FIELD OF INVENTION

[0001] The present invention relates to methods for detecting CALR mutant positive Myeloproliferative Neoplasms in a subject.

BACKGROUND OF INVENTION

[0002] Malignant blood diseases represent a significant burden for the healthcare systems. Several such conditions such as BCR-ABL negative Myeloproliferative Neoplasms (MPNs) are diseases of aged individuals, with many complications including thrombosis and bleeding necessitating major resources for treatment. MPNs can evolve to a very severe condition called secondary Acute Myeloid Leukemia (AML) which is almost always fatal. The three major MPNs types, namely Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF) can occur in children and young adults but is most common in aged individuals where incidence can reach 1/5,000 persons per year.

[0003] About 25% of ET and MF cases are due to acquired mutations in calreticulin (CALR). CALR mutant positive MPNs are thus a subset of MPN constituting an interesting target for therapy and diagnosis.

[0004] The inventors previously elucidated how CALR mutants bind to the extracellular domain of thrombopoietin receptor (TPOR) to induce its activation in the absence of its ligand, the thrombopoietin (THPO or TPO), leading to persistent activation of the JAK2- STAT5 pathway in HSCs and megakaryocytes (Chachoua et al., Blood, 2016; Pecquet et al., Blood, 2019).

[0005] The inventors also discovered that CALR mutant are secreted and that their levels in the plasma of patients correlate both with allele burden and symptoms severity (Pecquet et al., Blood, 2018). Surprisingly, the stability of CALR mutant in the plasma was much higher than this expected from measuring the stability of recombinant CALR mutant proteins in culture medium, suggesting it was stabilized by another protein in the plasma. The soluble form of the Transferrin Receptor 1 (TFRC) was identified as being the protein involved in stabilization of plasma CALR mutants.

[0006] It is worth mentioning that, in other blood cancers distinct from CALR mutant positive MPNs, certain studies report a decreased or increased expression of TFRC in acute myelomonocytic leukemia (or myeloid leukemia, AML), acute erythroid leukemia, B-cell acute lymphoblastic leukemia (B-ALL) (Liu Qian et al., 2012; Liu Qian et al., 2014; W02008086182) while another study reports that AML, ALL and CML leukemia cells induce higher TFRC levels (Wang Changlong et al. 2021). However, these documents do not tackle CALR mutant positive MPNs.

[0007] Further investigation on the effect of CALR mutant on TFRC processing and subcellular localization revealed that TFRC expression at the cell surface could be used as a selective marker to target cells from the clone expressing CALR mutations, in CALR mutant positive MPNs.

[0008] Diagnostic for the MPNs pathology is done by sequencing of CALR exon 9 to detect the presence of frameshift mutations and define the allele burden. While this approach will remain required, the present invention provides means to use the expression of cell surface TFRC as an additional tool for diagnostic, risk stratification between patients and separation of healthy from malignant cells.

SUMMARY

[0009] The present invention relates to an in vitro method for detecting blood cancer cells in a subject, comprising the steps of:

(i) measuring the level of surface exposed Transferrin Receptor Protein 1 (TFRC) in a biological sample obtained from said subject, (ii) comparing the measured level of TFRC with the determined level, and detecting blood cancer cells when the measured level is at or less the determined level.

[0010] In some embodiments, the biological sample is selected from the group consisting of blood (such as peripheral blood), bone marrow, and umbilical cord blood.

[0011] In some embodiments, the biological sample comprises blood cells or bone marrow cells.

[0012] In some embodiments, the biological sample comprises hematopoietic stem cells (HSCs), LSK (Lineage negative, Sca-1 positive, c-kit positive) cells, or bone marrow progenitor cells.

[0013] In some embodiments, the level of TFRC is measured at the surface of the cells in said biological sample.

[0014] In some embodiments, the blood cancer is a myeloproliferative neoplasm (MPN) or Acute Myeloid Leukemia (AML).

[0015] In some embodiments, the blood cancer is a MPN.

[0016] In some embodiments, the MPN is Polycythemia Vera (PV), Essential Thrombocythemia (ET) or Myelofibrosis (MF).

[0017] In some embodiments, the MPN is due to at least one Calreticulin (CALR) mutation.

[0018] In some embodiments, CALR mutant has a positively charged amino acid sequence in the C-terminus tail.

[0019] In some embodiments, the at least one CALR mutant comprises at least one mutation compared to the amino acid sequence of SEQ ID NO: 2.

[0020] In some embodiments, the measurement of TFRC level at step (i) is performed using a reagent that specifically binds to TFRC, wherein said reagent is selected from the group comprising or consisting of antibodies or antigen binding fragments thereof, peptide, chemical probe or nucleic acid probe.

[0021] In some embodiments, the measured level is at or less the determined level when the measured level of TFRC is at least a 1.1-fold less compared to said determined level.

[0022] In some embodiments, the method further comprises the detection of at least one other surface marker, preferably selected in the group comprising CALR mutant, thrombopoietin receptor (also known as TPOR, MPL or CD 110), Glucose 6-phosphate (G6P) and the like.

[0023] The present invention further relates to a method of separating healthy cells from a biological sample obtained from a subject with blood cancer, the method comprising the steps of:

(i) staining said biological sample with a substance that specifically binds to or reacts with Transferrin Receptor Protein 1 (TFRC) or a fragment thereof, wherein the substance binds to or reacts with TFRC or a fragment thereof on the healthy cells in said biological sample,

(ii) selecting the cells that bind to or react with the substance, thereby isolating a healthy cells population.

[0024] In some embodiments, TFRC is present on the cell surface.

[0025] The present invention further relates to a healthy cell population isolated by the isolation method according to the invention.

[0026] The present invention further relates to the healthy cell population according to the invention, for use for immunotherapy, such as for stem cell transplantation.

[0027] The present invention further relates to a method for treating blood cancer in a subject in need thereof, comprising the steps of:

(i) separating and isolating healthy cells from a biological sample obtained from the subject using the method according to the invention, and (ii) performing an autologous stem cell transplantation of the isolated healthy cells population in said subject.

[0028] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of at least one anticancer agent.

[0029] The present invention further relates to a reagent for detecting healthy cells in a biological sample obtained from a subject with blood cancer, comprising a substance that specifically binds to or reacts with Transferrin Receptor Protein 1 (TFRC) or a fragment thereof, wherein the substance that binds to or reacts with TFRC or a fragment thereof on the healthy cells in said biological sample.

[0030] The present invention further relates to a method for screening a therapeutic agent for the treatment of blood cancer comprising the steps of:

(a) culturing cells with a candidate substance; and

(b) determining the effect of the candidate substance on increasing the level of Transferrin Receptor Protein 1 (TFRC) on the surface level of said cells.

DEFINITIONS

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

[0032] The term “about”, when preceding a figure, means plus or less 10% of the value of said figure.

[0033] The term “amino acid substitution” refers to the replacement in a polypeptide of one amino acid with another amino acid. In one embodiment, an amino acid is replaced with another amino acid having similar structural and/or chemical properties, e.g., conservative amino acid replacements. “Conservative amino acid substitution” may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (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; negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. For example, amino acid substitutions can also result in replacing one amino acid with another amino acid having different structural and/or chemical properties, for example, replacing an amino acid from one group (e.g., polar) with another amino acid from a different group (e.g., basic). Amino acid substitutions can be generated 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 of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful.

[0034] The terms “at least one” and “one or more”, used herein interchangeably, mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000, or more.

[0035] The terms “determined level” or “determined surface level” of TFRC, herein used interchangeably, refer to the standard or the norm of level of expression for TFRC at the surface of a given cell type. In some embodiments, the determined surface levels of TFRC are known from the state of the art, typically the determined surface levels of TFRC are inferior or equal to normal or healthy surface levels of TFRC. In some embodiments, normal or healthy surface levels of TFRC are comprised within a certain range. In some embodiments, the determined surface levels of TFRC are established by measuring surface levels of TFRC on a healthy cell, population of cell or tissue. In some embodiments, the healthy cell, population of cell or tissue used to establish the determined surface levels of TFRC are of the same type (e.g., same cell type) as the biological sample used in the method according to the invention. In some embodiments, the healthy cell, population of cell or tissue used to establish the determined surface levels of TFRC are obtained from a different subject than the biological sample used in the method according to the invention. In some embodiments, the healthy cell, population of cell or tissue used to establish the determined surface levels of TFRC are obtain from at least one healthy subject, preferably more than one healthy subject. In some embodiments, the healthy cell, population of cell or tissue used to establish the determined surface levels of TFRC are obtained from healthy cells of the same subject as the biological sample used in the method according to the invention. Within the scope of the present invention, a healthy subject is defined herein as an individual who does not suffer from cancer, blood cancer, MPN, or CALR mutant positive MPN, preferably CALR mutant positive MPN. It is to be understood that the determined surface levels of TFRC, as defined within the scope of the present invention, do not necessarily reflect the normal or healthy surface levels of TFRC. In some embodiments, the determined surface levels of TFRC are inferior or equal to the lowest value the normal or healthy surface levels of TFRC. In some embodiments, the determined surface levels of TFRC are inferior to the lowest value the normal or healthy surface levels of TFRC. In some embodiments, the determined surface levels of TFRC are comprised with the lowest half, third, quarter or tenth of the value range for the normal or healthy surface levels of TFRC. In other words, the determined surface levels of TFRC according to the invention reflects the threshold below which the surface levels of TFRC correspond to an abnormal or pathological situation, /.< ., the determined level is a range of values defining a lower limit and an upper limit, beyond which the surface levels of TFRC correspond to an abnormal or pathological situation.

