Methods and means for the diagnosis and risk stratification of juvenile myelomonocytic leukemia

Abstract

This invention relates to the diagnosis and evaluation of juvenile myelomonocytic leukemia (JMML). In particular, the invention relates to a method for diagnosing JMML in a subject, comprising: a) at least one biomarker present on or in hematopoietic stem cells and progenitor cells (HSPCs) in a biological sample, i) CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP The present invention relates to a method comprising the steps of: 1) determining the amount of at least one biomarker selected from each of the following groups: 1) Group I consisting of CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34; and 2) Group II consisting of IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G; b) comparing the determined amount in step a) with a reference; and c) diagnosing JMML based on the comparison in step b). Furthermore, the present invention relates to a method for classifying subjects suffering from JMML into a low- or high-risk JMML group. Furthermore, the present invention relates to the use of at least one biomarker present on or in HSPC in a biological sample for diagnosing JMML to a low- or high-risk JMML group in subjects who have JMML or are at risk of developing it. Furthermore, the present invention relates to a kit for diagnosing JMML in a subject or for classifying a subject suffering from JMML into a low- or high-risk JMML group.Furthermore, the present invention relates to an inhibitor for use in the treatment and / or prevention of JMML that specifically inhibits at least one biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G, which are present on or in hematopoietic stem cells and progenitor cells (HSPCs). The present invention further relates to a pharmaceutical composition for use in the treatment and / or prevention of JMML, comprising at least two inhibitors according to the present invention. Finally, the present invention envisions a method for treating and / or preventing JMML.

Description

[Technical Field]

[0001] This invention relates to the diagnosis and evaluation of juvenile myelomonocytic leukemia. In particular, the present invention relates to a method for diagnosing JMML in a subject, comprising: a) determining the amount of at least one biomarker present on or in hematopoietic stem cells and progenitor cells (HSPCs) in a biological sample, i) selected from each of Group I consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34, and ii) determining the amount of Group II consisting of IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G. The present invention relates to a method comprising the steps of: b) comparing the determined amount from step a) with a reference, and c) diagnosing JMML based on the comparison from step b). Furthermore, the present invention relates to a method for classifying subjects suffering from JMML into a low- or high-risk JMML group. Furthermore, the present invention relates to the use of at least one biomarker present on or in HSPC in a biological sample for diagnosing JMML into a low- or high-risk JMML group in subjects who have JMML or are at risk of developing it. Furthermore, the present invention relates to a kit for diagnosing JMML in a subject or classifying subjects suffering from JMML into a low- or high-risk JMML group.Furthermore, the present invention relates to an inhibitor for use in the treatment and / or prevention of JMML that specifically inhibits at least one biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G, which are present on or in hematopoietic stem cells and progenitor cells (HSPCs). The present invention further relates to a pharmaceutical composition for use in the treatment and / or prevention of JMML, comprising at least two inhibitors according to the present invention. Finally, the present invention envisions a method for treating and / or preventing JMML. [Background technology]

[0002] Juvenile myelomonocytic leukemia (JMML) is a rare, aggressive hematological cancer that occurs in early childhood, with a median age of less than 2 years at diagnosis (Niemeyer et al., 1997, Blood). According to the latest WHO classification, JMML is defined as a myeloproliferative neoplasm (MPN) (Khoury et al., 2022 Leukemia) and shares some features with chronic myelomonocytic leukemia in adults. The disorder originates from pluripotent hematopoietic stem cells / progenitor cells and is characterized by the overproduction of mature and immature myeloid cells. Hyperactive RAS signaling is presumed to be the main promoting factor in JMML. The vast majority of patients (approximately 90%) carry somatic mutations in the KRAS, NRAS, PTPN11, NF1, and / or CBL genes, defining genetically and clinically distinct subtypes (Schonung et al., Clin Cancer Res. 2021 Jan 1; 27(1):158-168).

[0003] JMML is also primarily characterized by its heterogeneity. For example, the clinical course of the disease is highly heterogeneous and can only be partially predicted using clinical features. This clinical heterogeneity is reflected in therapeutic considerations ranging from observation to early allogeneic hematopoietic stem cell transplantation (HSCT) (Niemeyer & Flotho 2019, Blood). The disease eventually resolves spontaneously in about 15% of cases, but more than 50% of patients relapse after HSCT (Locatelli et al., 2005 Blood).

[0004] This clinical heterogeneity highlights the urgent need for diagnostic techniques that enable the direct identification of high-risk patients. Furthermore, there is a need to develop and test novel, risk-based, molecularly targeted treatment options. On the one hand, clinical studies on JMML have identified gene subgroups defined by driver mutations that lead to hyperactivation of the RAS pathway (Neubauer et al., 1991 Blood; Flotho et al., 1999 Leukemia). On the other hand, gene expression-related signatures have revealed various non-genetic features in JMML (Bresolin et al., 2010 Journal of Clinical Oncology; Helsmoortel et al., 2016 Blood). While both genetic and non-genetic features have been shown to carry some predictive power, these biomarkers have not been able to summarize all the clinical heterogeneity observed in JMML. As a result, clinical studies on JMML have long lacked meaningful and robust prognostic biomarkers.

[0005] The missing links between genetic and non-genetic signatures for a detailed analysis of clinical heterogeneity in JMML were considered to be either gene regulatory programs or epigenetic programs, respectively. Indeed, stratification of JMML patients by DNA methylation patterns demonstrated a significant correlation between epigenetics and prognosis in JMML (Olk-Batz et al., 2011, Blood). The use of genome-wide array-based DNA methylation analysis established epigenetic subgroups in JMML (Lipka et al., 2017, Nature Communications; Murakami et al., 2018, Blood; Stieglitz et al., 2017, Nature Communications), and DNA methylation was shown to be the only significant factor predicting overall survival in JMML. Furthermore, DNA methylation appears to predict treatment response. Treatment with hypomethylating agents was considered particularly effective in low- and intermediate-risk DNA methylation subgroups (Niemeyer et al., 2021 Blood Advances). However, given this weak response to low-methylating agents and the high relapse rate after HSCT, there is a lack of treatment options that can target high-risk JMML (Loh 2011, British Journal of Haematology).

[0006] Preclinical studies using patient-derived xenograft mouse models have shown that transplanted JMML cells can reconstitute the entire hematopoietic system (Lapidot et al., 1996 Blood; Iversen et al., 1997 Blood; Krombholz et al., 2016 Haematologica; Krombholz et al., 2019 Leukemia). Furthermore, DNA methylation patterns characteristic of epigenetic subgroups in JMML were re-established in xenograft mice. This suggests that hematopoietic stem cells (HSCs) can be considered leukemia-initiating cells in JMML, gradually increasing interest in this cell population in the field (Louka et al., 2021 Journal of Experimental Medicine). Currently, clinical outcomes can only be adequately explained by clinical or genetic features, but dysregulation of DNA methylation status has been shown as a non-random, subgroup-specific prognostic feature. To date, there is no systematic and subgroup-stratified characterization of HSPCs in JMML.

[0007] In principle, JMML is a rare, invasive clonal neoplasm (DeVos 2022, Mayerhofer 2021). The incidence of JMML is estimated at 1.3 cases per million children per year in children aged 0-14 years (WHO tumor classification). Therefore, in contrast to other neoplasms, the number of patient samples available is very limited. Furthermore, samples biobanked into the European JMML Biobank are typically DNA samples rather than living frozen cells. Nevertheless, there is still a need for treatment for childhood cancers, especially invasive cancers such as JMML that occur in infancy, for which treatment options are currently limited (Laetsch 2021).

[0008] Thus, prognostic and diagnostic tools are needed for risk stratification of JMML and for the development of novel subgroup-specific therapies. [Overview of the Initiative]

[0009] The technical problem underlying the present invention can be seen as providing means and methods that meet the above-mentioned needs. This technical problem is solved by the embodiments characterized below in the claims and in the present specification.

[0010] The present invention is a method for diagnosing juvenile myelomonocytic leukemia (JMML) in a subject, comprising: a) determining the amount of at least one biomarker on hematopoietic stem and progenitor cells (HSPCs) in a biological sample, wherein the at least one biomarker is selected from each of: i) Group I consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34, and ii) Group II consisting of IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G; b) comparing the determined amount in step a) with a reference; and c) diagnosing JMML based on the comparison in step b).

[0011] It relates to a method. It should be understood that in this specification and in the claims, "a" or "an" may mean one or more of the items referred to below, depending on the context in which it is used. Thus, for example, a reference to "a" item may mean that at least one item can be used.

[0012] ​When used hereinafter, the terms "have", "comprise", or "include" are used in a non-exclusive manner. Thus, these terms may refer to both situations where, in addition to the features introduced by these terms, no further features are present in the entity described in this context, and situations where one or more further features are present. As an example, the expressions "A has B", "A comprises B", and "A includes B" may refer to both situations where, in addition to B, no other elements are present in A (i.e., the situation where A consists solely and exclusively of B), and situations where, in addition to B, one or more further elements, such as element C, element C and element D, or further elements are present in entity A.

[0013] The terms "in particular", "more specifically", "typically", and "more typically" or similar terms are used with additional and / or alternative features without limiting alternative possibilities. Thus, the features introduced by these terms are additional and / or alternative features and are not intended to limit the scope of the claims in any way. As will be recognized by those skilled in the art, the present invention may be implemented by using further alternative features. Similarly, features introduced by "in an embodiment of the present invention" or similar expressions are intended to be additional and / or alternative features, without any limitation regarding alternative embodiments of the present invention, without any limitation regarding the scope of the present invention, and without any limitation regarding the possibility of combining the features thus introduced with other additional and / or alternative or non-additional and / or alternative features of the present invention.

[0014] Furthermore, when used hereinafter, the terms "preferably", "more preferably", "most preferably", "in particular", "more particularly", "typically", and "more typically" are used with features indicating that these features are preferred features, i.e., these terms indicate that alternative features can also be envisaged in accordance with the present invention.

[0015] Furthermore, the term "at least one" as used herein means that one or more items referred to after this term may be used in accordance with the invention. For example, where this term indicates the use of at least one item, this can be understood as one item or more items, i.e., two, three, four, five, or any other number of items. Depending on the item that this term refers to, a person skilled in the art will understand the upper limit of what the item may refer to, if any.

[0016] As used herein, the term "about" means that, with respect to any number following the term, the interval accuracy exists within a range that allows for the achievement of the technical effect. Therefore, in the context of this invention, the term "about" means ±20%, ±10%, ±5%, ±2%, or ±1% from the indicated parameter or value. This also takes into account normal deviations caused by measurement techniques, etc.

