhibit the constitutive activation of JAK / STAT that induces MPN.

[0168] MPNs are also frequently associated with mutations in CALR, a soluble protein that resides in the endoplasmic reticulum (ER) and acts as a chaperone in the ER protein quality control machinery, binding to glycans on nascent N-glycosylated proteins and retaining these glycoproteins within the ER until they are properly folded.

[0169] Briefly, frameshift mutations in CALR can result in (i) loss of its ER retention, (ii) aberrant activation of TPOR, and (iii) the presence of CALR on the cell surface and circulating CALR in the blood. One consequence of these mutations is the formation of a basic amino acid-rich carboxyl-terminal (C-ter) tail in CALR that does not contain its ER retention signal sequence.

[0170] In one embodiment, MPNs are derived, at least in part, by one or more mutations in the gene encoding calreticulin (CALR). In one embodiment, the amino acid sequence of the CALR mutant comprises at least one mutation compared to SEQ ID NO:8, the amino acid sequence of wild-type human calreticulin (UniProt ID number P27797).

[0171] In one embodiment, the one or more mutations consist of an insertion and / or deletion in exon 9, resulting in the production of a positively charged amino acid sequence at the C-terminus of CALR.

[0172] As used herein, positively charged amino acids, interchangeably referred to as basic amino acids, are amino acids that carry a positive charge in their side chains at neutral pH and include arginine, lysine, histidine, and all positively charged unnatural amino acids.

[0173] In some embodiments, the MPN-inducing CALR mutant comprises the sequence of SEQ ID NO:9.

[0174] In some embodiments, the one or more mutations in CALR result in a C-tertiary having an amino acid sequence comprising or selected from the group consisting of SEQ ID NO:10 to SEQ ID NO:45.

[0175] In some embodiments, the one or more mutations in CALR result in a C-tertail having the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:11.

[0176] In one embodiment, the one or more mutations in CALR result in a C-tertail having the amino acid sequence set forth in SEQ ID NO: 10, which is the CALR mutant L367fs, also known as CALR del52. * In another embodiment, the one or more mutations in CALR result in a C-tertiary amino acid sequence as set forth in SEQ ID NO: 11, which corresponds to the CALR mutant K385fs, also known as CALR ins5. * Corresponds to 47.

[0177] In some embodiments, a polypeptide or fusion protein according to the invention binds to a mutant of CALR as described herein above.

[0178] In some embodiments, the MPN is induced by (i) one or more CALR mutations and (ii) one or more mutations in another gene. In some embodiments, the one or more CALR mutations amplify the effect of one or more mutations in the other gene.

[0179] In one embodiment, the one or more CALR mutations and the one or more mutations in the other gene occur in the same cell, hi another embodiment, the one or more CALR mutations and the one or more mutations in the other gene occur in separate cells or populations of cells.

[0180] In some embodiments, MPN is induced by one or more CALR mutations and one or more mutations of the gene encoding TPOR.In some embodiments, the mutation of TPOR is selected from the group consisting of or includes R102P, P106L, G509N, and K39N.In some embodiments, the mutation of TPOR is R102P.

[0181] In some embodiments, a polypeptide, fusion protein, or pharmaceutical composition of the invention inhibits the proliferation of cells expressing a CALR mutant or cells expressing both a CALR mutant and a TPOR mutant.

[0182] In some embodiments, a polypeptide, fusion protein, nucleic acid, vector, or pharmaceutical composition of the invention is administered in combination with an additional anti-cancer agent or vaccine.

[0183] In certain embodiments, an additional anti-cancer agent or vaccine is administered in combination with the polypeptide, fusion protein, nucleic acid, vector, or pharmaceutical composition of the present invention, either simultaneously or sequentially.

[0184] In one embodiment, the additional anti-cancer agent or vaccine is administered simultaneously with the polypeptide, fusion protein, nucleic acid, vector, or pharmaceutical composition of the present invention (i.e., simultaneous administration, optionally in a co-formulation). In one embodiment, the additional anti-cancer agent or vaccine is administered at a different time than the polypeptide, fusion protein, nucleic acid, vector, or pharmaceutical composition of the present invention (i.e., sequential administration, in which the additional anti-cancer agent or vaccine is administered before or after administration of the polypeptide). In some embodiments, the additional anti-cancer agent or vaccine can be administered in the same manner as the polypeptide, fusion protein, nucleic acid, vector, or pharmaceutical composition of the present invention, or using the usual route of administration for the additional anti-cancer agent or vaccine.

[0185] In some embodiments, a polypeptide, nucleic acid, vector, or pharmaceutical composition of the invention is administered orally, parenterally, topically, by aerosol inhalation, rectally, 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.

[0186] In preferred embodiments, the polypeptides, nucleic acids, vectors, or pharmaceutical compositions of the invention are administered parenterally, subcutaneously, intravenously, or via an implanted reservoir.

[0187] In some embodiments, a polypeptide, nucleic acid, vector, or pharmaceutical composition of the invention is in a form suitable for injection, such as, for example, intraocular, intramuscular, subcutaneous, intradermal, transdermal, or intravenous injection or infusion.

[0188] Examples of forms suitable for injection include, but are not limited to, liquid solutions, such as sterile aqueous solutions, dispersions, emulsions, suspensions, solid forms suitable for use in preparing solutions or suspensions by adding a liquid prior to use, such as powders, liposomal forms, and the like.

[0189] Treatment may consist of a single dose or multiple doses over a period of time. The polypeptide, nucleic acid, vector, or pharmaceutical composition of the present invention may be formulated into a sustained release formulation to provide sustained release over an extended period of time, for example, at least 2 or 4 or 6 or 8 weeks. Preferably, sustained release is provided for at least 4 weeks.

[0190] 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 substance selected for administration, whether the administration is a single dose or multiple doses, and subject parameters, including age, physical condition, size, weight, sex, and the severity of the cancer being treated.

[0191] 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.

[0192] A "therapeutically effective amount" is an amount sufficient to administer a therapeutically effective dose to an individual without causing significant negative or adverse side effects. It means a level or amount necessary and sufficient to delay or stop the progression, worsening, or progression of one or more symptoms of cancer; or to alleviate the symptoms of cancer; or to cure cancer. In certain embodiments, the effective amount of the polypeptide of the present invention may be in the range of about 0.001 mg to about 3,000 mg per dosage unit, preferably about 0.05 mg to about 1,000 mg per dosage unit.

[0193] 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, 0.4 mg, 0.6 mg, 0.8 mg, 0. ... 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, 3 0mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550m g, 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, 1,850m g, 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.

[0194] In certain embodiments, the polypeptides of 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 the subject's body weight per day.

[0195] In some embodiments, the effective amount of the nucleic acid or vector administered is about 1×10 5 ~Approx. 1×10 15 Copies / dosage unit may range.

[0196] Within the scope of the present invention, about 1×10 5 ~Approx. 1×10 15 Copies are 1 x 10 per dosage unit 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9, 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1×10 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , 9×10 13 , 1×10 14 , 2×10 14 , 3×10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , 9×10 14 , and 1 × 10 15 Includes copy.

[0197] Preparations suitable for parenteral administration include aqueous and non-aqueous sterile injections that may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickening agents. The preparations may be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state that requires 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. Preparations for use in the present invention may further contain other agents conventional in the art, taking into account the type of preparation in question, for example, agents suitable for oral administration may include flavoring agents.

[0198] Another object of the present 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) a means for administering said polypeptide, fusion protein, nucleic acid, vector or pharmaceutical composition. In one embodiment, the kit is for treating and / or preventing a disease, preferably a hematological cancer, more preferably an MPN.

[0199] In certain embodiments, a means for administering a polypeptide, fusion protein, nucleic acid, vector, or pharmaceutical composition of the invention may include a syringe, trocar, catheter, cup, spatula, or the like.

[0200] In one embodiment, the kit further comprises an anti-cancer drug or vaccine, preferably for treating hematological cancer, more preferably for treating MPN.