[0036] The term “fragment” refers to any subset of a protein, as a shorter peptide. In one embodiment, a fragment of a protein is a peptide of at least 5, 10, 20, 30 amino acids in length, or more. In one embodiment, a fragment of a protein retains a domain or motif that can be bound, recognized, or otherwise detected by a suitable reagent.

[0037] The terms “healthy cell” or “population of healthy cells”, used herein interchangeably, refer to cells of the same type (e.g., same cell type or lineage) as the CALR mutant positive MPN cells, in a biological sample. In some embodiments, the healthy cells do not comprise any mutation inducing a CALR mutant positive MPN, or associated with a CALR mutant positive MPN. In some embodiment, a healthy cell is not a CALR mutant positive MPN but shares the same cell type, functionalization and/or lineage, and are comprised in the same biological sample. [0038] The term “identity” refers to a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. Methods to determine identity and similarity are codified in computer programs. Computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package, the GAP program. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include an average up to five point-mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0039] The term “myeloproliferative neoplasm” or “MPN” refers to a blood cancer type caused by a pathological constitutive activation of the JAK/STAT pathway that affects primarily hematopoietic stem cells and induces an abnormal expansion of cells of the myeloid lineage. In some embodiments, the MPN is Essential Thrombocythemia (ET) or Myelofibrosis (MF).

[0040] The term “calreticulin mutant-positive myeloproliferative neoplasms” or “CALR mutant positive MPNs” refers to a subset of MPNs wherein calreticulin (CALR) comprises at least one amino acid mutation. CALR typically has the sequence of SEQ ID NO: 2 in humans. The skilled artisan knows that mutations (alleles) may be present in a population and the sequences may vary. In some embodiments, the at least one mutation on CALR induces a partial or complete loss of CALR function. In some embodiments, the at least one mutation on CALR induces an alteration of CALR function, including, e.g., a gain of function. In some embodiments, the at least one mutation on CALR induces an alteration of CALR localization in the cell and in the organism. In some embodiments, the mutant of CALR losses partially or completely its endoplasmic reticulum (ER) localization. In some embodiments, the at least one mutation on CALR induces a conformational change of the protein. In some embodiments, the at least one mutation on CALR induces an alteration of its chemical properties. In some embodiments, CALR mutant has a positively charged amino acid sequence in the C- terminus tail. In one embodiment, the at least one mutation consists of insertions and/or deletions in exon 9, resulting in the generation of a positively charged amino acid sequence in the C-terminus of CALR.

[0041] The term “treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the disease or condition, preferably blood cancer, more preferably MPN, even more preferably CALR mutant positive MPN. Those in need of treatment include those already affected with the disease or condition, preferably blood cancer, more preferably MPN, even more preferably CALR mutant positive MPN, as well as those prone to have the disease or condition, preferably blood cancer, more preferably MPN, even more preferably CALR mutant positive MPN, or those in whom the disease or condition, preferably blood cancer, more preferably MPN, even more preferably CALR mutant positive MPN, is to be prevented. A subject or mammal is successfully "treated" for the disease or condition, preferably blood cancer, more preferably MPN, even more preferably CALR mutant positive MPN, if, after receiving a therapeutic amount of an inhibitor according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of pathogenic cells; reduction in the percent of total cells that are pathogenic; and/or relief to some extent, one or more of the symptoms associated with the disease or condition, preferably blood cancer, more preferably MPN, even more preferably CALR mutant positive MPN; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease or condition, preferably blood cancer, more preferably MPN, even more preferably CALR mutant positive MPN, are readily measurable by routine procedures familiar to a physician.

DETAILED DESCRIPTION

[0042] The present invention relates to an in vitro method for detecting calreticulin (CALR) mutant positive myeloproliferative neoplasms (MPNs) cells in a subject, comprising the steps of:

(i) measuring the level of surface exposed Transferrin Receptor Protein 1 (TFRC) in a biological sample obtained from said subject,

(ii) comparing the measured level of TFRC with the determined level, and

(iii) detecting CALR mutant positive MPN cells when the measured level is at or less the determined level.

[0043] Another object of the present invention relates to an in vitro method for detecting CALR mutant positive MPNs in a subject, comprising the steps of:

(i) measuring the level of surface exposed Transferrin Receptor Protein 1 (TFRC) in a biological sample obtained from said subject,

(ii) comparing the measured level of TFRC with the determined level, and

(iii) detecting CALR mutant positive MPN when the measured level is at or less the determined level.

[0044] In humans, TFRC typically has an amino acid sequence as set forth in SEQ ID NO: 1. The skilled artisan knows that mutations (alleles) may be present in a population and the sequences may vary.

[0045] In some embodiments, the biological sample is collected from the subject within the scope of a medical procedure or intervention. In some embodiments, the subject has symptoms and/or biological markers that would lead a medical practitioner, e.g., a physician, to diagnose cancer or a blood disease, preferably a CALR mutant positive MPN, or to suspect the existence of a cancer or blood disease, preferably a CALR mutant positive MPN. In another embodiment, the subject has no symptoms related to CALR mutant positive MPN, and the biological sample is collected from the subject as part of a routine medical examination.

[0046] In some embodiments, the biological sample is a biopsy. In some embodiments, collection of the biological sample is performed in sterile conditions.

[0047] In some embodiments, collection of the biological sample is performed by surgery, blood sampling or marrow sampling. In some embodiments, collection of the biological sample is performed by surgery. In some embodiments, collection of the biological sample is performed by blood sampling. In some embodiments, collection of the biological sample is performed by marrow sampling.

[0048] In some embodiments, the biological sample is selected from the group of tissues, population of cells, and isolated cells. In one embodiment, the population of cells is homogenous; in certain embodiments, the population of cells is sorted prior to their use in the method according to the invention. In another embodiment, the population of cells is heterogenous.

[0049] In some embodiments, the biological sample is selected from the group consisting of blood (such as peripheral blood), bone marrow, and umbilical cord blood.

[0050] In some embodiments, the biological sample comprises blood cells or bone marrow cells.

[0051] In some embodiments, the biological sample comprises blood cells. In some embodiments, the biological sample consists of blood cells.

[0052] In some embodiments, blood cells comprise or consist of red blood cells, white blood cells and platelets. In some embodiments, red blood cells comprise or consist of erythrocytes and erythroblasts. In some embodiments, white blood cells comprise or consist of basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes, monocytes (Including macrophages and dendritic cells) and lymphocytes. [0053] In some embodiments, the biological sample comprises marrow cells. In some embodiments, the biological sample consists of marrow cells. In some embodiments, the biological sample consists of bone marrow cells.

[0054] In some embodiments, the biological sample comprises hematopoietic stem cells (HSCs), LSK (Lineage negative, Sca-1 positive, c-kit positive) cells, or bone marrow progenitor cells.

[0055] In some embodiments, the biological sample comprises or consists of HSCs. In some embodiments, the biological sample comprises or consists of LSK cells. In some embodiments, the biological sample comprises or consists of bone marrow progenitor cells.

[0056] In some embodiment the biological sample is used fresh. In some embodiment the biological sample is used immediately, wherein immediately means at most 48 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours or 1 hour after collection of the biological sample.

[0057] In some embodiment the biological sample is stored in a suitable preservation medium at suitable temperature for later use.

[0058] In some embodiment, the biological sample is frozen at a temperature from about -20°C to about -200°C. In some embodiment the biological sample is flash-frozen in liquid nitrogen. In some embodiments, the biological sample is contacted with at least one cryoprotectant agent, such as, e.g., glycerol, prior to freezing.

[0059] In some embodiments, the biological sample is stored briefly in a suitable sterile biological buffer or medium at a temperature comprised between 0°C and 15°C.

[0060] In some embodiments, the biological sample is fixed. As used herein, “fixed” means that the biological sample has been contacted with at least one fixative agent, such as, e.g., paraformaldehyde.