[0017] The method of the present invention may consist of the steps described above, or may include additional steps such as a step for further evaluation of the evaluation obtained in step (c), a step for recommending therapeutic means such as treatment, or the like. Furthermore, it may include a step prior to step (a), such as a step relating to the pretreatment of a sample. However, preferably, the method is envisioned to be an ex vivo method that does not require any steps performed on the human or animal body. Furthermore, the method is assisted by automation. Typically, the determination of biomarkers can be assisted by robotic equipment, while comparison and evaluation can be assisted by data processing equipment such as a computer.

[0018] As used herein, the term “diagnosing” means assessing the health status of a subject. Therefore, as used herein, “diagnosing” means determining whether a subject has juvenile myelomonocytic leukemia (JMML), predicting the risk of developing JMML, and / or, in particular, predicting any deterioration of the subject’s health status with respect to the signs and symptoms associated with JMML. The most common signs and symptoms of JMML include hepatosplenomegaly, lymphadenopathy and anemia, and thrombocytopenia, each resulting in pallor, fatigue, weakness and bleeding, and / or bruising. Patients may also present with bone and joint pain, abdominal pain, recurrent fever, infection, and / or dry cough and dyspnea.

[0019] As those skilled in the art will understand, such diagnoses are usually preferably accurate for 100% of the subjects surveyed, but this may not always be the case. However, this term requires that a statistically significant portion of the subjects can be accurately diagnosed. Those skilled in the art can determine whether a portion is statistically significant without further work using various well-known statistical evaluation tools, such as confidence interval determination, p-value determination, Student's t-test, and Mann-Whitney U test. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Typically, the assumed confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. The p-values ​​are typically 0.2, 0.1, or 0.05. It should be understood that the diagnostic method of the present invention assists physicians in making a final diagnosis. A physician may also consider further factors to increase the accuracy of their diagnosis, such as the presence of symptoms of JMML as described elsewhere in this specification.

[0020] The term “subject” as used herein may be an animal, preferably a mammal. More preferably, the subject is a human. The subjects investigated by the method of the present invention are preferably young subjects, i.e., children or young adults. More specifically, the subjects are at most 16 years old, at most 15 years old, at most 14 years old, at most 13 years old, at most 12 years old, at most 11 years old, at most 10 years old, at most 9 years old, at most 8 years old, at most 7 years old, or at most 6 years old, at most 5.5 years old, at most 5 years old, at most 2.5 years old, at most 2 years old, at most 1.5 years old, at most 1 year old, at most 6 months old, or less than 6 months old.

[0021] The subjects investigated by the method of the present invention are subjects who have JMML or are at risk of developing it. As used herein, having JMML means that the subject exhibits the clinical parameters, signs and / or symptoms of JMML. Thus, the subjects according to the present invention are typically subjects who have JMML or are suspected of having JMML. As used herein, being at risk of developing JMML means an apparently healthy subject who does not yet exhibit the clinical signs or symptoms of JMML but is at increased risk of developing JMML, or a subject who exhibits mild signs or symptoms of the disease but is at risk of developing the disease or an exacerbation of related signs or symptoms.

[0022] The term “biomarker” means, according to the present invention, the presence, absence, or abundance of a biomolecule that indicates a health status. According to the present invention, such health status may be the absence of JMML or JMML, low-risk JMML, and / or high-risk JMML. The biomarker according to the present invention may be a protein or fragment thereof selected from proteins that are mentioned in more detail elsewhere herein. Furthermore, the biomarker may be the presence, absence, or abundance of a transcribed nucleic acid molecule that can be used as a substitute for a protein. Preferably, such a transcribed nucleic acid molecule is any precursor or variant thereof, including a messenger RNA molecule (mRNA) or mRNA for premRNA or splice variants. These RNA nucleic acid molecules may also be determined as biomarkers according to the present invention. Thus, for example, when CD52 is determined as a biomarker according to the present invention, it is understood that either the CD52 protein or a transcribed nucleic acid molecule encoding the CD52 protein, such as CD52 mRNA, can be determined. The same applies to all other biomarkers described herein, except those otherwise specified. In this specification, it refers to proteins, but those skilled in the art will be well aware of the transcribed nucleic acid molecules and encoding genes belonging to such proteins.

[0023] The biomarkers used in accordance with the present invention include biomarkers associated with high-risk JMML or low-risk JMML.

[0024] High-risk JMML biomarkers are those of Group I as referred to in accordance with the present invention, and are selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34. Preferably, the high-risk JMML biomarker is of Group I as referred to in accordance with the present invention, and is selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34. Preferably, the high-risk JMML biomarker is of group I as referred to in accordance with the present invention, and is selected from the group consisting of CD52, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, CD82, IGHM, RALA, HLA-DRA, SELL, CLEC7A, CLEC9A, and HCST. More preferably, the high-risk biomarker of group I is selected from the group consisting of CD52, CD69, CD164, IGHM, RALA, and HLA-DRA.

[0025] Low-risk JMML biomarkers are those of Group II as referred to in the present invention, and are selected from the group consisting of IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G. More preferably, low-risk biomarkers of Group II are selected from the group consisting of IGLL1 and HLA-G.

[0026] Thus, in order to diagnose JMML in a subject, it is necessary to determine at least one biomarker derived from the previously mentioned high-risk JMML biomarkers (Group I) and at least one biomarker derived from the previously mentioned low-risk JMML biomarkers (Group II). More preferably, all of the above biomarkers from either Group I or Group II may be determined. This can improve the diagnosis of JMML, including all disease stages from low-risk to high-risk.

[0027] As used herein, the term "CD52" refers to the group of 52 glycoproteins of differentiation antigens encoded by the CD52 gene in humans, also known as the CAMPATH-1 antigen. CD52 is typically localized on the surface of mature lymphocytes, monocytes, and dendritic cells. It is involved in the positive regulation of cytoplasmic calcium ion concentration. CD52 is a 61-amino acid peptide fixed to glycosylphosphatidylinositol (GPI). It is highly negatively charged and present on spermatidycetes and lymphocytes, so it is presumed to function as an anti-adhesion protein that allows cells to move freely. Several orthologues of CD52 have been reported in various animal species.

[0028] The CD52 protein referred to in accordance with the present invention is preferably human CD52 having the amino acid sequence deposited as UniProt accession number P31358. The term “CD52” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the CD52 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the human CD52 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the particular amino acid sequence of the said CD52 protein.

[0029] As used herein, the term "RAMP1" refers to the receptor-modified protein 1 encoded by the RAMP1 gene in humans. It belongs to the RAMP family of single-pass transmembrane domain proteins, called receptor (calcitonin)-modified proteins, which include three members: RAMP1, RAMP2, and RAMP3. RAMP is considered a type I transmembrane protein with an extracellular N-terminus and a cytoplasmic C-terminus. One important function of RAMP1 is to regulate the glycosylation of the calcitonin receptor (CRL) and thus its transport to the cell membrane. RAMP1 is widely expressed in the brain, spinal cord, gastrointestinal tract, adrenal glands, perivascular nerves, and arterial smooth muscle. Several orthologues of RAMP1 have been reported in various animal species.

[0030] The RAMP1 protein referred to in accordance with the present invention is preferably human RAMP1 having an amino acid sequence deposited as UniProt accession number O60894. The term “RAMP1” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the RAMP1 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the human RAMP1 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the particular amino acid sequence of the said RAMP1 protein.

[0031] As used herein, the term "LTB" (LTβ) refers to the lymphotoxin-beta protein, also known as tumor necrosis factor C (TNF-C), encoded in humans by the LTB gene. It is a type II membrane protein of the TNF superfamily and the primary ligand for the lymphotoxin-beta receptor. LTB interacts with two ligands: the membrane heterotrimer lymphotoxin alpha (LTα) and the homotrimer LIGHT. Typically, it is expressed by epithelial cells, stromal cells, receptor cells (DCs), and macrophages, but is not present on lymphocytes. LTB is known to be an important regulator of lymphoid tissue formation and inflammation. Furthermore, LTB has been established to have protumoric function. For example, mice exhibiting overexpression of LTα or LTβ have shown increased tumor growth and metastasis in several models of cancer. For LTB, there are two known isoforms in humans (UniProt accession numbers Q06643-1 and Q06643-2). Several orthologues of RAMP1 have been reported in various animal species.

[0032] The LTB protein referred to in accordance with the present invention is preferably a human LTB having the amino acid sequence deposited as UniProt accession number Q06643. The term “LTB” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the LTB protein described herein. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the human LTB protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0033] As used herein, the term "LST1" refers to the leukocyte-specific transcript 1 protein encoded by the LST1 gene in humans. It is a membrane protein that may play a role in the modulation of the immune response. To date, 13 isoforms produced by alternative splicing have been described. For example, isoforms 1 and 2 have inhibitory effects on lymphocyte proliferation. Several orthologues of LST1 have been reported in various animal species.

[0034] The LST1 protein referred to in accordance with the present invention is preferably human LST1 having the amino acid sequence deposited as UniProt accession number O00453. The term “LST1” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the LST1 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the human LST1 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the particular amino acid sequence of the said LST1 protein.

[0035] As used herein, the term "JAML" refers to the junction adhesion molecule-like protein or AMICA1 encoded by the JAML gene in humans. JAML is a transmembrane protein of the leukocyte cell membrane that controls its migration and activation through interaction with CXADR, a cell membrane receptor found on adjacent epithelial and endothelial cells. The interaction between the two receptors mediates the activation of gamma-delta T cells, a subpopulation of T cells present in epithelium and involved in tissue homeostasis and repair. Upon binding to epithelial CXADR, JAML induces downstream cellular signaling events in gamma-delta T cells via PI3 kinase and MAP kinase. This results in cytokines and growth factors by T cells, which then stimulate epithelial tissue repair. It also controls leukocyte reversion within epithelial and endothelial tissues through adhesion interactions with epithelial and endothelial CXADR. Typically, JAML is located in bicellular tight junctions, the nucleus, and the cell membrane. Four isoforms of JAML have been described, and several orthologues of these four isoforms have been reported in various animal species.

[0036] The JAML protein referred to in accordance with the present invention is preferably human JAML having an amino acid sequence deposited as UniProt accession number Q86YT9. The term “JAML” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the JAML protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assays referred to herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the human JAML protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0037] As used herein, the term "IFITM3" refers to interferon-inducible transmembrane protein 3, encoded by the IFITM3 gene in humans. The IFITM3 protein is a family of interferon-inducible antiviral proteins. This family includes five members, including IFITM1, IFITM2, and IFITM3, and belongs to the CD225 superfamily. This protein restricts cell entry by a variety of viral pathogens, including influenza A virus, Ebola virus, and SARS-CoV-2. Several orthologues of IFITM3 have been reported in various animal species.

[0038] The IFITM3 protein referred to in accordance with the present invention is preferably human IFITM3 having the amino acid sequence deposited as UniProt accession number Q01628. The term “IFITM3” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the IFITM3 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the human IFITM3 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the particular amino acid sequence of the said IFITM3 protein.