[0201] In some further aspects, the present invention also relates to the use of a polypeptide, a fusion protein, a nucleic acid, a vector, or a pharmaceutical composition according to the invention for the manufacture or preparation of a medicament.

[0202] The present invention also relates to a polypeptide, a fusion protein, a nucleic acid, a vector or a pharmaceutical composition according to the invention for the manufacture of a medicament for treating and / or preventing a hematological cancer, preferably a myeloproliferative neoplasm (MPN), preferably an MPN induced by one or more mutations in the gene encoding calreticulin (CALR).

[0203] The present invention also relates to a method for treating and / or preventing hematological cancer, preferably myeloproliferative neoplasms (MPN), preferably MPN induced by one or more mutations in the gene encoding calreticulin (CALR), in a subject in need thereof, comprising the step of administering to said subject a therapeutically effective amount of a polypeptide, fusion protein, nucleic acid, vector or pharmaceutical composition according to the invention.

[0204] The present invention also relates to a polypeptide, a fusion protein, a nucleic acid, a vector or a pharmaceutical composition according to the invention for use in inhibiting the growth of cancer cells.

[0205] The present invention also relates to a method of inhibiting the growth of cancer cells in an individual in need thereof, comprising at least the step of administering to the individual a therapeutically effective amount of a polypeptide, fusion protein, nucleic acid, vector, or pharmaceutical composition according to the present invention.

[0206] The present invention also relates to a polypeptide, a fusion protein, a nucleic acid, a vector or a pharmaceutical composition according to the invention for use in improving the overall survival of an individual with cancer.

[0207] The present invention also relates to a method of improving the overall survival of an individual having cancer, comprising at least the step of administering to the individual a therapeutically effective amount of a polypeptide, fusion protein, nucleic acid, vector or pharmaceutical composition according to the present invention.

[0208] The present invention also relates to a polypeptide, a fusion protein, a nucleic acid, a vector or a pharmaceutical composition according to the invention for use in improving the prognosis of an individual with cancer.

[0209] The present invention also relates to a method of improving the prognosis of an individual having cancer, comprising the step of administering to the individual at least a therapeutically effective amount of a polypeptide, fusion protein, nucleic acid, vector, or pharmaceutical composition according to the present invention. [Brief description of the drawings]

[0210] [Figure 1]Figure 1A-1I show that the CALR del52 mutant tail directly interacts with the TpoR extracellular domain. Figure 1A: Proliferation assay. Cells BaF3 cells stably expressing hTpoR in pMX-IRES-GFP were infected with the indicated CALR variants or empty vector (pMSCV-IRES-mCherry) and sorted by FACS. 250,000 cells were washed, plated in 10 mL of complete culture medium without cytokines, and counted daily using an automated cell counter (n=3). Values ​​represent the mean (±SD) of three independent experiments with triplicate technical replicates (n=6-9). Figure 1B: NanoBRET between NanoLuc-TpoR and CALR del52-HaloTag full-length or truncated from the N-terminus. Means (±SD) of five independent experiments are shown (N=5, n=20). Two-way ANOVA followed by SIDAK multiple comparison test. ****: p<0.0001, ns: not significant. Figures 1C-1E: Theoretical profile of specific vs. nonspecific interactions in Donor Saturation Assay (DSA) experiments. HEK293T were transfected with increasing amounts of HaloTag constructs and a fixed amount of NanoLuc donor. Negative control is empty NanoLuc vector (n=4). Figure 1C: DSA of CALR del52 full-length. Figure 1D: DSA of CALR del52 PC domain. Figure 1E: DSA of CALR del52 C domain. Figure 1F: Representative co-immunoprecipitation of HA-hTpoR with CALR del52-FLAG full-length or the indicated N-terminal truncated forms. Figure 1G: ELISA of CALR del52 species from supernatants of HEK293T cell cultures. Values ​​represent the mean of three ELISA experiments. One-way ANOVA followed by Tukey's multiple comparison test. ****: p<0.0001. Fig. 1H: Surface localization of CALR del52 full-length or PC domain in the presence of hTpoR or empty vector. Flow cytometry analysis using anti-FLAG antibody and anti-mouse secondary antibody coupled to APC on HEK293T cells co-transfected with the indicated constructs.Figure 1I: Transcriptional activity of STAT5 by hTpoR and the indicated CALR truncations. HEK293T were transiently transfected with HA-hTpoR and CALR truncations together with cDNAs encoding STAT5, JAK2, and the SpiLuc firefly luciferase reporter reflecting STAT5 transcriptional activity, and normalized to a control reporter (pRLTK) containing Renilla luciferase. Means (± SD) of three independent experiments performed in triplicate are shown (N=3, n=9). Two-way ANOVA followed by SIDAK multiple comparison test. ***: p<0.001, ****: p<0.0001. [Diagram 2] 2A-2F: CALR del52-TpoR interaction and dimerization driven by the mutant tail. 2A-C: Deuterium uptake of representative peptides from CALR del52 alone or complexed with TpoR ECD corresponding to the C-terminus of CALR del52 at five different exchange time points. The peptide shown in FIG. 2A corresponds to the end of the C-domain common to CALR WT and CALR del52, which shows no differential uptake. The peptide shown in FIG. 2B contains the mutant tail and shows high differential uptake. The peptide shown in FIG. 2C corresponds to the last amino acid of the mutant tail. FIG. 2D: Non-denaturing Western blots of the indicated CALR mutants in the presence or absence of a reducing agent (DTT). Stained with Coomassie Blue. FIG. 2E: Co-immunoprecipitation of CALR del52-HA full-length with CALR del52-FLAG full-length or truncated forms to assess dimerization. Representative Western blots under denaturing conditions are shown. Figure 2F: Cross-linking assay of hTpoR dimerization in the presence of Tpo, CALR del52 full-length, or C-terminal truncated forms. Representative Western blots under denaturing and reducing conditions are shown, showing cross-linked dimers of hTpoR monomers and o-PDM under the indicated conditions. [Diagram 3]Figures 3A-3G show the interaction of CALR del52 with TpoR D1D2. Figure 3A: Representative co-immunoprecipitation of HA-hTpoR ECD domain with CALR del52-FLAG or CALR del52 Y109F / D135L-FLAG as indicated. Figure 3B: Quantification of relative co-immunoprecipitation of TpoR species with CALR del52 (with or without mutations). Western blot quantification was performed with ImageJ. The ratio of TpoR species in CALR del52 normalized with respect to the expression of TpoR species in total lysates (+SD) is shown (N=2, n=6). Two-way ANOVA followed by SIDAK multiple comparison test. ***: p<0.001, *: p<0.05 ns: no significant difference. Figure 3C: NanoB RET between NanoLuc-TpoR subdomain and CALR del52-HaloTag full-length or N-terminal truncated forms. Mean values ​​(±SD) of two independent experiments are shown (N=3, n=12). Figures 3D-3F: Deuterium uptake of representative peptides from CALR del52 alone or CALR del52-D1D2 complex corresponding to key regions described herein. The peptide shown in Figure 3D corresponds to amino acids 50-74 of the N domain. The peptide shown in Figure 3E contains residues C105 and Y109 involved in glycan attachment. The peptide shown in Figure 3F contains the C-terminus of the mutant. Figure 3G: Relative differential deuterium uptake between CALR del52 complexed with sD1D2 containing immature glycans and CALR del52 alone at 60 min incubation in deuterium. Dots represent individual peptides detected by mass spectrometry. The Y-axis represents the relative deuterium exchange difference. Positive values ​​indicate regions of CALR del52 that are more protected in the presence of sD1D2, and negative values ​​represent regions that are less protected in the presence of sD1D2. [Figure 4]Figures 4A-4C relate to the interaction site between CALR del52 mutant tail and TpoR D1D2. Figure 4A: Relative deuterium uptake analysis between TpoR full ECD with mature glycans in the absence or presence of CALR del52 upon 1 h incubation in deuterium. Protected dots indicate significant differential deuterium uptake (p<0.001) by peptide-level significance test (hybrid mode) as described (Lau et al., 2021). Figure 4B: Deuterium uptake of FSRTFED peptides from mature TpoR full ECD alone (D1D4) or mature TpoR full ECD complexed with CALR del52 (CALR del52_D1D4) at five different exchange time points. Figure 4C: Transcriptional activity of STAT5 by hTpoR and the indicated CALR truncation forms. HEK293T were transiently transfected with truncated forms of HA-hTpoR and CALR together with cDNAs encoding STAT5, JAK2, and the SpiLuc firefly luciferase reporter reflecting the transcriptional activity of STAT5 and normalized to a control reporter (pRLTK) containing Renilla luciferase. Means (± SD) of three independent experiments performed in triplicate are shown. Two-way ANOVA followed by SIDAK multiple comparison test. ***: p<0.001, ****: p<0.0001. [Diagram 5]Figures 5A-5D are a set of graphs proving the principle of inhibition of CALR del52 bound to a fusion protein containing the TPOR domain (TPOR-Fc) in myeloproliferative neoplasms. Competition experiments with increasing concentrations of soluble D1D2D3D4-Fc (Figure 5A), D1D2-Fc (Figure 5B) and D1-Fc (Figure 5C). Experiments were performed in cells co-expressing equivalent cDNAs of TpoR / TPOR coupled to NanoLuciferase and CALR del52 coupled to HaloTag. The interaction between TPOR and CALR del52 is measured based on the BRET ratio. Figure 5D illustrates the NanoBRET signal used as a proxy for binding between TpoR full-length and CALR del52 in the presence of increasing amounts of the human D1 domain with the N117Q mutation. [Figure 6] Figure 6 is a histogram showing in trans inhibition of CALR mutant-TPOR interaction in live cells, showing detection of CALR del52 at the cell surface of cells following incubation with the indicated Fc-fusion proteins or controls. [Figure 7] 7 is a graph showing the in vivo efficacy of soluble D1 as an inhibitor of CALR mutants. The graph represents blood platelet concentrations in the indicated mice from 4 to 24 weeks. [Figure 8] FIG. 8 is a graph illustrating the NanoBRET signal used as a surrogate for binding between TPOR full length and CALR del52 in the presence of increasing amounts of human D1 WT and mutants of the FFPLHLWLV motif. [Figure 9] FIG. 9 is a three-dimensional structural model depicting the interaction of TPOR and CALR del52 mutant during the formation of the tetrameric complex. [Figure 10] FIG. 10 illustrates the NanoBRET signal used as a surrogate for binding between TPOR full length and CALR del52 in the presence of increasing amounts of human D1 WT, and mutants of the motifs QDV, SDSE, FSR, WDEE, or EAAP.