[0061] In some embodiment, the cells comprised in the tissue sample are isolated from each other, i.e., they are not forming tight junctions, or the like. In some embodiments, the cells comprised in the tissue sample are resuspended in a suitable biological buffer or medium.

[0062] Within the scope of the present invention, the term “blood cancer”, interchangeably used with the terms “malignant blood diseases” and “hematologic cancer” refers to a cancer of blood cells, blood-forming tissue cells (e.g., bone marrow) or immune system cells, typically erythroid lineage cells. Non limitative examples of blood cancer include myeloproliferative neoplasm (MPN), leukemia and lymphoma. In a very preferred embodiment, the “blood cancer” is a MPN, preferably a CALR mutant positive MPN.

[0063] As used herein, MPN refers to a blood cancer type caused by a pathological constitutive activation of the JAK/STAT pathway that affects primarily hematopoietic stem cells and induces an abnormal expansion of cells of the myeloid lineage.

[0064] In some embodiments, the MPN is Essential Thrombocythemia (ET) or Myelofibrosis (MF). In some embodiments, the MPN is ET. In some embodiments, the MPN is MF.

[0065] In some embodiments, the MPN in characterized by a constitutive activation of the JAK/STAT pathway.

[0066] In some embodiments, the MPN is caused by, or associated with, at least one amino acid mutation of calreticulin (CALR). In some embodiments, the MPN is caused by at least one Calreticulin (CALR) mutation. Thus, such MPN is hereby referred to as a “CALR mutant positive MPN”. Accordingly, in some embodiments, the CALR mutant positive MPN is a CALR mutant positive Essential Thrombocythemia or a CALR mutant positive Myelofibrosis.

[0067] In some embodiments, the CALR mutant positive MPN is induced by (i) at least one CALR mutation and (ii) one or more mutation(s) in another gene(s). In some embodiments, the at least one CALR mutation amplify the effect of the one or more mutation(s) in other gene(s). [0068] In one embodiment, the at least one CALR mutation and the one or more mutation(s) in other gene(s) occurs in the same cell. In another embodiment, the at least one CALR mutation and the one or more mutation(s) in other gene(s) occurs in separate cells or groups of cells.

[0069] In some embodiments, the at least one CALR mutation comprises at least one mutation compared to the amino acid sequence of SEQ ID NO: 2, wherein SEQ ID NO: 2 is the amino acid sequence of wild type human calreticulin (UniProt ID number P27797). Typically, mutations include substitutions, deletions and insertions.

[0070] In some embodiments, the at least one mutation on CALR induces a partial or complete loss of CALR function. In some embodiments, the at least one mutation on CALR induces an alteration of CALR function, including, e.g., a gain of function.

[0071] In some embodiments, the at least one mutation on CALR induces an alteration of CALR localization in the cell and in the organism. In some embodiments, the mutant of CALR losses partially or completely its endoplasmic reticulum (ER) localization.

[0072] In some embodiments, the at least one mutation on CALR induces a conformational change of the protein. In some embodiments, the at least one mutation on CALR induces an alteration of its chemical properties. As used herein, the term “chemical properties” is meant to designate, e.g., the hydrophobicity, polarity, charge, redox state, acide-base state, and the like. Typically, the chemical properties of a protein reflect the chemical properties of the side chains of amino acid comprised in the protein. Thus, in some embodiments, the chemical properties of the CALR mutant are modified by the addition, deletion or substitution of at least one amino acid having a definite set of chemical properties.

[0073] In some embodiments, CALR mutant has a positively charged amino acid sequence in the C-terminus tail.

[0074] In one embodiment, the at least one mutation consists of insertions and/or deletions in exon 9, resulting in the generation of a positively charged amino acid sequence in the C-terminus of CALR. [0075] As used herein, positively charged amino acids, interchangeably referred to as basic amino acids, are amino acids harboring a positive charge on their side chain at neutral pH, which encompass arginine, lysine, histidine, and all positively-charged unnatural amino acids.

[0076] In some embodiments, the CALR mutant inducing the MPN comprises the sequence SEQ ID NO: 3.

[0077] In some embodiments, the one or more mutation(s) of CALR results in a C-ter tail having an amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 4 to SEQ ID NO: 35. In some embodiments, the at least one amino acid mutation of CALR results in a C-ter tail having an amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 4 to SEQ ID NO: 35.

[0078] In some embodiments, the one or more mutation(s) of CALR results in a C-ter tail having an amino acid sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 5.

[0079] In one embodiment, the one or more mutation(s) of CALR results in a C-ter tail having an amino acid sequence as set forth in SEQ ID NO: 4, corresponding to CALR mutant L367fs*46, also referred to as CALR del52. In another embodiment, the one or more mutation(s) of CALR results in a C-ter tail having an amino acid sequence as set forth in to SEQ ID NO: 5, corresponding to CALR mutant K385fs*47, also referred to as CALR ins5. In some embodiments, the mutant of CALR is CALR del52 or CALR Ins5.

[0080] In some embodiments, the CALR mutant positive MPN is not acute myeloid leukemia (AML). In some embodiments, the subject does not suffer from AML. In some embodiments, the method of the invention is not for detecting AML without CALR mutant. In some embodiments, the method of the invention is not for treating AML without CALR mutation.

[0081] In some embodiments, the level of TFRC is measured at the surface of the cells in the biological sample. In some embodiments, the level of TFRC is measured at the surface of the cells by any suitable method known in the art. [0082] Techniques for assessing the surface level of a protein are known in the art and comprise, inter alia, flow cytometry, immunostaining, cell surface biotinylation assay, immunoprecipitation, affinity binding, cellular fragmentation or labelling of the protein of interest using exogenous expression or mutagenesis.

[0083] In some embodiments, the level of TFRC is measured at the surface of the cells by the means of a technique selected from the group comprising or consisting of flow cytometry, immunostaining, cell surface biotinylation assay, cellular fragmentation, immunoprecipitation and affinity binding.

[0084] In a preferred embodiment, the level of TFRC is measured at the surface of the cells by flow cytometry.

[0085] Flow cytometry is a routine technique well known by the skilled artisan. It is to be understood that all variants (e.g., mass cytometry, Raman cytometry), subtypes and possible applications of flow cytometry (e.g., cell sorting (FACS), cell counting) are comprised in the definition of flow cytometry as used herein.

[0086] In some embodiments, the level of TFRC is measured at the surface of the cells by immunostaining.

[0087] Immunostaining is a routine technique well known by the skilled artisan. In certain embodiments, the immunostaining does not comprise a permeabilization step, i.e., the plasma membrane of the cell is intact.

[0088] In some embodiments, the detection of surface levels of TFRC by immunostaining further comprises the use of optic microscopy, such as, e.g., epifluorescence microscopy, confocal microscopy, light sheet microscopy and the like for fluorescent labels, or bright field for chromogenic labels. In certain embodiments, he detection of surface levels of TFRC by immunostaining further comprises the use of electronic microscopy, such as, e.g, scanning electron microscopy or transmission electron microscopy for suitable labels such as gold beads.

[0089] In some embodiments, the detection of surface levels of TFRC by immunostaining further comprises the use of a quantification method, preferably computer-assisted quantification, in order to calculate the quantity of signal (e.g., fluorescence signal) expressed at the surface of the cell. In some embodiments, the surface levels of TFRC are expressed in arbitrary units (e.g., fluorescence units).

[0090] In some embodiments, the detection of surface levels of TFRC by immunostaining further comprises the co-detection, optionally co-immunostaining, of a surface marker other than TFRC, in order to distinguish surface TFRC from intracellular TFRC. In some embodiments, the detection of TFRC, in particular the quantification of TFRC signal, is limited to areas defined by using the surface marker.

[0091] In some embodiments, the level of TFRC is measured at the surface of the cells by cell surface biotinylation assay. Commercially available kits such as, e.g., Pierce™ Cell Surface Biotinylation and Isolation Kit (Thermo Scientific™ Cat. number: A44390) may be used to implement the technique.

[0092] In some embodiments, the detection of surface levels of TFRC by cell surface biotinylation assay further comprises a step of Western Blot. Typically, biotinylated proteins are detected in Western Blot by binding to avidin or streptavidin labeled with an enzyme (e.g., a peroxidase), a fluorochrome or the like; preferably, a reagent for the detection of TFRC is used (e.g., an anti-TFRC antibody) to discriminate TFRC-specific signal.

[0093] In some embodiments, the level of TFRC is measured at the surface of the cells by immunoprecipitation. In some embodiments, the cells expressing TFRC at their surface are isolated. In some embodiments, TFRC is measured at the surface of isolated cells. In some embodiments, immunoprecipitation is combined with another technique (e.g., flow cytometry, Western Blot and the like).