[0039] As used herein, the term "CD7" refers to the differentiation antigen group 7 protein encoded by the CD7 gene in humans. CD7 is a transmembrane protein found on thymocytes and mature T cells. It belongs to the immunoglobulin superfamily and plays an essential role in T cell interactions and T cell / B cell interactions during early lymphoid development. To date, there are five known potential isoforms, and several orthologues of CD7 have been reported in various animal species.

[0040] The CD7 protein referred to in accordance with the present invention is preferably human CD7 having the amino acid sequence deposited as UniProt accession number P09564. The term “CD7” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the CD7 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the particular amino acid sequence of the CD7 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0041] As used herein, the term "CD69" refers to the group 69 differentiation antigen protein encoded by the CD69 gene in humans. It is a disulfide-linked homodimeric protein having two distinct subunits. Each subunit consists of a single transmembrane domain and an extracellular C-type lectin domain (CTLD) connected to it, followed by a short cytoplasmic tail. It is an early activation marker expressed in hematopoietic stem cells, T cells, and many other cell types in the immune system. Activation of T lymphocytes and natural killer (NK) cells, both in vivo and in vitro, induces CD69 expression. It is involved in lymphocyte proliferation and functions as a signaling receptor in lymphocytes. It is also involved in T cell differentiation and lymphocyte retention in lymphoid tissues. To date, there is one known potential isoform, and several orthologues of CD69 have been reported in various animal species.

[0042] The CD69 protein referred to in accordance with the present invention is preferably human CD69 having the amino acid sequence deposited as UniProt accession number Q07108. The term “CD69” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the CD69 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the CD69 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the particular amino acid sequence of the said CD69 protein.

[0043] As used herein, the term "CD164" refers to the differentiation antigen group 164 protein or sialomucin core protein 24 (also known as endrin), encoded by the CD164 gene in humans. This gene encodes transmembrane sialomucin and cell adhesion molecules that regulate the proliferation, adhesion, and migration of hematopoietic progenitor cells. The encoded protein also interacts with CXC chemokine receptor 4 (CXCR4) and may regulate muscle development. Elevated expression of this gene has been observed in human patients with Sézary syndrome, a type of blood cancer, and mutations in this gene may be associated with hearing impairment. To date, five isoforms are known, and several orthologues of CD164 have been reported in various animal species.

[0044] The CD164 protein referred to in accordance with the present invention is preferably human CD164 having the amino acid sequence deposited as UniProt accession number Q04900. The term “CD164” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the CD164 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the CD164 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0045] As used herein, the term "CD74" refers to the differentiation antigen group 74 protein or HLA class II histocompatibility antigen gamma chain (also known as the HLA-DR antigen-associated invariant chain) encoded by the CD74 gene in humans. The protein encoded by this gene is an important chaperone associated with major histocompatibility complex (MHC) class II and modulates antigen presentation for the immune response. It also acts as a cell surface receptor for cytokine macrophage migration inhibitors (MIFs), and when bound to the encoded protein, it initiates survival pathways and cell proliferation. Multiple alternatively spliced ​​transcript variants encoding five different isoforms have been identified, and several orthologues of CD74 have been reported in various animal species.

[0046] The CD74 protein referred to in accordance with the present invention is preferably human CD74 having the amino acid sequence deposited as UniProt accession number P04233. The term “CD74” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the CD74 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the CD74 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the particular amino acid sequence of the said CD74 protein.

[0047] As used herein, the term "TNF" refers to tumor necrosis factor protein encoded by the TNF gene in humans. TNF belongs to the TNF superfamily, which consists of various transmembrane proteins with homologous TNF domains. It is an adipokine and cytokine, produced by a wide range of cell types, including lymphoid cells, mast cells, endothelial cells, cardiomyocytes, adipose tissue, fibroblasts, and neurons. As an adipokine, TNF promotes insulin resistance and is associated with obesity-induced type 2 diabetes. As a cytokine, it is involved in cellular signaling in the regulation of various biological processes, including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. This cytokine is involved in a variety of diseases, including autoimmune diseases, insulin resistance, psoriasis, rheumatoid arthritis, ankylosing spondylitis, tuberculosis, autosomal dominant polycystic kidney disease, and cancer. Mutations in this gene affect susceptibility to cerebral malaria, septic shock, and Alzheimer's disease. Knockout studies in mice have also suggested neuroprotective functions of this cytokine.

[0048] The TNF protein referred to in accordance with the present invention is preferably human TNF having the amino acid sequence deposited as UniProt accession number P01375. The term “TNF” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the TNF protein described herein. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion, and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the TNF protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%. Several orthologues of TNF have been reported in various animal species.

[0049] As used herein, the term "TFPI" refers to the tissue factor pathway inhibitor protein encoded by the TFPI gene in humans. This gene encodes a Kunitz-type serine protease inhibitor that modulates the tissue factor (TF)-dependent pathway of blood coagulation. Specifically, TFPI is a single-stranded polypeptide that can reversibly inhibit factor Xa of the coagulation cascade. Two distinct isoforms have been identified, and several orthologues of TFPI have been reported in various animal species.

[0050] The TFPI protein referred to in accordance with the present invention is preferably human TFPI having an amino acid sequence deposited as UniProt accession number P10646. The term “TFPI” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the TFPI protein described herein. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the particular amino acid sequence of the TFPI protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0051] As used herein, the term "DLK1" refers to the delta homolog 1 protein encoded by the DLK1 gene in humans. This gene encodes a transmembrane protein containing multiple epidermal growth factor repeats that functions as a regulator of cell proliferation. Soluble DLK1, which is cleaved and removed by ADAM17, is involved in the inhibition of lipid synthesis and the differentiation of preadipocytes into adipocytes. DKL1 is a member of the EGF-like family of homeoproteins. Two distinct isoforms have been identified, and several orthologues of DKL1 have been reported in various animal species.

[0052] The DLK1 protein referred to in accordance with the present invention is preferably human DLK1 having the amino acid sequence deposited as UniProt accession number P80370. The term “DLK1” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the DLK1 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the particular amino acid sequence of the DLK1 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0053] As used herein, the term "CD82" refers to the differentiation antigen group 82 protein encoded by the CD82 gene in humans. CD82 belongs to the tetraspanin / transmembrane 4 superfamily. This protein acts as a metastasis suppressor. The expression of this gene has been shown to be downregulated in tumor progression of human cancer and can be activated by p53 via a consensus binding sequence in the promoter. Its expression is strongly correlated with that of p53, and loss of expression of these two proteins is associated with low survival rates in prostate cancer patients. Two alternatively spliced ​​transcript variants encoding different isoforms have been identified, and several orthologues of CD82 have been reported in various animal species.

[0054] The CD82 protein referred to in accordance with the present invention is preferably human CD82 having the amino acid sequence deposited as UniProt accession number P22701. The term “CD82” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the CD82 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the CD82 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0055] As used herein, the term "IGHM" refers to the Ig muon chain C-region protein encoded by the IGHM gene in humans. The IGHM gene encodes the C-region of the muon heavy chain, which defines the IgM isotype. Naive B cells express transmembrane IgM and IgD on their surface. During an antibody response, activated B cells can switch to expressing individual downstream heavy chain C-region genes through a process of somatic recombination known as isotype switching. IGHM is associated with agammaglobulinemia-1. Two alternatively spliced ​​transcript variants encoding two different isoforms have been identified, and several orthologues of IGHM have been reported in various animal species.

[0056] The IGHM protein referred to in accordance with the present invention is preferably human IGHM having the amino acid sequence deposited as UniProt accession number P01871. The term “IGHM” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the IGHM protein described herein. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the particular amino acid sequence of the IGHM protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0057] As used herein, the term "CALCRL" refers to the calcitonin gene-associated peptide type 1 receptor protein encoded by the CALCRL gene in humans. It is a G protein-coupled receptor associated with the calcitonin receptor. Typically, CALCRL is located in the endoplasmic reticulum, endosomes, and lysosomes, and is likely active at the cell membrane. This protein has been suggested to modulate various bodily functions in all major systems (e.g., respiratory, endocrine, gastrointestinal, immune, and cardiovascular). More specifically, it is presumed to be involved in several processes, including G protein-coupled receptor signaling pathways, cellular responses to sucrose stimulation, and receptor internalization. Several orthologues of CALCRL have been reported in various animal species.

[0058] The CALCRL protein referred to in accordance with the present invention is preferably human CALCRL having the amino acid sequence deposited as UniProt accession number Q16602. The term “CALCRL” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the CALCRL protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the CALCRL protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0059] As used herein, the term "RALA" refers to the Ras-related protein Ral-A, encoded by the RALA gene on chromosome 7 in humans. RALA is one of two paralogs of the Ral protein, the other being RalB. The product of this gene belongs to the small GTPase superfamily, the Ras family of proteins. As a Ras GTPase, RalA functions as a molecular switch, becoming active when bound to GTP and inactive when bound to GDP. RalA can be activated by RalGEF, which then activates effectors in signaling pathways that lead to biological outcomes. Other downstream functions include exocytosis, receptor-mediated endocytosis, tight junction biogenesis, filopodialysis, mitochondrial fission, and cytokinesis. Several orthologues of RALA have been reported in various animal species.

[0060] The RALA protein referred to in accordance with the present invention is preferably human RALA having the amino acid sequence deposited as UniProt accession number P11233. The term “RALA” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the RALA protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assays referred to herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the particular amino acid sequence of the RALA protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0061] As used herein, the term “SLC2A5” (also known as GLUT5) refers to the solute carrier family 2, facilitated glucose transporter member 5 protein encoded by the SLC2A5 gene in humans. Typically, SLC2A5 is expressed at the apical boundary of intestinal cells in the small intestine, as well as in skeletal muscle, testes, kidneys, adipose tissue, and the brain. The protein encoded by this gene is a fructose transporter responsible for fructose uptake by the small intestine. SLC2A5 is also required for elevated blood pressure resulting from high dietary fructose consumption. Two alternatively spliced ​​transcript variants encoding two different isoforms have been identified, and several orthologues of SLC2A5 have been reported in various animal species.

[0062] The SLC2A5 protein referred to in accordance with the present invention is preferably human SLC2A5 having the amino acid sequence deposited as UniProt accession number P22732. The term “SLC2A5” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the SLC2A5 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the SLC2A5 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the particular amino acid sequence of the said SLC2A5 protein.

[0063] As used herein, the term "HSPA5" refers to the heat shock 70kDa protein 5 encoded by the HSPA5 gene in humans. It is also known as the binding immunoglobulin protein (BiP) or the 78kDa glucose regulatory protein (GRP-78). Typically, it is located in the lumen of the endoplasmic reticulum (ER), where it acts as the HSP70 chaperone involved in protein folding and assembly, and is an excellent regulator of ER homeostasis. Increased expression of this protein and its atypical translocation to the cell surface have been reported in viral infections and certain types of cancer cells. Several orthologues of HSPA5 have been reported in various animal species.