[0211] Working Example The present invention is further illustrated by the following examples.

[0212] As used herein, the term "mutant tail" refers to the C-terminal tail of CALR del52 having the amino acid sequence of SEQ ID NO:50.

[0213] Example 1: The mutant tail of CALR del52 directly interacts with the TpoR extracellular domain and mediates dimerization of TpoR and CALR material and method Mutagenesis: All CALR del52 mutants were generated using the QuickChange (Agilent), KLD enzyme mix (NEB) procedure according to the manufacturer's instructions, or purchased from Genscript. All constructs were verified by sequencing.

[0214] Ba / F3 proliferation assay: Ba / F3 were first transduced with pMX-IRES-GFP and human TpoR in CALR variant or empty vector (pMSCV-IRES-mCherry) and sorted by FACS for similar levels of GFP and mCherry. 250,000 cells were washed and seeded in 10 mL of cytokine-free RPMI, 10% FBS and counted daily in triplicate on a Coulter automated cell counter. This experiment was performed in three different biological replicates (N=3).

[0215] BRET: cDNAs encoding TpoR and erythropoietin receptor (EpoR) were cloned into a modified pNL-N vector (Promega) to generate N-terminal fusions of Nano-luciferase to the receptor. cDNAs encoding WT and del52 CALR mutants were cloned into the pHT-C vector to generate HaloTag fusion constructs (Promega). HEK-EBNA cells transiently transfected with these constructs were analyzed for Bioluminescence Resonance Energy Transfer (BRET) on a GloMax Discover multiplate reader (Promega) at 37°C using 450BP (donor) and 600LP (acceptor) built-in filters.

[0216] Co-immunoprecipitation and Western Blot: HEK293T were transiently transfected with cDNAs encoding the indicated constructs in 10 cm culture dishes. Confluent cells were lysed with NP-40 buffer 48 h after transfection. After pre-washing, samples were incubated with 2 μg / ml of anti-FLAG antibody (Genscript, Cat. No. A00187) or the corresponding isotype control (Genscript Cat. No. A01730) at 4°C overnight. Bound proteins were eluted with 40 μL / mL of rProtein G Agarose (ThermoFisher, 20397) for 3 h at 4°C. Samples were then centrifuged and washed three times, and immunoprecipitated proteins were analyzed by SDS-PAGE followed by revelation with anti-HA antibody (Roche, 3F10) for HA-hTpoR, HA-CALR del52, or anti-CALR mutant tail (SAT602).

[0217] ELISA: To measure the levels of soluble CALR mutant species from HEK293T supernatants 48 hours after transfection, a polyclonal rabbit antibody (SAT602 provided by Myelopro, Vienna, Austria) was used to coat ELISA plates. This antibody was raised against a peptide derived from the C-terminal sequence of the CALR mutant. After blocking (5% BSA + 0.05% Tween-20 in PBS), the plates were probed with samples of culture medium diluted in blocking buffer, and purified mutant-CALR protein produced in Expi-293F cells (Thermo Fisher Scientific, Merelbeke, Belgium) was included as a standard for quantification. For detection, anti-CALR antibody (FMC75, Abcam, Cambridge, United Kingdom) combined with anti-mouse IgG-HRP antibody (Southern Biotech, Birmingham, AL) was used, and TMB (3,3',5,5'-tetramethylbenzidine, Thermo Fisher Scientific, Merelbeke, Belgium) was added as substrate. Absorbance was measured at 450 nm and 620 nm in a microplate reader (SpectraMax i3, Molecular Devices, Silicon Valley, CA).

[0218] Flow cytometry: For evaluation of cell surface localization of CALR del52 species, HEK293T were transiently transfected with the indicated CALR del52 species and full-length human TpoR (hTpoR) or empty vector 48 hours before the experiment. Cells were detached without trypsinization and stained with anti-FLAG antibody or IgG control (primary) and APC-coupled goat anti-mouse antibody (secondary). Cells transfected with a single fluorescent vector (GFP or mCherry) or compensation beads stained with APC-coupled antibody were used for compensation control. 30,000 events of cells co-transfected with the two constructs were acquired on a BD LSRFortessa™ Cell Analyzer for each condition. Post-acquisition analysis was performed on FlowJo. For evaluation of cell surface localization of TpoR mutants, HEK293T were transfected with the indicated mutants or empty control vector. 48 hours after transfection, cells were treated as described above and stained with anti-HA coupled to APC antibody. 30,000 events of transfected cells were acquired for each condition using FACSVerse™, and the percentage of cells staining positive for HA-APC was determined among transfected cells.