[0094] In some embodiments, the level of TFRC is measured at the surface of the cells by using affinity binding to a labeled molecule (e.g., a protein or peptide, a small molecule).

[0095] In some embodiments, the level of TFRC is measured at the surface of the cells by cellular fragmentation. [0096] In some embodiments, the cells transiently express or stably express TFRC coupled to a tag, preferably the cells transiently express TFRC coupled to a tag. In some embodiments, the tag is selected from the group comprising or consisting of fluorescent or fluorogenic molecules (e.g., Green Fluorescent Protein), chromogenic molecules, affinity tags (e.g., hemagglutinin or biotin) and epitopes. In some embodiments, the level of TFRC is measured at the surface of the cells by using any one of the techniques described hereinabove, wherein said technique relies on the detection of the tag coupled to TFRC.

[0097] In some embodiments, the measure of TFRC level at step (i) is performed using a reagent that specifically binds to TFRC, wherein the reagent is selected from the group comprising or consisting of antibodies or antigen binding fragments thereof, peptides, chemical probes or nucleic acid probes.

[0098] In a preferred embodiment, the reagent is suitable for use in at least one of the techniques selected from the group comprising or consisting of flow cytometry, immunostaining, cell surface biotinylation assay, immunoprecipitation and affinity binding. In a more preferred embodiment, the reagent is suitable for use in flow cytometry.

[0099] In some embodiments, the reagent that specifically binds TFRC is an antibody or antigen binding fragment thereof.

[0100] In a preferred embodiment, the antibody or antigen binding fragment thereof specifically detects TFRC, i.e., in a preferred embodiment, the antibody or antigen binding fragment thereof does not recognize any protein or peptide other than TFRC. In a preferred embodiment, the antibody or antigen binding fragment thereof detects specifically human TFRC, wherein human TFRC typically has the amino acid sequence as set forth in SEQ ID NO: 1.

[0101] In some embodiments, the antibody or antigen binding fragment thereof is an anti-TFRC antibody. Non-limitative examples of anti-TFRC antibodies include clone CY1G4 (BioLegend® ref. 334107). [0102] In some embodiments, the antibody or antigen binding fragment thereof is of an isotype selected from the group comprising or consisting of immunoglobulin (Ig) A, IgD, IgE, IgG, IgM, IgY, IgW, IgT and IgZ. In a preferred embodiment, the antibody or antigen binding fragment thereof is of an isotype selected from the group comprising or consisting of immunoglobulin IgA, IgD, IgE, IgG and IgM. In a less preferred embodiment, the antibody or antigen binding fragment thereof is of an isotype selected from the group comprising or consisting of IgY, IgW, IgT and IgZ.

[0103] In some embodiments, the antibody or antigen binding fragment thereof is monoclonal or polyclonal. In some embodiments, the antibody or antigen binding fragment thereof is monoclonal. In some embodiments, the antibody or antigen binding fragment thereof is polyclonal.

[0104] In some embodiments, the antibody or antigen binding fragment thereof is a nanobody.

[0105] In some embodiments, the reagent that specifically binds TFRC is a peptide.

[0106] In some embodiments, the peptide possesses chemical properties that confers the peptide the ability to bind at least one amino acid region on TFRC. As used herein, the term “chemical properties” is meant to designate, e.g., the hydrophobicity, polarity, charge, redox state, acid-base state, and the like. Typically, the chemical properties of a peptide reflect the chemical properties of the side chains of amino acid comprised in the protein.

[0107] In some embodiments, the peptide is a fragment of TFRC, /.< ., the peptide consists of an amino acid sequence comprised in TFRC. In some embodiments, the peptide is a modified fragment of TFRC, z.e., the peptide consists of an amino acid sequence comprised in TFRC and further comprises at least one amino acid mutation. In some embodiments, the peptide consists of an amino acid sequence that is not comprised on TFRC.

[0108] In some embodiments, the peptide is a ligand of TFRC or a fragment thereof. In some embodiments, the peptide is transferrin or a fragment thereof, preferably human transferrin. In some embodiments, the transferrin has at least 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 36. In some embodiments, the transferrin is comprised in a fusion protein further comprising at one other polypeptide. In some embodiments, the transferrin is fused to an immunoglobulin or a tag.

[0109] In some embodiments, the peptide is another ligand of TFRC or a fragment thereof.

[0110] In some embodiments, the reagent that specifically binds TFRC is a chemical probe. In a preferred embodiment, the chemical probe is organic. In another embodiment, the chemical probe is inorganic.

[0111] In some embodiments, the reagent that specifically binds TFRC is a nucleic acid probe.

[0112] In some embodiments, the nucleic acid probe is a DNA or RNA molecule. In some embodiments, the nucleic acid probe is a DNA molecule. In some embodiments, the nucleic acid probe is a RNA molecule.

[0113] In some embodiments, the nucleic acid probe is single stranded or double stranded. In some embodiments, the nucleic acid probe is single stranded. In some embodiments, the nucleic acid probe is double stranded

[0114] In some embodiments, the nucleic acid probe comprises at least one unnatural nucleotide.

[0115] In some embodiments, the reagent that specifically binds TFRC is coupled to another molecule. In some embodiments, the reagent that specifically binds TFRC is coupled to another molecule by the means of covalent or non-covalent liaison. In some embodiments, the reagent that specifically binds TFRC is coupled to another molecule in a fusion protein, optionally through a linker.

[0116] In some embodiments, said molecule is selected from the group comprising or consisting of fluorescent or fluorogenic molecules, enzymes, radiolabeled molecules, chromogenic molecules, immunoglobulins, and affinity tags. [0117] In some embodiments, said molecule is selected from the group comprising or consisting of fluorescent or fluorogenic molecules, and chromogenic molecules.

[0118] In a preferred embodiment, said molecule is a fluorescent or fluorogenic molecule. In some embodiments, fluorescent or fluorogenic molecule is selected from the group comprising or consisting of fluorophores, fluorescent proteins and organic dyes.

[0119] In some embodiments, fluorescent or fluorogenic molecule is a fluorophore. Non-limitative examples of commercially available fluorophores include Alexa Fluor dyes or Cy dyes.

[0120] In some embodiments, fluorescent or fluorogenic molecule is a fluorescent protein. Non-limitative examples of fluorescent protein include the Green Fluorescent protein (GFP), the Red Fluorescent Protein (RPF) or mCherry.

[0121] In some embodiments, fluorescent or fluorogenic molecule is an organic dye. Non-limitative examples of organic dyes include fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate.

[0122] In some embodiments, said molecule is selected from the group comprising or consisting of immunoglobulins, and affinity tags. In some embodiments, said molecule is an immunoglobulin selected from the group comprising or consisting of IgA, IgD, IgE, IgG, and IgM. In some embodiments, said molecule is an affinity tag. Non-limitative examples of commonly used affinity tags include influenza hemagglutinin (HA), maltose binding protein (MBP), poly histidine, or glutathione S-transferase.

[0123] In some embodiments, the region of TFRC recognized and/or bound by the reagent comprises at least 2, 3, 4, 5, 10, 20, 30, 40, 50 or more amino acids. In some embodiments, the region of TFRC recognized and/or bound by the reagent comprises from 2 to 100 amino acids, from 3 to 100 amino acids, from 4 to 100 amino acids, from 5 to 100 amino acids, from 10 to 100 amino acids, from 20 to 100 amino acids, from 30 to 100 amino acids, from 40 to 100 amino acids, from 50 to 100 amino acids. In some embodiments, the region of TFRC recognized and/or bound by the reagent comprises from 2 to 50 amino acids, from 2 to 40 amino acids, from 2 to 30 amino acids, from 2 to 20 amino acids, from 2 to 10 amino acids, from 2 to 5 amino acids, from 2 to 4 amino acids, from 2 to 3 amino acids.

[0124] In some embodiments, the region of TFRC recognized and/or bound by the reagent comprises the asparagine (Asn) residue at position 251 of TFRC, wherein position numbering is with respect to the amino acid of SEQ ID NO: 1.

[0125] In some embodiments, the asparagine residue is glycosylated, preferably the glycosylation is immature. In some embodiments, the glycosylation is selected from the group comprising or consisting of HexNac:2, Hex:7, HexNac:2, Hex:8, HexNac:2, Hex:9, HexNac:2, Hex: 10 or HexNac:3 and Hex:6.