[0064] The HSPA5 protein referred to in accordance with the present invention is preferably human HSPA5 having the amino acid sequence deposited as UniProt accession number P11021. The term “HSPA5” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the HSPA5 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the HSPA5 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0065] As used herein, the term "HLA-DRA" refers to the HLA class II histocompatibility antigen, DR alpha chain protein, encoded in humans by the HLA-DRA gene. This protein is a heterodimer consisting of an alpha chain and a beta chain, both of which are membrane-bound. Typically, it is expressed on the surface of various antigen-presenting cells, such as B lymphocytes, dendritic cells, and monocytes / macrophages, and plays a central role in the immune system and immune response by presenting peptides derived from extracellular proteins, particularly pathogen-derived peptides, to T cells. Several orthologues of HLA-DRA have been reported in various animal species.

[0066] The HLA-DRA protein referred to in accordance with the present invention is preferably human HLA-DRA having an amino acid sequence deposited as UniProt accession number P01903. The term “HLA-DRA” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the HLA-DRA protein described herein. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the HLA-DRA protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the particular amino acid sequence of the said HLA-DRA protein.

[0067] As used herein, the term "RAB11A" refers to the Ras-related protein Rab-11A, encoded by the RAB11A gene in humans. The protein encoded by this gene belongs to the Rab family, a small GTPase superfamily. It is involved in both constitutive and regulatory secretory pathways and can participate in protein transport. Rab-11a regulates the intracellular transport of the innate immune receptor TLR4, thereby also regulating receptor signaling. Two isoforms are known, and several orthologues of RAB11A have been reported in various animal species.

[0068] The RAB11A protein referred to in accordance with the present invention is preferably human RAB11A having the amino acid sequence deposited as UniProt accession number P62491. The term “RAB11A” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the RAB11A protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the RAB11A protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the particular amino acid sequence of the said RAB11A protein.

[0069] As used herein, the term "SELL" refers to the L-selectin protein, also known as CD62L, encoded in humans by the SELL gene. This gene encodes a cell surface adhesion molecule belonging to the family of adhesion / homing receptors. The encoded protein contains a type C lectin-like domain, a calcium-binding epidermal growth factor-like domain, and two short complement-like repeats. The gene product is necessary for the binding and subsequent rolling of leukocytes on endothelial cells, facilitating their migration to secondary lymphoid organs and inflammatory sites. Single nucleotide polymorphisms in this gene have been associated with a variety of diseases, including immunoglobulin A neuropathy. Two isoforms are known, and several orthologues of SELL have been reported in various animal species.

[0070] The SELL protein referred to in accordance with the present invention is preferably human SELL having the amino acid sequence deposited as UniProt accession number P14151. The term “SELL” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the SELL protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the particular amino acid sequence of the SELL protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0071] As used herein, the term "VAMP5" refers to vesicle-associated membrane protein 5, encoded by the VAMP5 gene in humans. Synaptobrevin / VAMP, syntaxin, and 25kD synaptosome-associated proteins are key components of protein complexes involved in the docking and / or fusion of vesicles and cell membranes. The VAMP5 gene is a member of the vesicle-associated membrane protein (VAMP) / synaptobrevin family and the SNARE superfamily. Members of this VAMP family may participate in vesicle transport events associated with myogenesis. Several orthologues of VAMP5 have been reported in various animal species.

[0072] The VAMP5 protein referred to in accordance with the present invention is preferably human VAMP5 having the amino acid sequence deposited as UniProt accession number O95183. The term “VAMP5” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the VAMP5 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the VAMP5 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0073] As used herein, the term "FCMR" refers to the FC fragment of the IgM receptor protein encoded by the FCMR gene in humans. This protein may play a role in immune system processes. It is thought to protect against apoptosis induced by FAS, TNF-alpha, and FADD without increasing the expression of the apoptosis inhibitors BCL2 and BCLXL, and to activate an inhibitory pathway that prevents CASP8 activation after FAS stimulation rather than blocking downstream apoptotic signaling. FCMR is also involved in inhibiting FAS-induced apoptosis by preventing CASP8 processing via upregulation of CFLAR. Three isoforms are known, and several orthologues of FCMR have been reported in various animal species.

[0074] The FCMR protein referred to in accordance with the present invention is preferably human FCMR having the amino acid sequence deposited as UniProt accession number O60667. The term “FCMR” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the above FCMR protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay referred to herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the particular amino acid sequence of the FCMR protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0075] As used herein, the term "CLEC7A" refers to the C-type lectin domain family 7 member A protein encoded by the CLEC7A gene in humans. This gene encodes a member of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. The encoded glycoprotein is a small type II membrane receptor with an extracellular C-type lectin-like domain folding structure and a cytoplasmic domain possessing an immune receptor tyrosine-based activation motif. It functions as a pattern recognition receptor recognizing various beta-1,3-linked and beta-1,6-linked glucans derived from fungi and plants, and thus plays a role in the innate immune response. To date, ten isoforms are known, and several orthologues of CLEC7A have been reported in various animal species.

[0076] The CLEC7A protein referred to in accordance with the present invention is preferably human CLEC7A having the amino acid sequence deposited as UniProt accession number Q9BXN2. The term “CLEC7A” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the CLEC7A protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assays referred to herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the CLEC7A protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the particular amino acid sequence of the said CLEC7A protein.

[0077] As used herein, the term "NDFIP1" refers to Nedd4 family interacting protein 1, encoded by the NDFIP1 gene in humans. The protein encoded by this gene belongs to a small group of evolutionarily conserved proteins possessing three transmembrane domains. It is a potential target for ubiquitination by Nedd4 family proteins. NDFIP1 has been proposed to be part of a family of endogenous Golgi membrane proteins. Eight isoforms are known to date, and several orthologues of NDFIP1 have been reported in various animal species.

[0078] The NDFIP1 protein referred to in accordance with the present invention is preferably human NDFIP1 having the amino acid sequence deposited as UniProt accession number Q96PU5. The term “NDFIP1” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the NDFIP1 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the NDFIP1 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0079] As used herein, the term "CLEC9A" refers to the C-type lectin domain family 9 member A protein encoded by the CLEC9A gene in humans. Typically, this protein is expressed by myeloid cells. CLEC9A is a group V C-type lectin-like receptor (CTLR) that functions as an intracellular receptor on a small subset of myeloid cells specialized for the uptake and processing of material from dead cells. It recognizes fibrillary morphology of actin associated with specific actin-binding domains of cytoskeletal proteins, including spectrin, which are exposed when the cell membrane is damaged and mediates cross-presentation of dead cell-associated antigens in a Syk-dependent manner.

[0080] The CLEC9A protein referred to in accordance with the present invention is preferably human CLEC9A having the amino acid sequence deposited as UniProt accession number Q6UXN8. The term “CLEC9A” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the CLEC9A protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assays referred to herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion, and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the CLEC9A protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%. Several orthologues of CLEC9A have been reported in various animal species.

[0081] As used herein, the term "HCST" refers to the hematopoietic cell signaling factor protein encoded by the HCST gene in humans. This gene encodes a transmembrane signaling adapter containing a YxxM motif in its cytoplasmic domain. The encoded protein can form part of an immunorecognition receptor complex with the C-type lectin-like receptor NKG2D. As part of this receptor complex, the protein can activate a phosphatidylinositol 3-kinase-dependent signaling pathway via its cytoplasmic YxxM motif. This receptor complex may play a role in cell survival and proliferation through the activation of NK and T cell responses. Two isoforms are known, and several orthologues of HCST have been reported in various animal species.

[0082] The HCST protein referred to in accordance with the present invention is preferably human HCST having an amino acid sequence deposited as UniProt accession number Q9UBK5. The term “HCST” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the HCST protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the particular amino acid sequence of the HCST protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0083] As used herein, the term "LPAR6" refers to lysophosphatidic acid receptor 6, encoded by the LPAR6 gene in humans. LPAR6 is also known as LPA6, P2RY5, and GPR87. The protein encoded by this gene belongs to the family of G protein-coupled receptors that are selectively activated by adenosine and uridine nucleotides. Mutations in this gene are involved in causing a rare, genotypical form of hair loss (simple trichothyroidism). Several orthologues of LPAR6 have been reported in various animal species.

[0084] The LPAR6 protein referred to in accordance with the present invention is preferably human LPAR6 having the amino acid sequence deposited as UniProt accession number P43657. The term “LPAR6” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the LPAR6 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the LPAR6 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0085] As used herein, the term "HLA-DQA1" refers to the major histocompatibility complex, class II, DQ alpha 1 protein encoded by the HLA-DQA1 gene on chromosome 6 in humans. HLA-DQA1 is a heterodimer consisting of an alpha chain (DQA) and a beta chain (DQB), both of which are membrane-bound. This protein is expressed in antigen-presenting cells such as B lymphocytes, dendritic cells, and macrophages. It plays a central role in the immune system by presenting peptides derived from extracellular proteins. Several orthologues of HLA-DQA1 have been reported in various animal species.

[0086] The HLA-DQA1 protein referred to in accordance with the present invention is preferably human HLA-DQA1 having the amino acid sequence deposited as UniProt accession number P01909. The term “HLA-DQA1” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the HLA-DQA1 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the HLA-DQA1 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the particular amino acid sequence of the said HLA-DQA1 protein.

[0087] As used herein, the term "HLA-DRB5" refers to the HLA class II histocompatibility antigen, DRB5 beta-chain protein, encoded by the HLA-DRB5 gene in humans. This class II molecule is a heterodimer consisting of an alpha chain (DRA) and a beta chain (DRB), both of which are immobilized on the membrane. It plays a central role in the immune system by presenting peptides derived from extracellular proteins. Diseases associated with HLA-DRB5 include pityriasis rosea and multiple epiphyseal dysplasia caused by type 9 collagen abnormalities. Several orthologues of HLA-DRB5 have been reported in various animal species.

[0088] The HLA-DRB5 protein referred to in accordance with the present invention is preferably human HLA-DRB5 having the amino acid sequence deposited as UniProt accession number Q30154. The term “HLA-DRB5” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the HLA-DRB5 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the HLA-DRB5 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the particular amino acid sequence of the said HLA-DRB5 protein.

[0089] As used herein, the term "CD34" refers to the differentiation antigen group 34 protein encoded by the CD34 gene in humans. This transmembrane phosphoglycoprotein can play a role as an adhesion molecule in early hematopoiesis by mediating the binding of stem cells to the extracellular matrix of bone marrow or direct binding to stromal cells. Alternatively spliced ​​transcript variants encoding two different isoforms have been identified, and several orthologues of CD34 have been reported in various animal species.