[0219] Hydrogen-Deuterium Exchange Mass Spectroscopy (HDx-MS): Hydrogen-Deuterium Exchange Mass Spectroscopy was performed on a Waters nanoAcquity UPLC with HDx technology equipped with a Synapt G2-Si. All purified recombinant proteins were used at a concentration of 20 μM in equilibration buffer (5 mM K2HPO4, 5 mM KH2PO4, pH 7 in H2O). For the analysis of the interaction between recombinant mature D1D2D3D4 and CALR del52, the proteins were first mixed together at 1:1 molar concentration for 30 min at room temperature and then for 3 h at 4°C. The proteins were then kept at 0°C. Labeling was performed at 20°C for six different incubation times (0, 0.25, 1, 5, 20, and 60 min) with 20-fold dilutions of samples in labeling buffer (5 mM K2HPO4, 5 mM KH2PO4, pH 7 in D2O). After incubation, the reaction was quenched using a 1:1 dilution in quench buffer (0.05M K2HPO4, 0.05M KH2PO4 with 30mM TCEP, pH 2.3) and then injected into a pepsin column (Enzymate BEH Pepsin 2.1x30 Column, Waters Cat. No. 186007233) at a dynamic flow rate of 150-75μL / min. All mixing was performed automatically by the robotic station of the PAL-RTC. Peptides resulting from pepsin digestion were captured on an ACQUITY BEH C18 1.7 μM VANGUARD Pre-column (Waters Cat. 186009375), separated on an ACQUITY UPLC C18 1.7 μM 1.0x100mm column (Waters Cat. 186002346), and electrosprayed into a Waters SYNAPT G2-Si quadrupole-time-of-flight mass spectrometer. Measurements were performed in HDMSe mode with ion mobility. Lock mass calibration was performed by injection of leucine-enkephalin (m / z=556.277). Peptides were identified in triplicate using PLGS3.0 software (Waters).Identified peptides were further analyzed by DynamX3.0 (Waters) using an error margin of 10 ppm, a maximum length of 35 a.a, a minimum product per amino acid of 0.2, and requiring each peptide to be identified three times out of three replicates. Data were statistically analyzed using Deuteros 2.0, which performs peptide-level significance testing ( Lau et al., 2021 ).

[0220] result Calreticulin domains include a globular N domain (positions 16–197), a central proline-rich P domain (positions 198–308), and an acidic C domain (positions 309–417). In some CALR mutants, especially CALR del52, the C-terminal tail is positively charged. To investigate the role played by the positively charged mutant tail, we tested a prototype type I CALR mutant, which is a stepwise truncation of the C-terminus of CALR del52. Truncation of the C-terminal part up to position 394, thus removing the last 18 amino acids, allowed autonomous growth of a cytokine-dependent hematopoietic cell line (Ba / F3) similar to that induced by full-length CALR del52 (Figure 1A, “CALR del52 A394” compared to “CALR del52”). * Further truncation up to position 387 still allowed low but autonomous growth (Figure 1A, "CALR del52 M387 * " ), and further truncation completely abolished CALR activity ( Fig. 1A , "CALR del52 M377 * ", CALR del52 M371 * " and "CALR EXON 9" for deletion of exon 9.

[0221] Importantly, the CALR del52 Y109F / D135L mutant, which abolishes glycan-dependent binding and disrupts the helicity of the mutant tail, did not allow autonomous growth of Ba / F3 (Figure 1A, "CALR del52 Y109F / D135L").

[0222] N-terminal truncated forms of CALR del52 were also generated to explore whether the mutant tail of CALR del52 could mediate direct binding to the receptor in the absence of immature glycans. Bioluminescence resonance energy transfer (BRET) was used to measure the direct interaction between TpoR and the N-terminal truncated forms of CALR del52 in live cells. Strictly speaking, complete deletion of the N-domain of CALR del52 (CALR del52 PC domain-HT) or complete deletion of the N-domain and P-domain (CALR del52 C-domain-HT) did not completely block the direct interaction with the receptor (Figure 1B).

[0223] The specificity of the interaction was verified by donor saturation assays (DSA) in which increasing amounts of ligand (i.e., CALR variants) led to rapid signal saturation due to occupancy of all receiver binding sites ( Figure 1C-E ).

[0224] When only the mutant tail was present, a small but significant binding to TpoR was observed, suggesting that the tail required at least a portion of the C domain for proper folding and stability when fused to the HaloTag construct. To validate these results and explore only the binding of the C-tail to TpoR, co-immunoprecipitation was performed with a CALR del52 construct containing a FLAG tag at the C-terminus. An interaction between TpoR and a C-terminal truncated form of CALR del52 down to a fragment corresponding to only the mutant tail was detected (Figure 1F).

[0225] We next tested C-terminal fragments of CALR del52 to see whether they behaved similarly to the full-length in functional assays. Using ELISA on cell supernatants, we measured the secretion of the CALR del52 PC domain or the mutant tail alone compared to full-length CALR del52. Interestingly, the PC domain was strongly secreted, but the mutant tail alone was not (Figure 1G).

[0226] We next used flow cytometry to assess the surface localization of CALR del52 full-length or PC domain in the presence or absence of TpoR. CALR del52 full-length and even PC domain showed strong surface expression (Figure 1H). Interestingly, both CALR del52 species (full-length and PC domain) had high surface localization in cells co-expressing TpoR, suggesting that TpoR retains them at the cell surface (Figure 1H).

[0227] Finally, using functional transcription assays, we showed that the P domain fused to the C domain of CALR del52 was sufficient to induce a small but significant STAT5 transcriptional activity through TpoR but not EpoR ( Fig. 1I ).

[0228] Taken together, these results indicate that direct binding of CALR del52 to TpoR is mediated by both the mutant tail and N-domain interactions with immature sugars, but activation is strictly dependent on the presence of the mutant tail, which is further enhanced by glycan-dependent interactions.

[0229] Next, HDx-MS was used to map all possible interaction sites between the three CALR del52 mutants (i.e., FGNETWGVTKAAE-SEQ ID NO:51; TKAAEKQMKDKQDEEQRTRRMMRTKM-SEQ ID NO:52; and QWGTEA-SEQ ID NO:53) and the complete extracellular domain of TpoR, including the mature glycan. These two recombinant proteins were produced independently and mixed in solution at a 1:1 molar ratio, followed by deuterium exchange. Results revealed significant (p<0.001) exchange differences in multiple peptides containing the mutant tails (Figure 2A), confirming a direct interaction between the mutant tails and the mature TpoR extracellular domain. This differential exchange was not observed for the very last residues encompassing residue 406QWGTEA411 (SEQ ID NO:53, Figure 2C), indicating that the very last portion of the mutant tail is not involved in this interaction.

[0230] Because oligomerization of CALR mutants is important for TpoR activation (Araki et al., 2019; Rivera et al., 2021), and truncated forms before the C-terminal cysteine ​​did not affect proliferation, we compared the oligomer profiles of CALR del52 species in the presence or absence of reducing agents under non-denaturing conditions. No differences in the oligomer profiles were observed in the presence or absence of reducing agents (DTT), and CALR del52 A394 * and CALR del52 full-length both yielded similar oligomeric species (Fig. 2D). In contrast, oligomeric species were completely abolished with CALR Δc-tail, supporting the fact that the mutant tail is required for oligomerization and is independent of the C-terminal cysteine ​​(Fig. 2D).

[0231] Using the same set of CALR del52 as in the functional assay (see Figure 1A), the role of the mutant tail in dimerization was further explored by co-immunoprecipitation of the same FLAG-tagged truncated form of CALR del52 used in the proliferation assay with HA-tagged full-length CALR del52. * and CALR del52 M387 * We observed strong co-immunoprecipitation of CALR del52 and CALR M377 (Figure 2E), consistent with the conserved ability of these truncations to induce Ba / F3-independent growth. Despite their strong increase in stability, further truncation forms (CALR del52 M377 * and CALR del52 M371 * ) led to a dramatic reduction in immunoprecipitation of full-length HA-CALR del52 ( Fig. 2E ), confirming that the mutant tail is directly involved in CALR del52 dimerization.

[0232] We next used a cross-linking approach to test dimerization of TpoR with CALR del52 full-length or C-terminal truncated forms. To detect TpoR dimerization via cysteine ​​cross-linking, we truncated the intracellular domain of the receptor after the JAK2 binding site, removed the intracellular cysteine, inserted the L508C point mutation, which is homologous to mouse TpoR L501C, and orientated the cysteine ​​residues in the dimer orientation shown to be active (Staerk et al., 2011). These TpoR truncation forms retain the ability to be activated. Consistent with the functional assays, truncations up to M387 (CALR del52, CALR del52 A394 * , and CALR del52 M387 * ) still allowed dimerization of the TpoR transmembrane domain, but further truncations (CALR del52 M377 * and CALR del52 M371 * ) did not allow dimer formation (Figure 2F).