[0126] In some embodiments, the region of TFRC recognized and/or bound by the reagent comprises the amino acid residues from position 201 to position 301 of TFRC, preferably from position 211 to position 291 of TFRC, more preferably from position 221 to position 281 of TFRC, even preferably from position 231 to position 271 of TFRC, even preferably from position 241 to position 261 of TFRC, even preferably from position 246 to position 256 of TFRC, even preferably from position 247 to position 255 of TFRC, even preferably from position 248 to position 254 of TFRC, even preferably from position 249 to position 253 of TFRC, wherein position numbering is with respect to the amino acid of SEQ ID NO: 1.

[0127] While TFRC is often found to be upregulated in cancer cells (including blood cancers), and while one would therefore expect MPNs cells to display higher rather than lower levels of surface TFRC, the Inventors surprisingly found that, in contrast, CALR mutant cells express lower level of TFRC than non-mutant cells for a given individual.

[0128] In some embodiments, the measured level is at or less the determined level when the measured level of TFRC is at least a 1.1-fold less compared to the determined level.

[0129] Within the scope of the present invention, “at least a 1.1-fold” means at least 1.1- fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30- fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold or more. [0130] In some embodiments, step (ii) of the method according to the invention comprises comparing the measured surface levels of TFRC with the determined surface levels of TFRC, wherein the determined surface levels of TFRC are known before the measurement of surface levels of TFRC.

[0131] Within the scope of the present invention, the “determined level” or “determined surface level” of TFRC, herein used interchangeably, refer to the standard or the norm of level of expression for TFRC at the surface of a given cell type. The determined levels may be known from the art, or measured on a healthy cell population of the same type as a MPN cell, preferably a CALR mutant positive MPN. In some embodiments, the determined level is a range of values defining a lower limit and an upper limit, beyond which the surface levels of TFRC correspond to an abnormal or pathological situation.

[0132] In some embodiments, the determined surface levels of TFRC are known from the state of the art, typically the determined surface levels of TFRC are inferior or equal to normal or healthy surface levels of TFRC. In some embodiments, normal or healthy surface levels of TFRC are comprised within a certain range.

[0133] In some embodiments, the determined surface levels of TFRC are established by measuring surface levels of TFRC on a healthy cell, population of cell or tissue. In some embodiments, the healthy cell, population of cell or tissue used to establish the determined surface levels of TFRC are of the same type (e.g., same cell type) as the biological sample used in step (i) of the method according to the invention. In some embodiments, the healthy cell, population of cell or tissue used to establish the determined surface levels of TFRC are obtained from a different subject than the biological sample used in step (i) of the method according to the invention. In some embodiments, the healthy cell, population of cell or tissue used to establish the determined surface levels of TFRC are obtain from at least one healthy subject, preferably more than one healthy subject. In some embodiments, the healthy cell, population of cell or tissue used to establish the determined surface levels of TFRC are obtained from healthy cells of the same subject as the biological sample used in step (i) of the method according to the invention. [0134] Within the scope of the present invention, a healthy subject is defined herein as an individual who does not suffer from cancer, blood cancer or MPN, preferably CALR mutant positive MPN. In some embodiments, “healthy cells” refers to cells of the same type (e.g., same cell type or lineage) as the CALR mutant positive MPN cells, in a biological sample. In some embodiments, the healthy cells do not comprise any mutation inducing a CALR mutant positive MPN, or associated with a CALR mutant positive MPN. In some embodiment, a healthy cell is not a CALR mutant positive MPN but shares the same cell type, functionalization and/or lineage, and are comprised in the same biological sample.

[0135] It is to be understood that the determined surface levels of TFRC, as defined within the scope of the present invention, do not necessarily reflect the normal or healthy surface levels of TFRC. In some embodiments, the determined surface levels of TFRC are inferior or equal to the lowest value the normal or healthy surface levels of TFRC. In some embodiments, the determined surface levels of TFRC are inferior to the lowest value the normal or healthy surface levels of TFRC. In some embodiments, the determined surface levels of TFRC are comprised with the lowest half, third, quarter or tenth of the value range for the normal or healthy surface levels of TFRC. In other words, the determined surface levels of TFRC according to the invention reflects the threshold below which the surface levels of TFRC correspond to an abnormal or pathological situation.

[0136] In some embodiments, step (iii) of the method according to the invention comprises concluding that the subject has CALR mutant positive MPN when the measured surface levels of TFRC are at or less then the determined surface level of TFRC. In some embodiments, step (iii) of the method according to the invention consists in providing a diagnostic. In some embodiments, step (iii) of the method according to the invention consists in assessing the severity of CALR mutant positive MPN.

[0137] In some embodiments, step (iii) of the method according to the invention is performed with the help of a computer. In some embodiments, step (iii) is automated. [0138] In some embodiments, the method further comprises the detection of at least one other surface marker.

[0139] In some embodiments, the at least one other surface marker is a blood cancer surface marker, preferably a MPN surface marker, more preferably a surface marker of a MPN comprising at least one CALR mutation (/.< ., a CALR mutant positive MPN).

[0140] In a preferred embodiment, the surface marker is selected in the group comprising CALR mutant, thrombopoietin receptor (also known as TPOR, MPL or CD110) mutant, Glucose 6-phosphate (G6P) and the like.

[0141] CALR mutants have been described hereinabove.

[0142] In some embodiments, the method further comprises the detection of TPOR mutant. In some embodiments, TPOR mutant comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the mutation of TPOR is selected from the group comprising or consisting of R102P, P106L, G509N, and K39N, preferably R102P, wherein the amino acid position is with respect to SEQ ID NO: 37.

[0143] In some embodiments, the method instead or further comprises the detection of G6P.

[0144] In a preferred embodiment, the detection of TFRC and the detection of the at least one other surface maker are performed concomitantly. In another embodiment, the detection of TFRC and the detection of the at least one other surface maker are performed separately.

[0145] In a preferred embodiment, the surface levels TFRC and of the at least one other surface marker are measured by using the same technique, e.g., the surface levels of TFRC and of the at least one other surface marker are both measured using flow cytometry. In another embodiment, the surface levels TFRC and of the at least one other surface marker are measured by using distinct techniques. [0146] It is to be understood that, regardless if the surface levels TFRC and of the at least one other surface marker are measured by using the same technique or distinct techniques, the reagents used to detect TFRC and the at least one other surface marker are distinct.

[0147] The present invention further relates to a method of separating healthy cells from a biological sample obtained from a subj ect with CALR mutant positive MPN, the method comprising the steps of:

(i) staining said biological sample with a substance that specifically binds to or reacts with Transferrin Receptor Protein 1 (TFRC) or a fragment thereof, wherein the substance binds to or reacts with TFRC or a fragment thereof on the healthy cells in said biological sample,

(ii) selecting the cells that bind to or react with the substance, thereby isolating a healthy cells population.

[0148] In some embodiments, the method comprises the steps of:

(i) contacting said biological sample with a substance that specifically binds to or reacts with Transferrin Receptor Protein 1 (TFRC) or a fragment thereof, wherein the substance binds to or reacts with TFRC or a fragment thereof on the healthy cells in said biological sample,

(ii) selecting the cells that bind to or react with the substance, thereby isolating a healthy cells population.

[0149] In some embodiments, TFRC is present on the cell surface.

[0150] In some embodiments, the MPN is Essential Thrombocythemia (ET) or Myelofibrosis (MF). In some embodiments, the MPN is ET. In some embodiments, the MPN is MF.

[0151] In some embodiments, the MPN is due to at least one Calreticulin (CALR) mutation. [0152] In some embodiments, the MPN is induced by (i) at least one CALR mutation and (ii) one or more mutation(s) in another gene(s). In some embodiments, the at least one CALR mutation amplify the effect of the one or more mutation(s) in other gene(s).

[0153] In one embodiment, the at least one CALR mutation and the one or more mutation(s) in other gene(s) occurs in the same cell. In another embodiment, the at least one CALR mutation and the one or more mutation(s) in other gene(s) occurs in separate cells or groups of cells.

[0154] CALR mutants have been described hereinabove.

[0155] In some embodiments, the substance that specifically binds to, or reacts with, TFRC or a fragment thereof is selected from the group comprising or consisting of antibodies or antigen binding fragments thereof, peptides, chemical probes or nucleic acid probes.

[0156] In a preferred embodiment, the substance does not bind to, or reacts with, any protein or peptide other than TFRC or fragment thereof.

[0157] In some embodiments, the antibody or antigen binding fragment thereof is an anti-TFRC antibody. In some embodiments, the antibody or antigen binding fragment thereof is an anti-TFRC antibody that recognized and/or bound a region of TFRC as described hereinabove.

[0158] Suitable methods for cell sorting are known in the art. In some embodiments, the method comprises the use of at least one technique selected in the group comprising or consisting of flow cytometry, immunoprecipitation and microfluidics.