[0090] The CD34 protein referred to in accordance with the present invention is preferably human CD34 having the amino acid sequence deposited as UniProt accession number P28906. The term “CD34” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the CD34 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the CD34 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0091] As used herein, the term "IGLL1" refers to the immunoglobulin lambda-like polypeptide 1 protein encoded by the IGLL1 gene in humans. This protein is involved in signaling for cell proliferation, differentiation from pro-B cells to pre-B cells, allele exclusion at the Ig heavy chain locus, and promotion of Ig light chain rearrangement. Mutations in this gene can result in B cell deficiency and agammaglobulinemia, autosomal recessive disorders in which little or no gamma globulin or antibody is produced. Two distinct isoforms have been identified, and several orthologues of IGLL1 have been reported in various animal species.

[0092] The IGLL1 protein referred to in accordance with the present invention is preferably human IGLL1 having the amino acid sequence deposited as UniProt accession number P15814. The term “IGLL1” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the IGLL1 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the IGLL1 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the particular amino acid sequence of the said IGLL1 protein.

[0093] As used herein, the term "BEST1" refers to the bethrophilin 1 protein encoded by the BEST1 gene in humans. It belongs to the bethrophilin family, which includes four related genes encoding endogenous membrane proteins. BEST1 is characterized by a highly conserved N-terminus with 4–6 transmembrane domains. Bethrophilin can form chloride ion channels or modulate voltage-gated L-type calcium ion channels. While bethrophilin is generally thought to form calcium-activated chloride ion channels in epithelial cells, it has also been shown to be highly permeable to bicarbonate ion transport in retinal tissue. Mutations in this gene cause juvenile-onset vitelliform macular dystrophy (VMD2), also known as BEST macular degeneration, in addition to adult-onset vitelliform macular dystrophy (AVMD) and other retinopathy. Mutations in the BEST1 gene have also been identified as the primary cause of at least five different retinal degenerative diseases. Two different isoforms have been identified, and several orthologues of BEST1 have been reported in various animal species.

[0094] The BEST1 protein referred to in accordance with the present invention is preferably human BEST1 having the amino acid sequence deposited as UniProt accession number O76090. The term “BEST1” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the BEST1 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the BEST1 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0095] As used herein, the term "EREG" refers to the epiregulin protein encoded by the EREG gene in humans. This gene encodes a member of the secreted peptide hormone and epidermal growth factor (EGF) family of proteins. The encoded protein is a ligand for the epidermal growth factor receptor (EGFR) and the structurally related erb-b2 receptor tyrosine kinase 4 (ERBB4). The encoded protein may be involved in a variety of biological processes, including inflammation, wound healing, egg maturation, and cell proliferation. Furthermore, the encoded protein may promote the progression of cancer in various human tissues. Several orthologues of EREG have been reported in various animal species.

[0096] The EREG protein referred to in accordance with the present invention is preferably human EREG having the amino acid sequence deposited as UniProt accession number O14944. The term “EREG” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the EREG protein described herein. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the particular amino acid sequence of the EREG protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0097] As used herein, the term "SLC5A3" refers to the sodium / myo-inositol cotransporter protein encoded by the SLC5A3 gene in humans. Typically, it is located in the cell membrane. SLC5A3 is a sodium / myo-inositol cotransporter. It is also said to act upstream of or within several processes, including peripheral nervous system development, positive regulation of reactive oxygen species biosynthesis processes, and regulation of respiratory gas exchange. Several orthologues of SLC5A3 have been reported in various animal species.

[0098] The SLC5A3 protein referred to in accordance with the present invention is preferably human SLC5A3 having the amino acid sequence deposited as UniProt accession number P53794. The term “SLC5A3” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the SLC5A3 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the SLC5A3 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the particular amino acid sequence of the said SLC5A3 protein.

[0099] As used herein, the term "SELK" refers to selenoprotein K, encoded by the SELK gene in humans. It is a transmembrane protein located in the endoplasmic reticulum (ER) and is involved in ER-associated degradation (ERAD) of misfolded glycosylated proteins. It also plays a role in protecting cells from ER stress-induced apoptosis. Knockout studies in mice showed Ca in immune cells 2+This demonstrates the importance of this gene in promoting cell fluidity and enhancing effective immune responses. SELK also plays a role in T cell proliferation and T cell and neutrophil migration. Several orthologues of SELK have been reported in various animal species.

[0100] The SELK protein referred to in accordance with the present invention is preferably human SELK having the amino acid sequence deposited as UniProt accession number Q9Y6D0. The term “SELK” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the SELK protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assays referred to herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the particular amino acid sequence of the SELK protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0101] As used herein, the term "PRRG3" refers to the transmembrane gamma-carboxyglutamate protein 3 encoded by the PRRG3 gene in humans. This gene encodes a protein containing a vitamin K-dependent carboxylation / gamma-carboxyglutamate domain. The encoded protein is a member of the family of vitamin K-dependent transmembrane proteins containing a glutamate-rich extracellular domain. Diseases associated with PRRG3 include hereditary combined deficiencies of vitamin K-dependent coagulation factors and vitamin K deficiency hemorrhage. Several orthologues of PRRG3 have been reported in various animal species.

[0102] The PRRG3 protein referred to in accordance with the present invention is preferably human PRRG3 having the amino acid sequence deposited as UniProt accession number Q9BZD7. The term “PRRG3” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the PRRG3 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the PRRG3 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0103] As used herein, the term "NINJ1" refers to the ninjurin-1 protein encoded by the NINJ1 gene in humans. It is upregulated after nerve injury in both dorsal root ganglion neurons and Schwann cells. NINJ1 is an alloaffinity transmembrane adhesion molecule involved in various processes such as inflammation, cell death, axonal growth, cell chemotaxis, and angiogenesis. Several orthologues of NINJ1 have been reported in various animal species.

[0104] The NINJ1 protein referred to in accordance with the present invention is preferably human NINJ1 having the amino acid sequence deposited as UniProt accession number Q92982. The term “NINJ1” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the NINJ1 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the NINJ1 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0105] As used herein, the term "MGST1" refers to the microsomal glutathione S-transferase 1 protein encoded by the MGST1 gene in humans. This gene encodes a protein that catalyzes the conjugation of glutathione to electrophiles and the reduction of lipid hydroperoxides. Typically, MGST1 localizes to the endoplasmic reticulum and the outer mitochondrial membrane, where it is thought to protect these membranes from oxidative stress. It is involved in cellular defense against toxic, carcinogenic, and pharmacologically active electrophiles. Two isoforms are known, and several orthologues of MGST1 have been reported in various animal species.

[0106] The MGST1 protein referred to in accordance with the present invention is preferably human MGST1 having the amino acid sequence deposited as UniProt accession number P10620. The term “MGST1” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the MGST1 protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the MGST1 protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0107] As used herein, the term "HLA-G" refers to the HLA-G histocompatibility antigen, class I, G protein, also known as human leukocyte antigen G, encoded by the HLA-G gene in humans. HLA-G belongs to the HLA class I heavy chain paralog. This class I molecule is a heterodimer (beta-2 microglobulin) consisting of a heavy chain and a light chain. The heavy chain is fixed to the membrane. HLA-G is expressed on fetal placental cells. It functions as a major immune checkpoint, for example, downmodulating the immune system response. HLA-G has also been shown to play a role in immune tolerance during pregnancy, modulation of the immune response to parasitic diseases, tumor avoidance in cancer, and allergic responses. Seven isoforms are known, and several orthologues of HLA have been reported in various animal species.

[0108] The HLA-G protein referred to in accordance with the present invention is preferably a human HLA-G having an amino acid sequence deposited as UniProt accession number P17693. The term “HLA-G” is also understood to refer to variants of the said protein. Such variants have at least the same essential biological and immunological properties as the HLA-G protein described above. In particular, they share the same essential biological and immunological properties if they are detectable by the same particular assay described herein. Furthermore, it should be understood that the variants referred to in accordance with the present invention have different amino acid sequences resulting from at least one amino acid substitution, deletion and / or addition, and the amino acid sequences of the variants are still preferably, each, identical to the specific amino acid sequence of the HLA-G protein over the entire length, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0109] The degree of identity between two amino acid sequences according to the present invention can be determined by algorithms well known in the art. Preferably, the degree of identity should be determined by comparing two optimally aligned sequences across a comparison window, where the amino acid sequence fragments in the comparison window may include additions or deletions (e.g., gaps or protrusions) compared to a reference sequence for optimal alignment (which includes neither additions nor deletions). The percentage is calculated by determining the number of positions where identical amino acid residues exist in both sequences, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The optimal alignment of sequences for comparison may be performed by Smith's local homology algorithm, Needleman's homology alignment algorithm, Pearson's similarity search method, computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by visual inspection. Given that two sequences are identified for comparison, GAP and BESTFIT are preferably used to determine their optimal alignment and thus the degree of identity. Preferably, default values ​​of 5.00 for gap weights and 0.30 for gap weight lengths are used. The variants mentioned above may be allele variants or any other species-specific homologs, paralogs, or orthologues.

[0110] As used in accordance with the present invention, the term "determining the amount of at least one biomarker" refers to the qualitative and quantitative determination of the biomarker, that is, the term includes determining the presence or absence of the biomarker or determining its absolute or relative amount. The term further includes measuring the amount or concentration, preferably semi-quantitatively or quantitatively.

[0111] As used herein, the term “quantity” means the absolute quantity of a biomarker, the relative quantity or concentration of the biomarker, and any values ​​or parameters that correlate with or can be derived from it. Such values ​​or parameters include intensity signal values ​​derived from all specific physical or chemical properties obtained from the biomarker or detection molecule and / or detectable label. Values ​​or parameters can be obtained by direct or indirect measurements. Direct measurements relate to measuring the quantity or concentration of the biomarker and the intensity that directly correlates with the number of biomarker molecules present in a sample, based on signals obtained from the biomarker molecule itself. Such signals, sometimes referred to herein as intensity signals, can be obtained, for example, by measuring intensity values ​​of specific physical or chemical properties of the biomarker molecule. Indirect measurements include measuring signals obtained from secondary components, i.e., components other than the biomarker molecule itself. It should be understood that values ​​correlated with the above quantities or parameters can also be obtained and / or modified by any standard mathematical operations.

[0112] The determination of the quantity in the method of the present invention can be carried out by any technique that enables the detection of the presence or absence or quantity of the biomarker. The preferred technique depends on the properties of the molecule and the characteristics of the biomarker. For example, a protein biomarker may be determined by measuring characteristics other than those of a transcribed nucleic acid molecular biomarker. Those skilled in the art are well aware of these differences in measurable characteristics. Furthermore, it is understood that a protein biomarker may be detected by using a different detection agent and / or technique than those used for transcribed nucleic acid molecular biomarkers. However, those skilled in the art are well aware of these different detection agents and / or techniques.