[0233] Example 2: TpoR D1D2 is sufficient to mediate full binding to CALR del52 via the glycan and mutant tail material and method For co-immunoprecipitation / Western blotting, BRET, HDx-MS, see Example 1.

[0234] result Previous findings showed that activation of TpoR by CALR del52 is mediated by the extracellular domain of TpoR and depends on glycosylation of Asn117, and to a lesser extent Asn178, present in the TpoR D1 and D2 domains, respectively (Chachoua et al., 2016; C. Pecquet et al., 2019). Using co-immunoprecipitation of C-terminal truncated forms of the TpoR ECD, we showed that deletion of D3D4 did not affect CALR del52 binding to the receptor (Figures 3A-B for CALR del52-FLAG, sD1D2). Deletion of the D2 domain resulted in a small but significant reduction in co-immunoprecipitation compared to the D1 domain alone (Figures 3A-B for CALR del52-FLAG, sD1).

[0235] We next used the CALR del52 Y109F / D135L mutant, which lacks glycan-binding ability (Kapoor et al., 2004). As expected, loss of glycan-dependent binding resulted in a drastic decrease in the rate of co-immunoprecipitation (Figure 3A-B for CALR del52 Y109F / D135L-FLAG). Strictly speaking, no difference in the rate of co-immunoprecipitation was detected between D1 alone and D1F2 in the absence of glycan binding (Figure 3A-B for CALR del52 Y109F / D135L-FLAG, compare sD1 with sD1D2), suggesting that the CALR del52 mutant tail interacts exclusively with TpoR D1.

[0236] We next performed BRET assays between CALR del52 truncations and subdomains of the TpoR ECD to measure their direct interaction in live cells. Confirming the co-immunoprecipitation results, we observed a decrease in the BRET signal between D1D2 and D1 for full-length CALR del52 (Figure 3C for CALR del52-HT, compare NL-sD1D2toNL-sD1). In contrast, binding of CALR del52 PC domain or C domain alone to the receptor ECD was similar or slightly higher after truncation of D3D4 or D2D3D4 (Figure 3C for CALR del52 PC domain-HT and CALR del52 C-domain-HT, NL-sD1D2 and NL-sD1).

[0237] Taken together with the above results, this confirmed that the CALR del52 mutant tail directly interacts with the D1 domain of the receptor.

[0238] Next, we purified CALR del52-D1D2 produced as a complex in Schneider cells. Briefly, the extracellular domains of human TpoR and human CALR mutant del52 produced in Drosophila S2 cells (Thermo Fisher Scientific) were fractionated by loading onto a Superdex 200 Increase10 / 300 column (GE Healthcare) using size-exclusion chromatography. Elution was performed with buffer TNG (Tris-NaCl-glycerol) pH 7.5 at 0.5 ml / min, and 0.5 ml fractions were collected and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. Proteins in the fractions were immunoprecipitated with anti-His5 antibody (Qiagen) and treated with endoglycosidase H (NEB) or N-glycosidase F (NEB). Using HDx-MS, we detected all the interaction sites between CALR del52 and TpoR D1D2 (sD1D2) produced in the same cells. Comparison of deuterium uptake between CALR del52 alone and the CALR del52-TpoR D1D2 complex indicated that the CALR mutant interacts with TpoR through two main domains. First, the highest deuterium uptake difference was observed at the putative glycan-binding site of calreticulin (Figure 3E). This region contains, among others, C105, Y109, D135 and W319, all of which have been reported to be important for the binding of sugar moieties (Gopalakrishnapai et al., 2006). Importantly, this region is not involved in the binding of mature TpoR as shown in, confirming that it is specifically involved in the interaction with immature glycans. Second, multiple peptides containing the CALR del52 mutant tail equally demonstrated a high degree of differential uptake between the CALR del52-sD1D2 complex and CALR del52 alone (data not shown).This confirmed that the mutant tail is involved in binding not only to the mature form of the receptor but also to immature TpoR, an interaction that was further strengthened by the strong interaction between the CALR glycan-binding pocket and the immature sugar moiety on Asn117 of TpoR D1D2 (Chachoua et al., 2016). Finally, the P domain of CALR del52 was more accessible to deuterium uptake when complexed with the TpoR D1D2 domain (sD1D2) (Figure 3G), demonstrating that it is not involved in the interaction with the receptor as previously suggested (Araki et al., 2016).

[0239] Example 3: A comprehensive model of the interaction of the TpoR extracellular domain with the CALR del52 mutant tail material and method Mutagenesis: All alanine scanning mutants were generated using the QuickChange (Agilent), KLD enzyme mix (NEB) procedure according to the manufacturer's instructions, or purchased from Genscript. All constructs were verified by sequencing.

[0240] Transcriptional Dual Luciferase Assay: Transcriptional Dual Luciferase was performed as described in Chachoua et al., 2016. Briefly, for alanine scanning, HEK293T and γ2a were transfected with plasmids encoding STAT5, mouse JAK2 and the indicated human TpoR mutants (all in pMX-IRES-GFP vector), as well as empty vector CALR WT, CALR del52 (in pMSCV-IRES-mCherry). For other assays, HEK29ET or human TpoR WT were transfected with the indicated CALR species. In both cases, the SpiLuc reporter was used as a readout for STAT5 transcriptional activation, and pRLTK was used as an internal control (Promega). Cells were stimulated or not with 25 ng / ml rhTpo (Milteneyi Biotec) as indicated.

[0241] Molecular Dynamics and Docking Simulations: The sequences of the TpoR extracellular and transmembrane domains of CALR Del52 were profiled by state-of-the-art predictors for secondary structure, intrinsic disorder, and accessibility propensity (Barik et al., 2020). The closest templates were retrieved by Phyre 2. Modeller 9.21, Alpha-Fold2, and Rosetta Folding were used to effectively build 3D models and identify the interaction interface between the two CALR del52 mutants. HDx-MS data was used to identify contacts between TpoR and CALR del52 in the formation of a tetrameric complex. The glycans of the ER-specific G3M9 of TpoR that contact CALR were modeled by Glycopack in a configuration consistent with the NMR data, and the remaining complex types were built in agreement with the SAGS database. The solid-state NMR data on the contacts identified by HDx-MS and the configuration of the TM region of the TpoR dimer were used as constraints in creating the overall 2CALR-2TpoR model. The glycoproteic tetramer was placed in an all-atom representation of the environment, with a lipid bilayer of 1607 POPC molecules that fit the TM region of TpoR, as well as TIP3P water molecules and 63 sodium ions that account for the solvent space hydrating the remainder of the tetramer. These entire systems, consisting of about 1.5 million atoms, were subjected to a complete molecular dynamics cycle using OpenMM. First, the system was minimized to a threshold of 1 kJ / molar concentration using the L-BFG algorithm. Heating was then performed gradually over a period of 13 ns from 0 to 300 K at 1 bar pressure and a membrane surface tension of 340 bar nm using the Langevin integrator with a time step of 2 fs in the NPT ensemble, constraining the hydrogen bonds to achieve this high time step. Production was then continued for 20 ns. Visualization and analysis were performed using VMD 1.9.3 using the MDTraj library and in-house Python 3.6 scripts.

[0242] result First, the interaction site between the CALR del52 mutant tail and the TPOR ECD was explored by performing a complete alanine scanning of the D1D2 domain of full-length TpoR by mutating 3-4 amino acids at a time in most mutants or 8 amino acids in three of these. Since the mutant tail is strictly required for activation but not for binding that also occurs through the glycan, the resulting mutants were explored for their ability to be activated by CALR del52 full-length or thrombopoietin (Tpo) ligand in a STAT5 transcriptional luciferase assay. Results revealed that the majority of mutants capable of inhibiting CALR del52-dependent activation of the full-length receptor were located in the D1 domain or the C-terminus of D2 (data not shown). The inhibitory mutations were particularly clustered at the N-terminus of TpoR and concentrated on the inner / lateral surface of the D1 domain.