[0159] In a preferred embodiment, the technique is flow cytometry. Fluorescence- activated cell sorting (FACS) is the flow cytometry application that is most commonly used in the art to perform cell sorting, although other variant may be used, such as, e.g., immunomagnetic cell sorting. Within the scope of the present invention, the use of flow cytometry is not intended to be limitative. [0160] In another embodiment, the technique is immunoprecipitation. In another embodiment, the technique is microfluidics.

[0161] The Inventors demonstrated that TFRC expression at the cell surface is strongly decreased in cells expressing a CALR mutant (see Figure 1 A-1B). Therefore, it is to be understood that healthy cell normally express TFRC at their surface and are therefore detected by the substance that specifically binds to or reacts with TFRC, while CALR mutant positive cancer cells (i.e., blood cancer cells, MPN cells and/or cells expressing a CALR mutant as defined hereinabove) express less TFRC so that cancer cells are not detected by the substance that specifically binds to or reacts with TFRC.

[0162] In some embodiments, the substance that specifically binds to, or reacts with, TFRC or a fragment thereof has a sensitivity that allows for the detection of healthy cells expressing normal surface levels of TFRC, but does not allow for the detection of cancer cells (i.e., blood cancer cells, MPN cells and/or cells expressing a CALR mutant as defined hereinabove). In some embodiments, the substance that specifically binds to, or reacts with, TFRC or a fragment thereof has a sensitivity that allows for the detection of healthy cells expressing normal surface levels of TFRC. In some embodiments, the substance that specifically binds to, or reacts with, TFRC or a fragment thereof has a sensitivity that does not allow for the detection of cancer cells (i.e., blood cancer cells, MPN cells and/or cells expressing a CALR mutant as defined hereinabove) expressing lower surface levels of TFRC compared to healthy cells.

[0163] As used herein, the term “sensitivity” represents the threshold below which a detection method will not enable the detection of the target, the target being TFRC or a fragment thereof.

[0164] In some embodiments, the method enables the obtention of two distinct cell populations, one population comprising or consisting of healthy cells, the other population comprising or consisting of cancer cells (i.e., blood cancer cells, MPN cells and/or cells expressing a CALR mutant as defined hereinabove).

[0165] The present invention further relates to a healthy cell population isolated by the isolation method according to the invention. [0166] In some embodiments, the healthy cell population is substantially free of contaminants, preferably cellular contaminants. In some embodiments, the healthy cell population is substantially free of contaminants with regard to cancer cells, preferably blood cancer cells, more preferably MPN cells, even more preferably CALR mutant positive MPN. In some embodiments, the healthy cell population is substantially free of contaminants with regard to cells comprising at least one mutation of CALR as described hereinabove.

[0167] In some embodiments, “substantially free of contaminants” means that the healthy cell population comprises less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001% or 0.0000001% of contaminants, preferably cellular contaminants. In some embodiments, the healthy cell population comprises less than 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001% or 0.0000001% of contaminants, preferably cellular contaminants.

[0168] In some embodiments, the healthy cell population comprises less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001% or 0.0000001% of cancer cells, preferably blood cancer cells, more preferably CALR mutant positive MPN cells. In some embodiments, the healthy cell population comprises less than 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001% or 0.0000001% of cancer cells, preferably blood cancer cells, more preferably CALR mutant positive MPN cells.

[0169] In some embodiments, less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001% or 0.0000001% of cells comprised in the healthy cell population carry a mutation of CALR as described hereinabove.

[0170] In some embodiments, the cells comprised in the healthy cell population express a CALR protein having an amino acid sequence that is not selected from the group comprising or consisting of SEQ ID NO: 4 to SEQ ID NO: 35. In some embodiments, the cells comprised in the healthy cell population express a CALR protein having an amino acid sequence that is not selected from the group comprising or consisting of SEQ ID NO: 4 and SEQ ID NO: 5. In some embodiments, the cells comprised in the healthy cell population express a CALR protein that does not have an amino acid sequence of SEQ ID NO: 4. In some embodiments, the cells comprised in the healthy cell population express a CALR protein that does not have an amino acid sequence of SEQ ID NO: 5.

[0171] In certain embodiments, the cells comprised in the healthy cell population express a CALR protein having at least 75% sequence similarity with the amino acid sequence SEQ ID NO: 2, wherein said CALR protein does not have an amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 4 to SEQ ID NO: 35. In certain embodiments, the cells comprised in the healthy cell population express a CALR protein having the amino acid sequence SEQ ID NO: 2.

[0172] In a preferred embodiment, the healthy cell population is isolated.

[0173] In some embodiments, the absence of cancer cells (i.e., blood cancer cells, MPN cells and/or cells expressing a CALR mutant as defined hereinabove) in the healthy cell population is confirmed by another method. In some embodiments, the other method is selected from the group comprising or consisting of sequencing, PCR and quantitative PCR.

[0174] In some embodiments, the healthy cell population is devoid of microbial contamination (e.g., contamination by bacteria, viruses, fungi, archaebacteria and the like).

[0175] In some embodiment the healthy cell population is stored in a suitable preservation medium at suitable temperature.

[0176] In some embodiment, the healthy cell population or part thereof is frozen at a temperature from about -20°C to about -200°C. In some embodiment the healthy cell population is flash-frozen in liquid nitrogen. In some embodiments, the healthy cell population is contacted with at least one cryoprotectant agent, such as, e.g. , glycerol, prior to freezing. [0177] In some embodiments, the healthy cell population is stored briefly in a suitable sterile biological buffer or medium at a temperature comprised between 0°C and 15°C.

[0178] In a less preferred embodiment, the healthy cell population is fixed. As used herein, “fixed” means that the healthy cell population has been contacted with at least one fixative agent, such as, e.g., paraformaldehyde.

[0179] In certain embodiments, the healthy cell population is cultured in a suitable medium. In certain embodiments, the healthy cell population is amplified in vitro.

[0180] The present invention further relates to the healthy cell population according to the invention, for use for immunotherapy, such as for stem cell transplantation.

[0181] In some embodiments, the healthy cell population is for use for cell transplantation. In a preferred embodiment, the healthy cell population is for use for autologous cell transplantation. In another embodiment, the healthy cell population is for use for heterologous cell transplantation.

[0182] In some embodiments, the healthy cell population is for use for cell transplantation in a subject in need thereof, wherein said subject suffers from blood cancer, preferably MPN, preferably MPN comprising at least one mutation of CALR as described hereinabove (/.< ., CALR mutant positive MPN).

[0183] The present invention further relates to a method for treating CALR mutant positive MPN in a subject in need thereof, comprising the steps of:

(i) separating and isolating healthy cells from a biological sample obtained from the subject using the method according to the invention, and

(ii) performing an autologous stem cell transplantation of the isolated healthy cells population in said subject.

[0184] In some embodiments, the MPN is Essential Thrombocythemia (ET) or Myelofibrosis (MF). In some embodiments, the MPN is ET. In some embodiments, the MPN is MF. [0185] In a preferred embodiment, the MPN is due to at least one Calreticulin (CALR) mutation (i.e., a CALR mutant positive MPN).

[0186] In some embodiments, the MPN is induced by (i) at least one CALR mutation and (ii) one or more mutation(s) in another gene(s). In some embodiments, the at least one CALR mutation amplify the effect of the one or more mutation(s) in other gene(s).

[0187] In one embodiment, the at least one CALR mutation and the one or more mutation(s) in other gene(s) occurs in the same cell. In another embodiment, the at least one CALR mutation and the one or more mutation(s) in other gene(s) occurs in separate cells or groups of cells.

[0188] In some embodiment, the at least one CALR mutation is as described hereinabove.

[0189] In some embodiments, the healthy cells are a substantially pure healthy cell population, preferably an isolated healthy cell population, as defined hereinabove. In some embodiments, the healthy cells are a healthy cell population according to the invention.

[0190] In some embodiments, the healthy cells are prepared in order to be suitable for transplantation in the subject. In some embodiments, the healthy cells are comprised in a composition suitable for transplantation in the subject. In some embodiment, all components of the composition are safe for human administration, /.< ., the composition does not induce any adverse effect in the subject.

[0191] In some embodiments, the healthy cells or the composition are free of microbial contamination, e.g., bacterial, viral, fungal contamination and the like. In some embodiments, the composition further comprises antibiotic, antiviral agents, antifungal agents or combinations thereof.

[0192] In some embodiments, the healthy cells are comprised in a suitable medium, wherein said medium is in the form of a liquid medium or a biocompatible gel. In some embodiment, the medium maintains the healthy cells alive. In some embodiment, the medium comprises nutrients. In some embodiment, the medium maintains a suitable osmotic pressure. In some embodiment, the medium, preferably in the form of a biocompatible gel, provides mechanical support to the healthy cells. In some embodiments, the medium is comprised in the composition.