[0113] According to the present invention, the determination of the amount of a biomarker can be achieved by any known means for determining such an amount in a sample. The means include immunoassay devices and methods that can use molecules labeled in various sandwich, competitive, or other assay formats. The assay will produce a signal indicating the presence or absence of the protein. Furthermore, preferably, the signal intensity can be directly or indirectly correlated (e.g., inversely proportional) with the amount of biomarker present in the sample. A more preferred method includes measuring physical or chemical properties specific to the biomarker. The method preferably includes a biosensor, an optical device coupled with the immunoassay, a biochip, or other analytical device such as a chromatography device, or a single-cell analytical device such as a FACS analyzer, or a device for PCR analysis such as a device for single-cell PCR, qPCR, or bulk PCR, or a sequencing device.

[0114] In a preferred embodiment of the method of the present invention, the amount of the biomarker is detected by flow cytometry. More preferably, the flow cytometry is fluorescence-activated cell sorting (FACS). Flow cytometry is a well-known method. By counting positive versus negative sorting events, protein biomarkers can be quantified proportionally within a cell population. Thus, the biomarker data for clinical samples is not a single value that represents the overall staining intensity, but rather a value that reflects the proportion of individual cells that exceed the intensity threshold for a particular biomarker. The gating criterion for positive sorting events can be set as a combination of desired signal intensities for the protein biomarker used.

[0115] Preferably, the biomarker determined according to the present invention may be determined as a protein. For this purpose, typically, a binding molecule is applied that can be detected by a detectable label present in the binding molecule, or by a secondary binding molecule that specifically binds to the first binding molecule and contains a detectable label. The binding molecule may be exposed to the biomarker in solution or while the binding molecule is immobilized on a solid-phase indicator.

[0116] In this context, a binding molecule refers to any molecule that can specifically bind to the biomarker to be detected. The binding molecule is selected based on the type of analysis to be performed. Examples of binding molecules, but not limited to, include aptamers, antibodies, adonectins, ankyrins, antibody mimetics and other protein scaffolds, small molecules, nucleic acids, lectins, aphibodies, nanobodies, avimers, and peptidomimetics. Preferably, such a binding molecule may be an antibody or its antigen-binding fragment.

[0117] The “antibody” according to the present invention may encompass any type of antibody that specifically binds to a biomarker protein. Preferably, the antibody of the present invention is a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a chimeric antibody, or any fragment or derivative of such an antibody that can still specifically bind to a biomarker protein.

[0118] The term "antigen-binding fragment" refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen. Examples of binding fragments included in the term "antigen-binding fragment" are fragment antigen-binding (Fab) fragments, Fab' fragments, F(ab')2 fragments, heavy chain antibodies, single chain antibodies (sdAb), single chain variable fragments (scFv), variable fragments (Fv), and V H Domain, V L Examples include domains, single-domain antibodies, nanobodies, IgNARs (immunoglobulin neoantigen receptors), di-scFvs, bispecific T cell engagers (BITEs), dual-affinity retargeting (DART) molecules, triple bodies, diabodies, single-chain diabodies, selective scaffold proteins, and fusion proteins thereof.

[0119] Specific binding, as used in the context of the antibodies of this invention, means that the antibody does not cross-react with other molecules present in the sample to be investigated. Specific binding can be tested by various well-known techniques. Antibodies or fragments thereof can generally be obtained by using methods described in standard textbooks, e.g., Harlow and Lane, "Antibodies, A Laboratory Manual," CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies can be prepared by techniques including the fusion of mouse myeloma cells to immunized mammalian cells, preferably spleen cells derived from immunized mice. Preferably, immunogenic peptides are applied to mammals. The peptides are preferably conjugated to carrier proteins such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). Depending on the host species, various adjuvants can be used to enhance the immune response. Such adjuvants preferably include Freund's adjuvants, mineral gels, e.g., aluminum hydroxide, and surfactants, e.g., lysolecithin, Pluronic® polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, and dinitrophenols. Subsequently, monoclonal antibodies that specifically bind to the analyte can be prepared using well-known hybridoma techniques, human B-cell hybridoma techniques, and EBV hybridoma techniques. Detection systems using antibodies are based on the highly specific binding affinity of the antibody to a particular antigen, i.e., a biomarker protein. The binding event results in a physicochemical change that can be detected as described elsewhere in this specification.

[0120] As used herein, "adonectin" refers to fibronectin type 10 ( 10Adnectin refers to a synthetic binding protein, also known as a monobody, based on the Fn3 domain. It is a member of the immunoglobulin superfamily and contains a "beta sandwich" protein folding structure that is remarkably similar to that of the antibody domain. Therefore, adnectin is a simple and robust alternative to antibodies for producing target-binding proteins. A major advantage of adnectin compared to conventional antibodies is that it can be easily used as a genetically encoded intracellular inhibitor; that is, a person skilled in the art can express an adnectin inhibitor in selected cells simply by transfecting them with an adnectin expression vector. Preferably, the adonectin used herein specifically binds to CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G.

[0121] As used herein, “ankyrin” refers to a family of proteins containing binding sites for various membrane proteins. Ankyrins contain four functional domains: (i) an N-terminal domain with 24 tandem ankyrin repeats responsible for the recognition of multiple membrane proteins; (ii) a central domain that binds to spectrin; (iii) a death domain that binds to proteins involved in apoptosis; and (iv) a C-terminal regulatory domain that is highly variable among different ankyrin proteins. In humans, ankyrins are encoded by three genes, which in turn produce multiple proteins through alternative splicing. Preferably, the ankyrins used herein bind specifically to at least one biomarker as described elsewhere herein.

[0122] As used herein, "antibody mimetic" refers to a compound that, like an antibody, can specifically bind to an antigen, but is not structurally related to an antibody. Typically, an antibody mimetic is an artificial peptide or protein with a molar mass of approximately 3 to 20 kDa that contains one or more exposed domains that specifically bind to the antigen. Examples include, in particular, LACI-D1 (lipoprotein-related coagulation inhibitor); affilins, e.g., human-γB crystalline or human ubiquitin; cystatin; Sac7D derived from Sulfolobus acidocaldarius; lipocalin and anticarin derived from lipocalin; DARPin (designed ankyrin repeat domain); SH3 domain of Fyn; Kunits domain of protease inhibitors; monobodies, e.g., the 10th type III domain of fibronectin; adnectin; nottin (cysteine ​​knot miniprotein); atrimers; organisms, e.g., binders based on CTLA4; affilos, e.g., Staphylococcus aureus Examples include a three-helix bundle derived from the Z domain of protein A (derived from *Aureus*); transbodies, e.g., human transferrin; tetranectin, e.g., monomeric or trimeric human C-type lectin domains; microbodies, e.g., trypsin inhibitor II; affilin; and armadillo repeat proteins. Nucleic acids and small molecules are also sometimes considered antibody mimetics (aptamers), but are not artificial antibodies, antibody fragments, or fusion proteins composed of these. General advantages over antibodies include good solubility, tissue penetration, thermal and enzyme stability, and relatively low production costs. Preferably, the antibody mimetic used herein binds specifically to at least one biomarker as described elsewhere herein.

[0123] As used herein, "scaffolding protein" refers to a specific protein whose primary function is mediating protein complexes. Scaffolding proteins typically have multiple protein domains that mediate binding to other proteins. Examples of scaffolding proteins include, but are not limited to, the protein inaD derived from the rod-like adduct of Drosophila melanogaster or the protein titin found in muscle.

[0124] As used herein, the term “lectin” refers to carbohydrate-binding proteins that are highly specific to sugar groups. They are naturally ubiquitous and can bind to soluble carbohydrates or carbohydrate moieties that are part of glycoproteins or glycolipids. Lectins typically cause aggregation of certain cells and / or sedimentation of sugar conjugates. For this reason, they are useful in medicine, particularly for blood typing. Lectins are also used in neuroscience for anterograde labeling to track the pathways of efferent axons. Preferably, the lectins used herein bind specifically to at least one biomarker as described elsewhere herein.

[0125] As used herein, "affibodies" are small, highly robust proteins with high affinity for target proteins. In contrast to antibodies, affibodies are composed of alpha helices and lack disulfide crosslinks. In particular, they are based on a 3-helix bundle with 58 amino acids and have a molar mass of approximately 6 kDa. They can be expressed in a variety of host cells in a soluble and proteolytically stable form, either as is or through fusion with other protein partners. Affibodies can be used as research reagents for protein purification, enzyme inhibition, protein capture and detection, diagnostic imaging, and targeted therapy. For example, the second-generation affibody ABY-025 selectively binds to the HER2 receptor with picomolar affinity. Preferably, the affibodies used herein bind specifically to at least one biomarker as described elsewhere herein.

[0126] As used herein, the term “nanobody” typically refers to a very small, recombinantly produced antigen-binding fragment consisting of a single monomeric variable antibody domain. Nanobodies lack light and heavy chain constant domains, but their antigen-binding ability remains similar to that of conventional antibodies. Typically, the complementarity-determining region 3 (CDR3) of a nanobody is similar to, or even longer than, that of the human variable domain (VH) of an immunoglobulin heavy chain. They can form finger-like structures that recognize cavities or hidden epitopes unavailable to monoclonal antibodies, which are features that enhance the binding affinity and specificity of the nanobody. Preferably, the nanobodies used herein bind specifically to at least one biomarker as described elsewhere herein.

[0127] As used herein, “avimer” (short for avidity multimer) refers to an artificial protein having multiple binding sites for specific binding to a particular antigen. They are structurally unrelated to antibodies and are thus classified as antibody mimetic. Typically, they consist of two or more peptide sequences of 30-35 amino acids linked by a linker peptide. Each sequence is derived from the A domain of various membrane receptors and has a rigid structure stabilized by disulfide bonds and calcium. Each A domain can bind to a specific epitope of the target protein. A combination of domains that bind to different epitopes of the same protein increases its affinity for that protein, an effect known as avidity. Avimers are widely used in early detection, treatment, and carcinogenesis studies in tissue imaging. Preferably, the avimers used herein bind specifically to at least one biomarker as described elsewhere herein.

[0128] As used herein, the term “peptidomimetic” means a compound that mimics one or more structural aspects or bioactivity of naturally occurring polypeptides, but which contains one or more non-peptides or chemical structures or linkages that do not exist naturally. Peptidomimetic compounds are often used to mimic the biological action of peptides, and thus they may be small protein-like chains designed to mimic one or more peptides. Peptidomimetic compounds are often synthesized based on existing peptides of interest that have one or more modifications that alter the structure or properties of the molecule. Modifications can alter the stability, half-life, bioactivity, absorption, or side effects of the peptide molecule (e.g., toxicity, solubility, hydrophobicity, side-chain charge, or flexibility). Peptidomimetic compounds may be useful as drugs or drug-like compounds developed based on modifications of existing peptides that reasonably, known, or putatively have bioactivity. Preferably, peptidomimetic compounds as used herein bind specifically to at least one biomarker as described elsewhere herein.