[0243] Given the important role of D1 in binding the mutant tail, we further explored the interaction of the CALR del52 PC domain with the D1 alanine mutant and found similar results to those obtained with full-length CALR del52 (data not shown), suggesting that they act by preventing binding or activation mediated by the mutant tail.

[0244] Considering the very high proportion of basic residues (Arg) in the CALR del52 mutant tail, we investigated the acidic residues of TpoR that may be involved in the interaction with the mutant tail. One stretch rich in acidic residues at the very N-terminus of TpoR, including residue 37 PLKCFSRTFEDLTCFWDEEE56 (SEQ ID NO: 7), appeared to be potentially important for the activation of TpoR. Of the 62 mutants tested, all that inhibited CALR del52-mediated activation equally prevented TPo-induced activation of TpoR, but the reverse was not true (data not shown). This suggests that all sites used by Tpo for activation are also used by CALR del52, explaining the reduced action of Tpo in cells co-expressing TpoR and CALR del52, but also suggests that CALR del52 uses additional sites that are not used by Tpo ligands.

[0245] To further explore the binding site of the CALR del52 mutant tail on the TpoR ECD, we performed HDx-MS to measure the differential deuterium exchange ratio between the complete mature TpoR ECD (labeled D1D4) or the CALR del52-D1D4 complex, which we showed interacted via the mutant tail (see Figure 2A) described above. Among the spanning regions of the TpoR ECD, three peptides, namely 41FSRTFED47 peptide (SEQ ID NO: 59), 51WDEEEAAPGST62 peptide (SEQ ID NO: 61), and 272WSLPVT277 peptide (SEQ ID NO: 64), exhibited significant differential deuterium exchange in the presence of CALR del52 (Figure 4A). Consistent with the alanine scanning data, the 41FSRTFED47 peptide (SEQ ID NO: 59) containing the 44TFED47 fragment (SEQ ID NO: 60) showed the strongest deuterium exchange difference (Figure 4A-B), followed by the highly negatively charged 51WDEEEAAPGST62 fragment (SEQ ID NO: 61), but the differential deuterium exchange was due to the 51WDEE54 fragment (SEQ ID NO: 62), since another peptide containing 55EAAPGST62 (SEQ ID NO: 63) showed no significant effect difference between the two conditions (not shown). Surprisingly, a small but significant exchange difference was also detected in the 272WSLPVT277 peptide (SEQ ID NO: 64) present at the C-terminus of the D2 domain. The other detected peptides did not show significant deuterium reduction exchange between the two conditions, but the absence of other interaction sites between the two proteins could not be excluded due to incomplete sequence coverage.

[0246] To further explore the interaction of TpoR with the CALR del52 mutant tail, molecular dynamics (MD) was used to obtain the structure of the TpoR D1D2 domain. After the model was generated, two complex glycans were attached to the glycosylation sites Asn117 and Asn178. To evaluate the stability of the model, five MD simulation trials were performed in explicit solvent, each with a length of 1 μs. The final model was consistent with that recently published by AlphaFold 2.0. Sequence analysis indicates that TpoR D1D2 has an unbalanced charged composition with an excess of 11 negatively charged and acidic amino acids (data not shown). Furthermore, the model identifies one extended (S1) and a second more localized (S2) negatively charged region on the TpoR surface. These acidic regions are particularly susceptible to interactions with the highly basic C-terminal tail of the CALR mutant, which was shown to adopt an extensive helical structure up to residue 394. The mutant tail is indeed enriched in basic (Lys / Arg) and hydrophobic (Met) residues.

[0247] To assess the role of hydrophobic residues relative to acidic residues in the activation of TpoR, all basic residues (arginine (R) and lysine (K) residues) or all hydrophobic residues (methionine (M) residues) were mutated to Gly (G) or Asn (Q), providing CALR del52 R / K tG, CALR del52 R / K tQ, CALR del52 MtG, and CALR del52 MtQ, respectively. Results showed that mutation of hydrophobic residues to either Gly or Asn resulted in a decrease in activation of TpoR quantitatively similar to that observed after modulation of helical orientation (Figure 4C, CALR del52 MtG and CALR del52 MtQ). In contrast, mutation of basic residues to either Asn or Gly (CALR del52 R / K tG and CALR del52 R / K tQ) resulted in a complete loss of the ability of CALR del52 to activate TpoR (Figure 4C). These results are consistent with previously published data ( Elf et al., 2016 ) and suggest that the interaction between the mutant tail and the TpoR ECD essentially relies on basic-acidic interactions.

[0248] Next, in silico simulations were performed to explore the most stable interaction site of the mutant tail alone with the TpoR D1D2 domain. After modeling the CALR del52 mutant tail, docking trials were performed using three main starting configurations of the complex based on the acidic region of TpoR, which were set as input in HADDOCK for the TpoR D1D2-CALR del52 mutant tail complex optimization search. The top configurations were further optimized using a 500 ns molecular dynamics trial to obtain three final poses in which the last residue of the mutant was unfolded (data not shown), confirming the FTIR data.

[0249] The free energy (ΔG) was further estimated using Prodigy. There was a large number of microstates of the complex that showed very large negative values ​​of free energy (ΔG<-9 kcal / mol, data not shown), consistent with the high binding affinity of the CALR del52 mutant tail for TpoR D1D2. Thus, even the additional small negative cluster region found in the N-terminal region of TpoR-D1 alone has significant affinity for the CALR del52 mutant tail. Interestingly, MD experiments showed that in the process of binding this TpoR-D1 region, the CALR del52 mutant tail can induce the rearrangement and unfolding of the helix found in the N-terminus of TpoR. Computational work showed that the CALR del52 mutant tail has the ability to bind TpoR with very high affinity in a large number of microconfigurations that target both the continuous acidic region found mainly in D1 (and partially in D2), as well as the small acidic patch in the N-terminal region of D1. The "anaconda" effect by which CALR del52 can bind its TpoR "pray" in so many ways, both through its lectin and mutant tail, is mainly due to the synergistic delocalization effect induced by charge complementarity, which, in combination with the significant flexibility of the CALR del52 mutant tail, can be expanded over a much larger area of ​​the two interactors and in this way mold a more rigid shape to TpoR. This suggested that local intervention (e.g., by point mutation) simply modulates the global interaction between the two molecular objects and does not completely disrupt it. 44 The T, E, and D residues of the TFED47 motif are predicted to directly interact with negatively charged residues on the CALR del52 mutant tail by MD simulations with the model displayed as pose 2, in agreement with the alanine scanning and HDX-MS data. Similarly, pose 3 predicted an interaction between Arg of the CALR del52 mutant tail and 53EE54 of 51WDEE54 (SEQ ID NO: 62), as well as Q64 of the 64QLLY68 (SEQ ID NO: 65) motif.Mutation of both motifs to alanine resulted in loss of CALR del52-induced activation.

[0250] MD simulations using only the mutant tail showed that the latter can bind to the receptor in various microconfigurations. However, binding of TpoR to full-length CALR del52 also involves a strong interaction between the N domain and the immature glycan on Asn117. Thus, AlphaFold 2.0 predictions were used to complete the modeling of TpoR, resulting in the complete extracellular domain of the receptor inserted into the POPC lipid bilayer. TpoR monomers dimerized through their TM domain with residue L508 at the interface, similar to the active configuration in the presence of CALR del52 (Figure 2F). The CALR del52 dimer was docked to the dimer of TpoR through the glycan-binding domain and the mutant tail based on experimental data and energy minimization (data not shown). This final structure places the mutant tail in a configuration in which the main interaction site is located around the 44TFED47 (SEQ ID NO: 60) motif, in agreement with the HDx-MS and alanine scanning data described above. Similarly, the immature glycan on Asn117 of the receptor, which interacts with an N-domain pocket containing key residues, was involved in binding of glycans containing C105, Y109, D135, and W319.