[0193] In some embodiment, the medium or the composition further comprises one or more agents that are useful for the transplantation, selected from the group comprising or consisting of anti-inflammatory agents, analgesic agents, antipyretic agents, immunosuppressive agents, sedatives, and combinations thereof.

[0194] In some embodiments, the transplantation is performed in the context of a medical intervention, e.g., a surgery, by a medical practitioner. In some embodiments, the transplantation is performed in sterile conditions.

[0195] In some embodiments, the method further comprises the step of eliminating the cancer cells (i.e., blood cancer cells, MPN cells and/or cells expressing a CALR mutant as defined hereinabove) in the subject prior to the transplantation.

[0196] In some embodiments, cancer cells are selectively eliminated in the subject.

[0197] It is to be understood that the selective elimination of cancer cells, while preferable, is not always possible. Thus, in some embodiments, all cells sharing the same cell type as the cancer cells are eliminated in the subject. In some embodiments, the bone marrow of the subject is removed entirely.

[0198] In some embodiments, the cells or tissue (e.g. , bone marrow) are reconstituted by the healthy cell population. In some embodiments, the cells or tissue (e.g., bone marrow) are reconstituted within 1 day, 1 week, 1 month, 1 year or more after the transplantation. The follow-up of the subject well-being is typically performed by a medical practitioner.

[0199] In some embodiments, the method comprises transplanting in said subject a number of healthy cells comprised from about 100 to about 10 ⁹ healthy cells, from about 100 to about 10 ⁸ healthy cells, from about 100 to about 10 ⁷ healthy cells, from about 100 to about 10 ⁶ healthy cells, from about 100 to about 10 ⁵ healthy cells, from about 100 to about 10 ⁴ healthy cells, from about 100 to about 10 ³ healthy cells. In some embodiments, the method comprises transplanting in said subject a number of healthy cells comprised from about 10 ³ to about 10 ⁹ healthy cells, from about 10 ⁴ to about 10 ⁹ healthy cells, from about 10 ⁵ to about 10 ⁹ healthy cells, from about 10 ⁶ to about 10 ⁹ healthy cells, from about 10 ⁷ to about 10 ⁹ healthy cells, from about 10 ⁸ to about 10 ⁹ healthy cells. Methods to count cells in a cell population are widely known in the art and typically involves the use of an automatic cell counter.

[0200] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of at least one anticancer agent.

[0201] As used herein, the term “anticancer agent” comprises, e.g., antitumoral peptides and proteins, pharmacological agents and drugs for treating cancer, and anticancer vaccines.

[0202] Anticancer agents are known from the state of the art. Non-limitative examples of anticancer agents include acalabrutinib, alectinib, alemtuzumab, anastrozole, avapritinib, avelumab, belinostat, bevacizumab, bleomycin, blinatumomab, bosutinib, brigatinib, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin copanlisib, cytarabine, daunorubicin, decitabine, dexamethasone, docetaxel, doxorubicin, encorafenib, erdafitinib, etoposide, everolimus, exemestane, fludarabine, 5-fluorouracil, gemcitabine, ifosfamide, imatinib Mesylate, leuprolide, lomustine, mechlorethamine, melphalan, methotrexate, mitomycin, nelarabine, paclitaxel, pamidronate, panobinostat, pralatrexate, prednisolone, ofatumumab, rituximab, temozolomide, topotecan, tositumomab, trastuzumab, vandetanib, vincristine, vorinostat, zanubrutinib, and the likes.

[0203] By “therapeutically effective amount”, it is meant a level or amount that is necessary and sufficient for slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of CALR mutant positive MPN; or alleviating the symptoms of CALR mutant positive MPN; or curing CALR mutant positive MPN, without causing significant negative or adverse side effects to the individual. In certain embodiments, an effective amount of the anticancer agent may range from about 0.001 mg to about 3,000 mg, per dosage unit, preferably from about 0.05 mg to about 1,000 mg, per dosage unit. [0204] In some embodiments, the method for detecting CALR mutant positive MPN in a subject according to the invention is performed after the transplantation in order to control if the method for treating CALR mutant positive MPN according to the invention was successful, wherein after means 1 day, 1 week, 1 month, 1 year or more after the transplantation.

[0205] The present invention further relates to a reagent for detecting healthy cells in a biological sample obtained from a subject with a CALR mutant positive MPN, comprising a substance that specifically binds to or reacts with Transferrin Receptor Protein 1 (TFRC) or a fragment thereof, wherein the substance that binds to or reacts with TFRC or a fragment thereof on the healthy cells in said biological sample.

[0206] In a preferred embodiment, the substance is suitable for use in at least one of the techniques selected from the group comprising or consisting of flow cytometry, immunostaining, cell surface biotinylation assay, cellular fragmentation, immunoprecipitation and affinity binding. In a more preferred embodiment, the substance is suitable for use in flow cytometry.

[0207] In a preferred embodiment, the substance is an antibody or antigen binding fragment thereof.

[0208] In a preferred embodiment, the antibody or antigen binding fragment thereof specifically detects TFRC, /.< ., in a preferred embodiment, the antibody or antigen binding fragment thereof does not recognize any protein or peptide other than TFRC. In a preferred embodiment, the antibody or antigen binding fragment thereof detects specifically human TFRC, wherein human TFRC typically has the amino acid sequence as set forth in SEQ ID NO: 1.

[0209] In some embodiments, the antibody or antigen binding fragment thereof is an anti-TFRC antibody. Non-limitative examples of anti-TFRC antibodies include clone CY1G4 (BioLegend® ref. 334107).

[0210] In some embodiments, the antibody or antigen binding fragment thereof is of an isotype selected from the group comprising or consisting of immunoglobulin (Ig) A, IgD, IgE, IgG, IgM, IgY, IgW, IgT and IgZ. In a preferred embodiment, the antibody or antigen binding fragment thereof is of an isotype selected from the group comprising or consisting of immunoglobulin IgA, IgD, IgE, IgG and IgM. In a less preferred embodiment, the antibody or antigen binding fragment thereof is of an isotype selected from the group comprising or consisting of IgY, IgW, IgT and IgZ.

[0211] In some embodiments, the antibody or antigen binding fragment thereof is monoclonal or polyclonal. In some embodiments, the antibody or antigen binding fragment thereof is monoclonal. In some embodiments, the antibody or antigen binding fragment thereof is polyclonal.

[0212] In some embodiments, the antibody or antigen binding fragment thereof is an anti-TFRC antibody that recognized and/or bound a region of TFRC as described hereinabove.

[0213] In some embodiments, the antibody or antigen binding fragment thereof is a nanobody.

[0214] In some embodiments, the substance is a peptide.

[0215] In some embodiments, the peptide possesses chemical properties that confers the peptide the ability to bind at least one amino acid region on TFRC.

[0216] In some embodiments, the peptide is a fragment of TFRC, /.< ., the peptide consists of an amino acid sequence comprised in TFRC. In some embodiments, the peptide is a modified fragment of TFRC, z.e., the peptide consists of an amino acid sequence comprised in TFRC and further comprises at least one amino acid mutation. In some embodiments, the peptide consists of an amino acid sequence that is not comprised on TFRC.

[0217] In some embodiments, the substance is a chemical probe. In a preferred embodiment, the chemical probe is organic. In another embodiment, the chemical probe is inorganic.

[0218] In some embodiments, the substance is a nucleic acid probe. [0219] In some embodiments, the nucleic acid probe is a DNA or RNA molecule. In some embodiments, the nucleic acid probe is a DNA molecule. In some embodiments, the nucleic acid probe is a RNA molecule.

[0220] In some embodiments, the nucleic acid probe is single stranded or double stranded. In some embodiments, the nucleic acid probe is single stranded. In some embodiments, the nucleic acid probe is double stranded

[0221] In some embodiments, the nucleic acid probe comprises at least one unnatural nucleotide.

[0222] In some embodiments, the substance is coupled to another molecule.

[0223] In some embodiments, said molecule is selected from the group comprising or consisting of fluorescent or fluorogenic molecules, enzymes, radiolabeled molecules or chromogenic molecules. In some embodiments, said molecule is selected from the group comprising or consisting of fluorescent or fluorogenic molecules, and chromogenic molecules.

[0224] In a preferred embodiment, said molecule is a fluorescent or fluorogenic molecule. In some embodiments, fluorescent or fluorogenic molecule is selected from the group comprising or consisting of fluorophores, fluorescent proteins and organic dyes.

[0225] In some embodiments, fluorescent or fluorogenic molecule is a fluorophore. Non-limitative examples of commercially available fluorophores include Alexa Fluor dyes or Cy dyes.