[0129] Examples of "detectable labels" used in accordance with the present invention, as referred to herein, include gold particles, latex beads, acridan esters, luminol, ruthenium, enzyme activity labels, radioactive labels, magnetic labels, such as magnetic beads including paramagnetic and superparamagnetic labels, and fluorescent labels. Examples of enzyme activity labels include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, luciferase, and their derivatives. Suitable substrates for detection include di-amino-benzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, and NBT-BCIP (4-nitrobluetetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate). Suitable enzyme-substrate combinations can be measured according to methods known in the art (e.g., using photosensitive film or a suitable camera system), resulting in colored reaction products, fluorescence, or chemiluminescence. The criteria given above are similarly applicable to the measurement of enzyme reactions. Typical fluorescent labels include, for example, fluorescent proteins (GFP and its derivatives), Cy3, Cy5, Texas Red, fluorescein, Alexa dye, brilliant violet, or brilliant ultraviolet. The use of quantum dots as fluorescent labels is also considered. Typical radioactive labels include 35S, 125I, 32P, and 33P. Radioactive labels can be detected by any known and suitable method, such as a photosensitive film or a phosphor imager. Preferred labels may also include, or contain, tags such as biotin, digoxigenin, His-, GST-, FLAG-, GFP-, MYC- tags, influenza A virus hemagglutinin (HA), and maltose-binding proteins.

[0130] The amount of a biomarker can be detected using a biomarker / binding molecule complex. Alternatively, the amount can be detected indirectly from the biomarker / binding molecule complex, for example, as a result of a reaction that depends on the formation of the biomarker / binding molecule complex, but after the biomarker / binding molecule interaction. In some cases, the amount of a biomarker can be detected directly from the biomarker in the biological sample. The amount of a biomarker can also be detected using a multiplexing configuration that allows for the simultaneous detection of two or more biomarkers in the biological sample. In the multiplexing configuration, the binding molecules are fixed directly or indirectly, covalently or non-covalently, at separate positions on a solid-phase indicator.

[0131] Preferably, the biomarker determined according to the present invention may be determined as a transcribed nucleic acid molecule. For this purpose, a binding molecule, such as a nucleic acid molecule that specifically hybridizes to a nucleic acid molecule transcribed by a reverse transcription reaction or any molecule derived therefrom, may be applied for detection. The hybridizing binding molecule itself may contain a detectable label, or it may specifically bind to a secondary molecule containing such a detectable label.

[0132] Alternatively, the amount of a biomarker can also be detected by PCR-based techniques such as quantitative polymerase chain reaction (qPCR). "Quantitative PCR" or "real-time PCR" is a well-known technique used for the detection and quantification of nucleic acids (DNA or RNA) in a sample. Typically, a fluorescent reporter dye is used as an indirect measure of the amount of nucleic acid present between each amplification cycle. The increase in the fluorescent signal is directly proportional to the exponentially increased amount of PCR product molecules (amplicons) produced during the reaction's repeat phase.

[0133] More preferably, the amount of a biomarker can be detected by transcriptome sequencing, also known as single-cell sequencing (scRNA-seq). Transcriptome sequencing is a well-known method that refers to the sequencing of a single-cell genome or transcriptome to obtain genomic, transcriptome, or other multi-omics information. Thus, as used herein, “transcriptome sequencing” refers to a method for analyzing RNA expression from a large population of cells, preferably a population of hematopoietic stem cells and progenitor cells. Typically, the method involves isolating a single cell and its RNA, followed by reverse transcription, amplification, library generation, and sequencing. These techniques include, but are not limited to, droplet-based and plate-based scRNA-seq techniques. Plate-based methods require single-cell isolation, for example, by FACS. However, droplet-based methods use lipid droplet formation to isolate single cells by phase separation in a bulk sample of single cells in suspension. The latter technique allows for the analysis of thousands of cells, while plate-based methods are typically suitable for hundreds of cells.

[0134] When a biomarker indicates an abnormal process or disease in a subject, it is generally stated that the biomarker is overexpressed compared to the level or amount of a biomarker indicating the absence of a normal process or disease or other condition in the subject. "Overexpression" means a level or amount of the biomarker in a biological sample that is higher than the level or amount of the biomarker typically detected in a similar biological sample from a healthy or normal subject.

[0135] The biomarkers of the present invention are present on or in hematopoietic stem and progenitor cells (HSPC). For example, biomarkers presented on the surface of HSPC in the form of surface proteins, receptors, lipids, etc. are present on HSPC according to the present invention. Biomarkers produced inside HSPC, such as transcribed nucleic acid molecules, and intended to remain intracellularly are thus present in HSPC. Hematopoietic stem and progenitor cells (HSPC) are a heterogeneous cell population responsible for the production of substantially all types of mature blood and immune cells. HSPC includes a series of low-primed undifferentiated cells, including hematopoietic stem cells (HSC) (Velten et al., 2017 Nature Cell Biology), which are considered to be the origin of postnatal hematopoietic differentiation. HSC can self-renew and gradually acquire lineage biases along multiple directions within the HSPC compartment, giving rise to committed or unilineage-restricted progenitor cells, respectively. For example, myeloid progenitor cells ultimately give rise to differentiated cells such as monocytes, macrophages, neutrophils, or dendritic cells, while lymphoid progenitor cells ultimately give rise to, for example, T cells, B cells, or natural killer cells. As used herein, the term "HSPC" refers to CD34 + cells, preferably CD34 + CD38 - cells, more preferably Lin - CD34 + CD38 - cells. Said cells can preferably be isolated from a sample referred to herein, preferably a tissue sample or a body fluid sample, by, for example, fluorescence-activated cell sorting (FACS) or other cell sorting techniques known from the literature. Lineage-negative (Lin - ) cells are a mixture of all cells that express little or no mature cell lineage markers. These lineage markers can preferably be selected from the group consisting of CD4, CD8, CD11b, CD14, CD19, CD20, CD56, and CD235a. More preferably, these lineage markers consist of CD4, CD8, CD11b, CD14, CD19, CD20, CD56, and CD235a. In the latter case, Lin - CD34+ CD38 - The cell is CD4 - CD8 - CD11b - CD14 - CD19 - CD20 - CD56 - CD235a - CD34 + CD38 - It corresponds to a cell.

[0136] The term "sample" refers to any sample containing HSPC obtained from the subject to be investigated. Preferably, the sample may be a tissue or body fluid sample. Preferably, a tissue sample is a connective tissue sample, preferably a bone marrow sample, or a spleen sample. Preferably, a body fluid sample is a peripheral blood sample or an umbilical cord blood sample. A blood sample also includes its fractions. For example, a blood sample may be fractionated into serum, plasma, or fractions containing specific types of blood cells such as red blood cells or white blood cells (leucocytes). The term "sample" also includes materials containing homogenized solid material derived from tissue samples or tissue biopsies, etc. Body fluid samples can be obtained by well-known techniques and preferably include blood samples. Tissue or organ samples, such as bone marrow samples, can be obtained, for example, by biopsy. Cells separated by separation techniques such as centrifugation or cell sorting can be obtained from body fluids or tissues or organs. Therefore, this term refers to the biological sample itself as used directly during the determination of the amount of biomarker. However, this term may also refer to a sample that must undergo various steps prior to determining the amount of biomarker, such as the isolation of cells from the biomaterial.

[0137] The determined amount of the biomarker is compared to a reference according to the method of the present invention. As used herein, the term “reference” refers to a quantity or value that enables the assignment of subjects to either a group of subjects who have or are at risk of developing the disease or condition, or a group of subjects who do not have or are not at risk of developing the disease or condition. Such a reference may be a threshold quantity that separates these groups from one another. Thus, the reference is a quantity that enables the assignment of subjects to either a group of subjects who have or are at risk of developing the disease or condition, or who do not. A suitable threshold quantity for separating the two groups can be calculated without further work by statistical tests referred to elsewhere herein, based on the amount of biomarker derived from either a subject or group of subjects known to have or be at risk of developing the disease or condition, or a subject or group of subjects known not to have or are not at risk of developing the disease or condition. The reference quantity applicable to individual subjects may vary depending on various physiological parameters such as age, sex, or subgroup. The reference may preferably be a reference for each biomarker derived from at least one subject known to have JMML. More preferably, the reference for each biomarker may be derived from at least one subject not known to have JMML. More preferably, the reference for each biomarker is derived from at least one subject diagnosed with JMML or belonging to either the high-risk or low-risk JMML group by DNA methylome analysis, as performed in the appendix examples below.

[0138] Reference quantities can, in principle, be calculated for a cohort of subjects based on the mean or average values ​​of a given parameter, such as biomarker levels, by applying standard statistical methods. In particular, the precision of tests, such as methods intended to diagnose events or not, is best described by their subject-operational characteristics (ROC) (see, in particular, Zweig 1993, Clin. Chem. 39:561-577). An ROC graph is a plot of all sensitivity / specificity pairs obtained by continuously varying the discrimination threshold over the entire range of observed data. The clinical performance of a diagnostic method depends on its precision, i.e., its ability to accurately assign subjects to a specific prognosis or diagnosis. An ROC plot shows the overlap between two distributions by plotting sensitivity versus 1-specificity over the full range of thresholds suitable for making distinctions. On the y-axis is sensitivity, or true positive rate, which is defined as the ratio of the number of true positive test results to the product of the number of true positive test results and the number of false negative test results. This, too, was referred to as positive in the presence of the disease or condition. It is calculated only from the affected subgroup. On the x-axis is the false positive rate, or 1-specificity, which is defined as the ratio of the number of false positive results to the product of the number of true negative results and the number of false positive results. It is an index of specificity and is calculated entirely from the unaffected subgroup. The ROC plot is independent of the prevalence of events in the cohort, as the true positive rate and false positive rate are calculated entirely independently by using test results from two different subgroups. Each point on the ROC plot represents a sensitivity / -specificity pair corresponding to a specific discrimination threshold. A test showing perfect discrimination (no overlap in the two distributions of results) has an ROC plot that passes through the upper left corner, where the true positive rate is 1.0, or 100% (perfect sensitivity) and the false positive rate is 0 (perfect specificity). A theoretical plot for a test that does not show discrimination (identical distribution of results for the two groups) is a 45-degree diagonal from the lower left corner to the upper right corner. Many plots lie between these two extreme values.If the ROC plot is completely below the 45-degree diagonal, this can be easily corrected by reversing the criteria for "positive" from "greater than" to "less than," or vice versa. Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test. Depending on the desired confidence interval, thresholds that enable diagnosis or prediction of a given event with an appropriate balance of sensitivity and specificity can be derived from the ROC curve. Thus, the reference to be used for the above method of the present invention, i.e., the threshold that enables the identification of subjects with coagulation disorders from those without, can usually be generated by establishing the ROC for the above cohort and deriving the threshold amount therefrom. Depending on the desired sensitivity and specificity for the diagnostic method, the ROC plot allows for the derivation of a suitable threshold. It is understood that optimal sensitivity is desirable for excluding high-risk subjects (i.e., rule out), while optimal specificity is assumed for subjects that should be assessed as high-risk (i.e., rule in).