[0251] Next, a 20 ns molecular dynamics simulation was performed on the entire tetramer. The complex was very stable during this time frame, except for the highly flexible P domain (video not shown). The intermolecular contacts of TPOR and CALR were analyzed over the course of the simulation. Most contacts were dependent on basic interactions, occurring both in cis and trans, further stabilizing the TpoR dimer.

[0252] Thus, we propose a model in which CALR del52 interacts with the TpoR D1 domain through two regions. First, the mutant interacts directly with multiple negatively charged residues on the inner / lateral side of the TpoR D1 domain. This interaction between TpoR and CALR del52 is further stabilized by a strong interaction between the CALR glycan-binding domain and the immature sugar moiety, mainly on Asn117 of TpoR. Thus, this anaconda effect of the CALR mutant protein binding to two different sites on TpoR explains both its high affinity and specificity for the receptor.

[0253] Example 4: Fc-Coupled TpoR ECD for Binding of CALR Mutants material and method Fusion of the Fc fragment of huIgG1 or muIgG2B (depending on the experiment) to different fragments of the TPOR extracellular domain was performed. Such methods have been successfully used to treat several pathologies, including rheumatoid arthritis, treated with Etanercept®, a fusion protein between the extracellular domain of tumor necrosis factor (TNF) and the Fc fragment, which can capture and block the pathological action of TNF (Zhao et al., 2019; Korth-Bradley JM et al, 2000). For inhibition of binding and activation of CALR mutants of TPOR, three Fc-coupled soluble species of the TPO receptor were developed. These soluble species contain either the entire extracellular domain of the receptor (designated D1D2D3D4), the first two domains of the receptor (designated D1D2), or exclusively the first domain of the receptor (designated D1). All are coupled to the Fc fragment via the C-terminus. Their mode of action is binary. First, they act as capture molecules for circulating CALR mutants, thereby preventing their labile cytokine action ( Pecquet et al., 2019 ), and second, they compete with surface-exposed bound TPOR for binding to CALR mutants.

[0254] Using bioluminescence resonance energy transfer (BRET), we tested the ability of these soluble species to prevent CALR mutant binding to TPOR (see Example 1). The technique allows us to directly test the interaction between NanoLuc tag molecules (herein, TPOR) and HaloTag coupling partners (here, CALR del52) in living cells. NanoLuc and HaloTag are in close proximity (<10 nm), and energy transfer between NanoLuc (donor) and HaloTag (acceptor) results in a shift in the BRET ratio, which is used as a readout for the proximity between the two partners.

[0255] result All three soluble fusion proteins, Fc-D1, Fc-D1D2, and Fc-D1D2D3D4, are able to inhibit the binding of CALR mutants to full-length TPOR (Figure 5A-C). The fact that the D1 domain alone can prevent this binding suggests that the interaction between CALR mutants and the receptor may occur exclusively through the D1 domain, even when contained in a fusion protein (here with the Fc fragment of IgG).

[0256] Importantly, Figure 5D shows that inhibition of CALR mutant interaction with TPOR is independent of the presence of N-glycosylation at position 117. Thus, although the presence of an immature N-glycan is required for full activation of TPOR by CALR mutants (Chachoua et al., Blood 2016), inhibition of CALR mutant-TPOR interaction is independent of immature N-glycosylation. Specifically, mutants of D1 lacking N117 remain capable of inhibiting CALR mutant binding to full-length TPOR (Figure 5D).

[0257] Example 5: TpoR ECD fragment D1 can inhibit binding of CALR mutants to TpoR in trans. material and method Soluble extracellular domain (ECD) species of TPOR fused to Fc, namely D1-Fc and D1D2D3D4-Fc, were collected after their secretion by cultured HEK293T cells. These were added to the culture medium of hematopoietic cell line Baf3 cells stably expressing human TPOR on their surface together with CALR mutants (Balligand et al., Leukemia 2016). The ability of Fc-coupled TPOR species to prevent the interaction between CALR mutants and TPOR was assessed by measuring the levels of cell surface CALR mutants in the presence of control vehicle or the same concentration of Fc fragments alone, D1-Fc or D1D2D3D4-Fc fragments. Measurements were performed by flow cytometry with an antibody that specifically recognizes mutant CALR on the cell surface of live cells.

[0258] result At the concentrations tested, the D1-Fc fusion protein was able to reduce the levels of CALR mutants on the cell surface by more than 50%. This was specific to D1-Fc, as the Fc control did not modulate the cell surface levels of CALR mutants (Figure 6). Compared to D1-Fc, D1D2D3D4-Fc was less able to inhibit the interaction between TPOR and CALR, with the inhibition being less than 50% (Figure 6). These results demonstrate that (i) D1-Fc can inhibit the binding of mutant CALR to TpoR in trans (acting from the extracellular space) and (ii) D1-Fc is a more potent inhibitor than D1D2D3D4-Fc.

[0259] Example 6: In vivo application of TpoR ECD fragments for the treatment of essential thrombocythemia material and method Next, a genetically engineered mouse model was used to express endogenous levels of CALR mutants. Mice exhibit an essential thrombocythemia phenotype as described in Balligand et al., 2020. Briefly, small guide RNAs (sgRNAs) were cloned into the px330 vector (Addgene #42230). The generated vectors were microinjected into the nuclei of fertilized B6D2 mouse zygotes. For the generation of del52 mutations, homologous recombination repair (HDR) was performed by coinjecting single-stranded oligo DNA as a template as described in Balligand et al., 2020. Selected pups were crossed with C57BL / 6-DBA / 2 (B6D22.F1) mice, and subsequent generations were selected based on the presence of del52 mutation in Calr exon 9 by PCR screening. Bone marrow cells from these mice were isolated and transduced with viral particles containing either the DNA of the soluble D1 species (not coupled to an Fc fragment) or an empty vector used as a control. Recipient mice were lethally irradiated and transplanted with the infected cells. The phenotype of these mice was followed for 24 weeks to assess the ability of the soluble D1 species to prevent the development of essential thrombocythemia in vivo by measuring platelet concentration, used as a readout for the ET phenotype.

[0260] result As expected, control mice with ET (CALR del52+pMEGIX) showed high levels of circulating platelets reminiscent of the ET phenotype observed in patients (Figure 7). In stark contrast, when cells were infected with the soluble D1 domain of TPOR (CALR del52+pMEGIX-soluble D1), they did not develop the ET phenotype and showed platelet levels similar to wild-type littermates (Figure 7). This demonstrated that inhibition of CALR mutants via soluble D1 molecules can prevent disease development in vivo.

[0261] Example 7: FFPLHLWV is not involved in the interaction material and method The FFPLtAAAA and HLWVtAAAA mutants of D1 were purchased from Genscript. All constructs were verified by sequencing.

[0262] Using bioluminescence resonance energy transfer (BRET), the ability of D1-Fc mutants ("FFPLtAAAA" and "HLWVtAAAA") to compete with the interaction between full-length TpoR and CALR del52 was tested, and their effectiveness was compared with that of non-mutant D1-Fc fusion protein (see Example 1). The technique allows the interaction between NanoLuc tag molecules (here TPOR) and HaloTag coupling partners (here CALR del52) to be directly tested in living cells. NanoLuc and HaloTag are in close proximity (<10 nm), and the energy transfer between NanoLuc (donor) and HaloTag (acceptor) results in a shift in the BRET ratio, which is used as a readout for the proximity between the two partners.

[0263] FFPLtAAAA mutant of D1 (SEQ ID NO: 87) QDVSLASDSEPLKCFSRTFEDLTCFWDEEEAAPSGTYQLLYAYPREKPRACPLSSQSMPHFGTRYVCQFPDQEEVRLAAAAHLWVKNVFLNQT

[0264] HLWVtAAAA mutant of D1 (SEQ ID NO: 88) QDVSLASDSEPLKCFSRTFEDLTCFWDEEEAAPSGTYQLLYAYPREKPRACPLSSQSMPHFGTRYVCQFPDQEEVRLFFPLAAAAKNVFLNQT

[0265] result These results demonstrate that polypeptides containing D1 can compete with TPOR independently of the FFPLHLWV motif. Indeed, mutation of this motif to alanine actually increases the ability of D1 to prevent TPOR-CALR mutant interaction (Figure 8).