[0226] In some embodiments, fluorescent or fluorogenic molecule is a fluorescent protein. Non-limitative examples of fluorescent protein include the Green Fluorescent protein (GFP), the Red Fluorescent Protein (RPF) or mCherry.

[0227] In some embodiments, fluorescent or fluorogenic molecule is an organic dye. Non-limitative examples of organic dyes include fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate. [0228] In some embodiments, the nucleic acid probe is covalently or non-covalently coupled to the other molecule, preferably the nucleic acid probe is covalently coupled to the other molecule.

[0229] In some embodiments, the region of TFRC recognized and/or bound by the substance comprises the asparagine (Asn) residue at position 251 of TFRC, wherein position numbering is with respect to the amino acid of SEQ ID NO: 1.

[0230] In some embodiments, the asparagine residue is glycosylated, preferably the glycosylation is immature.

[0231 ] The present invention further relates to a method for screening a therapeutic agent for the treatment of CALR mutant positive MPN comprising the steps of:

(a) culturing cells with a candidate substance; and

(b) determining the effect of the candidate substance on increasing the level of Transferrin Receptor Protein 1 (TFRC) on the surface level of said cells.

[0232] In some embodiments, the cultured cells comprise at least one mutation on CALR as described hereinabove. In some embodiments, the cultured cells express a CALR mutant having an amino acid sequence having at least 75% sequence similarity with the amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 4 to SEQ ID NO: 35, wherein the sequence is not 100% identical to SEQ ID NO: 2. In some embodiments, the cultured cells express a CALR mutant having an amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 4 to SEQ ID NO: 35. In a preferred embodiment, the cultured cells express a CALR mutant having the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In a preferred embodiment, the cultured cells express a CALR mutant having the amino acid sequence of SEQ ID NO: 4. In a preferred embodiment, the cultured cells express a CALR mutant having the amino acid sequence of SEQ ID NO: 5.

[0233] In some embodiments, 100% of the cultured cell express a mutant of CALR, i.e., the cultured cells are an isolated population of mutant cells. [0234] In some embodiments, the cultured cells are an heterogenous cell population. In some embodiments, the heterogenous cell population comprises healthy cells and cells expressing at least one mutation on CALR. In a less preferred embodiments, the heterogenous cell population further comprises other cell types.

[0235] In some embodiments, the therapeutic agent for the treatment of CALR mutant positive MPN is selected from the group comprising or consisting of pharmaceutical agents (including small molecules), antibodies (including antibody fragments or nanobodies), proteins, polypeptides, lipids, lipoproteins, sugars, glycoproteins, polymers, nucleic acids (including DNA and RNA), hormones, or combinations thereof.

[0236] In some embodiments, the MPN is Essential Thrombocythemia (ET) or Myelofibrosis (MF). In some embodiments, the MPN is ET. In some embodiments, the MPN is MF.

[0237] In some embodiments, the MPN is due to at least one Calreticulin (CALR) mutation (/.< ., a CALR mutant positive MPN).

[0238] In some embodiments, the MPN is induced by (i) at least one CALR mutation and (ii) one or more mutation(s) in another gene(s). In some embodiments, the at least one CALR mutation amplify the effect of the one or more mutation(s) in other gene(s).

[0239] In one embodiment, the at least one CALR mutation and the one or more mutation(s) in other gene(s) occurs in the same cell. In another embodiment, the at least one CALR mutation and the one or more mutation(s) in other gene(s) occurs in separate cells or groups of cells.

[0240] In some embodiment, the at least one CALR mutation is as described hereinabove.

[0241] In some embodiments, the method further comprises performing the method concomitantly on healthy cells. It is to be understood that the healthy cells are intended to be used as control, to assess, e.g., safety and/or side effects of the therapeutic agent. [0242] In some embodiments, the therapeutic agent for the treatment of CALR mutant positive MPN is determined as efficient when it restores the surface levels of TFRC in a cell population. By “restores the surface levels”, it is meant that the surface levels are increased up to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the surface levels of TFRC of healthy cells. In some embodiments, he therapeutic agent for the treatment of CALR mutant positive MPN is determined as efficient when it restores the surface levels of TFRC to about the same levels measure in healthy cells, in a cell population.

[0243] In some embodiments, the therapeutic agent for the treatment of CALR mutant positive MPN is determined as efficient when it eliminates at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the CALR mutant positive MPN cells, preferably MPN cells, more preferably cells expressing at least on mutation on CALR. By “eliminates” it is meant that the cells are dead or have triggered an apoptotic cascade. Methods to evaluate cell death are widely known in the art and comprise, e.g., vital stains.

[0244] In some embodiments, the method further comprises the use of a high throughput screening technique selected from the group comprising or consisting of transcriptomics, proteomics, metabolomics, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

[0245] Figure 1A-1H is a combination of graphs and plots showing that the Transferrin Receptor 1 cell surface expression is downregulated in CALR mutant expressing cells. Representative flow-cytometry analysis of cell surface expression of transferrin receptor 1 on murine pro-B cell lines (Ba/F3) (Fig. 1A), in megakaryoblastic human cell line (UT- 7/Tpo) (Fig. IB), primary mouse PBMC (Fig. 1C) and lineage positive mouse bone marrow cells (Fig. ID). Fig. IE shows mean fluorescence intensity (MFI) of cell surface TFRC from lineage positive bone marrow cells. Data represents mean +/- SD of 2-3 mice per condition. Fig. IF shows selection of LSK cells from lineage negative bone marrow cell. Fig. 1G shows representative flow-cytometry analysis of cell surface expression of transferrin receptor 1 on murine LSK. Fig. 1H shows mean fluorescence intensity (MFI) of cell surface TFRC from LSK cells. Data represents mean +/- SD of 2-3 mice per condition.

EXAMPLES

[0246] The present invention is further illustrated by the following example.

Cell surface TFRC is downregulated in CALR mutant positive cells.

Materials and Methods

[0247] HEK293T were from the American Type Culture Collection (ATCC). The Ba/F3 cell line (IL-3 dependent pro-B cells) were from the Ludwig Institute for Cancer Research, Brussels Branch. HEK293T were cultured in DMEM culture medium supplemented with 10% fetal bovine serum. Ba/F3 were cultured in RPMI culture medium supplemented with 10% fetal bovine serum and 0.5ng/ml of murine IL-3. The addition of murine IL-3 was omitted for growth experiments. UT-7/Tpo cell lines were described in Jia et al. (Blood, 2021, 137(14): 1920-1931). UT-7/Tpo CALR del52 cell lines were obtained by introducing the CALR mutation corresponding to SEQ ID NO: 3 in the endogeneous CALR gene (SEQ ID NO: 2) by CRISPR-Cas9. The cells were cultured in DMEM culture medium supplemented with 10% fetal bovine serum with 5ng/ml of human GM-CSF. Murine primary PBMCs were isolated from C57BL6 mice wild-type or CALR del52 knock-in described in Balligand et al. (Leukemia, 2020, 34(2):510-521). LSK cells are defined as cells lin-, Scal+, Kit-.

[0248] For flow cytometry analysis, samples were stained with PE-coupled anti-mouse TFRC (BioLegend, clone RI7217) or APC-coupled anti-human TFRC Biolegend, Clone CY1G4). Data was acquired on a BD FACS Verse flow cytometer and analysed with Flow Jo.

Results

[0249] Cell surface flow-cytometry was used to measure the surface expression of TFRC in Ba/F3 that were either retrovirally transduced with CALR del52 or expressing CALR mutants via the endogenous promotor which were engineered by CRISPR-Cas9. Strikingly, the cell surface expression of TFRC was strongly reduced in transduced cell lines and virtually inexistant in CRISPR cells (Figure 1A). This very strong downregulation of cell surface TFRC could thus be used as a selective marker to identify cells that express CALR mutants.

[0250] To further validate these findings, this pattern was reproduced in megakaryocytic human cell lines (UT-7/Tpo) that were modified by CRISPR for CALR del52 and in primary peripheral blood mononuclear cells (PBMCs) and lineage positive bone marrow cells (BMC) from our CALR del52 KI mouse models (Figure 1B-1E). In bone marrow cells, populations were subdivided between lineage positive population and lineage negative (progenitors) population. In the latter, the cell surface TFRC expression was analyzed in LSK (Lin-, Scal+, Kit+) that contain the earliest progenitors that are able to re-populate the bone marrow upon transplantation. Remarkably, the differential TFRC cell surface expression was conserved in these early progenitors (Figure 1E-1H). This finding strongly confirms that the TFRC is a suitable and robust selective marker to detect and select cells from the clone that express CALR mutant proteins. In addition, the fact that this selective marker is conserved in early progenitors suggests it can be used as a tool to separate healthy from malignant cells from an individual prior to autologous transplantation.