[0266] Example 8: Comprehensive model of the TPOR-CALR mutant complex material and method Using templates from AlphaFold2 and Rosetta Folding, a tetrameric 3D model was efficiently constructed and the interaction interface between the two CALR variants was identified. HDx-MS data was used to identify contacts between TPOR and CALR Del52 in the formation of a tetrameric complex. The ER-specific G1M9 glycan of TPOR in contact with CALR was modeled by Glycopack59 in a configuration consistent with the NMR data60, and the rest, which are complex-type, were constructed in line with the SAGS Database61,62. The contacts identified by HDx-MS and the solid-state NMR data in the TM region configuration of the TPOR dimer were used as constraints in creating the entire 2CALR-2TpoR model. This glycoproteic tetramer was placed into a full-atom representation of the environment using the CHARMM-GUI server63, in a lipid bilayer of 1907 POPC that fits the TM region of TpoR, as well as 478,479 TIP3P water molecules, 1328 chloride, and 1402 sodium ions that account for the solvent region hydrating the remainder of the tetramer. These entire systems, consisting of about one million atoms, were subjected to a gentle simulated annealing procedure consisting of a tart minimization, heating to 300 K, then cooling to 0 K, and finally a final extended minimization, using NAMD v.2.1364 CHARMM36 force field65-67. The same procedure was used to build the TPOR-CALR Ins5 complex. The TpoR-CALR-Del52 / ins5 model was further subjected to three molecular dynamics runs to explore the sample space of configurations.

[0267] result Based on our experimental data, we generated an atomic model of the complete TPOR-CALR mutant tetrameric complex. The data showed that the binding of the C-terminus of CALR del52 alone to TPOR can occur in various microconfigurations, but the extended S1 acidic region at the center of 44TFED47 is important for the interaction. In addition, the binding of TPOR to full-length CALR del52 also involves strong interactions between specific residues in the N-domain and the immature N-glycan on Asn117. Using AlphaFold 2.0, we completed the modeling of TPOR and generated the complete extracellular and transmembrane domains of the receptor. TPOR monomers dimerized with residue L508 through their TM domains at the interface, similar to the active configuration in the presence of CALR del52 (Figure 2F). Given the experimental data showing that binding occurs simultaneously between the immature N-glycan on Asn117 of TPOR and residues in the CALR N-domain, and between the TPOR S1 acidic region and the C-terminus of the CALR mutant, we docked the CALR del52 dimer to the TPOR dimer. The final structure places the C-terminus of the mutant in a configuration in which the major interaction site is located around the 44TFED47 motif, in agreement with the above results. Similarly, the immature N-glycan on Asn117 of TpoR interacts with the N-domain pocket that contains key residues involved in N-glycan binding, including C105, Y109, and W319 (Figure 9). We embedded this glycoprotein tetramer in a POPC lipid bilayer and subjected the water box (approximately 1 million atoms in total) to a 100 ns all-atom triplet molecular dynamics simulation. The complex remained stable during this time frame, except for the highly flexible P-domain. Most of the contacts identified during the simulations relied on basic-acidic interactions, occurring both in cis and trans, further stabilizing the TpoR dimer. These involved the TFED motif, but also other negative patches, including the extended negative patches present in D1, PDQEE and WEEP (Table 1).

[0268] [Table 1] JPEG2025511108000002.jpg226159JPEG2025511108000003.jpg225159JPEG2025511108000004.jpg41159

[0269] To assess whether this model is also compatible with CALR Ins5 (CALR2 variant), which possesses a similar but longer C-terminus, we also generated a CALR Ins5-TpoR tetrameric complex following the same procedure as CALR del52 and subjected this complex to triplicate all-atom molecular dynamics simulations. Similar to CALR del52, the complex remained stable over a 100 ns time frame. Analysis of interacting residues over the simulation time frame revealed that 44 TFED47 motifs, 96 PDQEE100 motifs, in addition to 26 QDV28 motifs, were conserved in the CALR-Ins5-TpoR tetrameric complex (Table 2).

[0270] [Table 2] JPEG2025511108000006.jpg223159JPEG2025511108000007.jpg222159JPEG2025511108000008.jpg220159JPEG2025511108000009.jpg64159

[0271] These experimental data and atomic simulations indicate that the CALR mutants interact essentially through two regions of TPOR located on the D1 domain: the C-terminus of the mutants directly interacts with multiple negatively charged residues on the inner / lateral surface of the TPOR D1 domain, represented by the S1 negative patch.

[0272] Example 9: Identification of residues from D1 involved in the binding of TPOR to CALR material and method See Example 4 for NanoBRET techniques.

[0273] Figure 10 illustrates the NanoBRET signal used as a proxy for binding between TPOR full-length and CALR del52 in the presence of increasing amounts of human D1 WT and mutants. These data indicate that the motif [ka] Figure 1 shows that charged residues in TPOR, including those in (a) and (b), are important for maximal ability of D1 to act as a competitor of CALR mutant-TPOR interaction. Critical residues identified via HDx-MS and / or molecular dynamics simulations are also underlined.

[0274] These results correlate with (i) the HDx-MS results showing an interaction between the C-terminus of the CALR mutant and a negative patch on the TpoR D1 domain, and (ii) molecular dynamics simulations identifying additional negatively charged residues as key interactors with mutant CALR. Loss of such residues results in a reduced ability of D1 to act as a competitor for the CALR mutant-TPOR interaction.

Claims

1. PLKCFSX ２ TFX １ X １ LTCFWX １ X １ X １ X １ AAPSGTYQLLYAYPREKPRACPLSSQSMPHFGTRYVCQFPX １ QX １ X １ VX ２ A polypeptide comprising an amino acid sequence having at least 75% sequence identity with (SEQ ID NO:66), １ is D (Asp, aspartic acid) or E (Glu, glutamic acid), ２ is R (Arg, arginine) or K (Lys, lysine), and the amino acid sequence of the polypeptide does not include SEQ ID NO:5; Polypeptides.

2. The polypeptide of claim 1, wherein the amino acid sequence has at least 75% sequence identity to SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:

4.

3. The polypeptide of claim 1 or 2, wherein the polypeptide binds to a mutant of calreticulin (CALR) having a positively charged amino acid sequence in its C-terminal tail.

4. 4. The polypeptide according to any one of claims 1 to 3, wherein said amino acid sequence comprises SEQ ID NO: 7 and / or at least one amino acid Asn at position 117, said positions being defined with respect to the amino acid sequence of SEQ ID NO:

1.

5. The polypeptide of claim 4, wherein the amino acid Asn at position 117 is not glycosylated.

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 increases the stability and / or reduces the immunogenicity of the first polypeptide.

8. 8. The fusion protein of claim 7, wherein the second polypeptide is an Fc region of an immunoglobulin or a functional equivalent thereof, preferably selected from the group consisting of IgG, IgA, IgD, IgE, or IgM.

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

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

11. 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- ceutically acceptable vehicle.

12. 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, a vector according to claim 10, or a pharmaceutical composition according to claim 11, for use as a medicament.

13. 13. A polypeptide, fusion protein, nucleic acid, vector or pharmaceutical composition as claimed in claim 12 for use in the treatment and / or prevention of myeloproliferative neoplasms (MPNs).

14. The polypeptide, fusion protein, nucleic acid, vector, or pharmaceutical composition of claim 13, wherein the MPN is derived from one or more mutations in calreticulin (CALR), preferably resulting in the production of a positively charged amino acid sequence at the C-terminus of CALR.

15. 15. A kit for treating and / or preventing myeloproliferative neoplasms (MPNs), comprising (i) a polypeptide, fusion protein, nucleic acid, vector, or pharmaceutical composition according to any one of claims 12 to 14, (ii) a means for administering said polypeptide, fusion protein, or pharmaceutical composition, and optionally (iii) a further anti-cancer agent or vaccine.

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