[0139] As used herein, the term “comparing” encompasses comparing a determined quantity of a biomarker referred to herein with a reference. As used herein, comparing means any type of comparison made between a quantity value and a reference. However, it should be understood that, preferably, values ​​of the same type are compared with each other, for example, if an absolute quantity is determined and compared in the method of the present invention, the reference is also an absolute quantity, and if a relative quantity is determined and compared in the method of the present invention, the reference is also a relative quantity. The comparison may be performed manually or with computer assistance. The values ​​of quantities or references may be compared with each other, for example, and the comparison may be performed automatically by a computer program that executes an algorithm for comparison. The computer program that performs the evaluation will provide the desired evaluation in a preferred output format.

[0140] The term "JMML" as used in this invention encompasses JMML, JMML-like neoplasms, and JMML associated with neurofibromatosis or Noonan syndrome-associated myeloproliferative disorder (CBL syndrome). Typical symptoms and signs at the time of diagnosis of JMML are splenomegaly, hepatomegaly, lymphadenopathy, pallor, and fever. Less frequent symptoms include infection, bleeding, cough, and skin rash. Rarely, café-au-lait spots, abdominal pain, xanthomas, bone pain, diarrhea, and CNS infiltration may occur (Arber et al., 2022 Blood). Typically, if specific diagnostic criteria for JMML can be validated by specific assays such as blood tests, bone marrow aspiration, cytogenetic testing, and molecular testing, the subject is known to have JMML.

[0141] The diagnostic criteria for JMML according to the present invention may preferably be those of the International Consensus Classification of Myeloneoplasms and Acute Leukemia (ICC). The ICC classifies JMML and related disorders as MDS / MPN and groups them with pediatric and / or germline mutation-related disorders (Arber, ibid.). JMML is a childhood clonal disorder characterized by constitutive activation of the RAS signaling pathway. More than 95% of patients carry mutations in the RAS pathway. Canonical mutations affect the PTPN11, NRAS, KRAS, NF1, CBL, and rarely the RRAS gene. Clonal disorders that phenotypically mimic JMML but do not carry one of these mutations are classified as JMML-like neoplasms. Polyclonal Noonan syndrome-associated myeloproliferative disorders can be clinically similar to JMML. All of these cases, namely JMML, JMML-like neoplasms, and Noonan syndrome-associated myeloproliferative disorders, share typical features.

[0142] The diagnostic criteria for JMML, as enumerated by the ICC, include genetic, as well as clinical and hematological features. The percentage of blast cells in peripheral blood and bone marrow must be less than 20%, and the BCR::ABL1 fusion (or Philadelphia chromosome) must be absent. Many cases present with splenomegaly (absent in approximately 3% of cases) and 1x10⁻¹⁰ 9They have a monocyte count of 1 / L or higher (approximately 7% of cases do not reach this number). Genetically, one of the following findings is usually observed: - Somatic mutations in PTPN11, KRAS, NRAS, or RRAS; - Or germline mutations and loss of heterozygosity in NF1 or clinical diagnosis of neurofibromatosis type 1; - Or mutations in CBL and loss of heterozygosity in CBL.

[0143] Furthermore, monosomy 7 or any other chromosomal abnormality, hemoglobin F levels higher than normal for the patient's age, myeloid progenitor cells in the blood, granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity in colony assays, and hyperphosphorylation of STAT5 may indicate JMML, but KMT2A rearrangements are excluded (Niemeyer & Flotho 2019 Blood). While GM-CSF hypersensitivity and hematological parameters were previously considered paramount, the current WHO classification emphasizes RAS pathway mutations as the most important criterion. JMML-like neoplasms lack RAS pathway mutations but are phenotypically similar to JMML. This group includes rearrangements such as ALK, ROS1, FIP1L1::RARA, or CCDC88C::FLT3 fusions (Arber, ibid.).

[0144] The diagnostic criteria for JMML according to the present invention may also preferably be those of the WHO, Chapter 5, Myelodysplastic / Myeloproliferative Neoplasms (Orazi et al., 2016). Therefore, all four of the following clinical and hematological criteria are required: - 1x10 9 Peripheral blood monocyte count of cells / L or greater; - Percentage of blast cells in peripheral blood and bone marrow less than 20%; - Splenomegaly; and - The Philadelphia (Ph) chromosome or BCR-ABL1 fusion is absent.

[0145] From the following genetic criteria, at least one criterion is sufficient: - Somatic mutations or germline mutations in PTPN11, KRAS, or NRAS (indicating Noonan syndrome) must be ruled out; - Clinical diagnosis of neurofibromatosis type 1 or NF1 mutation; and - Germline CBL mutations and LOH of CBL, as well as incidental cases, have heterozygous splice site mutations.

[0146] For cases that do not meet any of the above genetic criteria, the following criteria must be met in addition to the above clinical and hematological criteria: - Monosomy 7 or any other chromosomal abnormality, or - At least two of the following criteria: - High hemoglobin F for one's age; - Myeloid or erythrocyte progenitor cells on a peripheral blood smear; - Hypersensitivity to granulocyte-macrophage colony-stimulating factor (also known as CSF2) in colony assays; - High phosphorylation of STAT5.

[0147] More preferably, subjects infected with JMML are identified by DNA methylome analysis, for example, as described in the attached examples below.

[0148] In one embodiment, the reference is derived from at least one subject known to be affected by JMML.

[0149] Preferably, subjects suffering from JMML are identified by determining the amount of at least one biomarker on or in the HSPC in a biological sample, selected from each of the following: (i) Group I consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34; and ii) Group II consisting of IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G; and comparing the determined amount to a reference.

[0150] In embodiments of the method of the present invention, if the reference is derived from at least one subject known to have JMML, a determined amount of at least one of the markers, which is identical to or greater than that of the reference, indicates that the subject has JMML. A determined amount of at least one of the markers, which is lower than that of the reference, may indicate that the subject does not have JMML.

[0151] In another embodiment, the reference derives from at least one subject known not to have JMML.

[0152] Typically, if the above diagnostic criteria for JMML are not met, the subject is known not to have JMML. Preferably, subjects not suffering from JMML can be identified, for example, by DNA methylome analysis, as similarly described in the appended examples below.

[0153] Preferably, subjects known not to have JMML can also be characterized by the physiological abundance of a biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G in the HSPC of the subject's biological sample.

[0154] Preferably, subjects not affected by JMML are identified by determining the amount of at least one biomarker on or in the HSPC in a biological sample, selected from each of the following: (i) Group I consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34; and ii) Group II consisting of IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G; and comparing the determined amount to a reference.

[0155] In embodiments where the reference is derived from at least one subject known not to have JMML, a determined amount of at least one of the markers, which is identical to or less than the reference, indicates that the subject is not affected by JMML. A determined amount of at least one of the markers, which is greater than the reference, may indicate that the subject is affected by JMML.

[0156] The method of the present invention is advantageous in that JMML can be diagnosed based on the presence or abundance of one or more biomarkers of group I, i.e., high-risk JMML biomarkers, or one or more biomarkers of group II, i.e., low-risk JMML biomarkers, on or in the HSPC. The method of the present invention also provides assistance in diagnosing the absence of JMML, i.e., whether group I and group II biomarkers are not determined on or in the HSPC. Thus, by investigating group I and II biomarkers, a subject can be diagnosed for JMML. Thanks to the findings in the research on which the present invention is based, it is possible to efficiently screen for JMML in a population of young subjects or to make the diagnosis for individual subjects more reliable. Therefore, treatment efforts can be improved. As already discussed previously, JMML is a rare invasive cancer in children. Given the low incidence and prevalence of this disease, the availability of patient samples for investigation is limited, making the development of diagnostic and therapeutic agents difficult and cumbersome. In the research that forms the basis of this invention, we were able to isolate living cells from a limited number of patient samples that represent all known JMML subtypes. Among these living cells, the number of HSPCs (Lin-CD34+CD38-HSPCs) is typically small. Nevertheless, by applying single-cell techniques, including scRNA-seq or FACS, to the available materials, we were able to obtain a rich dataset.

[0157] Furthermore, by identifying biomarkers specific to high-risk JMML subjects and biomarkers for low-risk JMML subjects, the present invention enables the classification of subjects and the direct identification of high-risk or low-risk JMML subjects.

[0158] Therefore, and in light of the above, the present invention is a method for classifying subjects suffering from JMML into low- or high-risk groups for JMML, a) At least one biomarker present on or in hematopoietic stem cells and progenitor cells (HSPCs) in a biological sample, i) Group I consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34, and ii) Group II consisting of IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G A step of determining the amount of at least one biomarker selected from each of the following; b) A step of comparing the determined amount in step a) with a reference; and c) A step to classify subjects into low or high-risk groups for JMML based on the comparison in step b). We also intend to include methods that encompass this.

[0159] As used herein, “classifying” means assigning a subject to a group of subjects exhibiting similar or identical disease conditions. The disease conditions that a subject can be classified into by applying the method of the present invention are either the high-risk JMML group or the low-risk JMML group.

[0160] As used herein, the term “high-risk JMML group” refers to a group of subjects known to be at high risk of developing JMML and / or having a high risk of death from JMML if, without treatment, within a period of up to 8 years, up to 7 years, up to 6.5 years, up to 6 years, up to 5.5 years, up to 5 years, up to 4.5 years, up to 4 years, up to 3 years, up to 2.5 years, up to 2 years, up to 1.5 years, up to 1 year, up to 6 months, or less than 6 months after diagnosis. This term also refers to a group of subjects known to be at high risk of developing JMML and having a high risk of recurrence. Subjects known to develop JMML may also be classified as high-risk JMML if certain biomarkers on the HSPC are detected.

[0161] Thus, in one embodiment of the above method of the present invention, if at least one biomarker selected from group I is determined in step a), the subject is classified into the JMML high-risk group. In such cases, it is understood that, preferably, a reference for at least one biomarker of group I can be obtained from at least one subject known to originate from the high-risk JMML group. A determined amount of at least one biomarker from group I that is identical or increased compared to the reference indicates a high-risk subject. Furthermore, it is understood that, preferably, a reference for at least one biomarker of group I can be obtained from at least one subject known not to originate from the JMML high-risk group. A determined amount of at least one biomarker from group I that is identical or decreased compared to the reference indicates that the subject is not a high-risk subject.

[0162] As used herein, the term “JMML low-risk group” refers to a group of subjects known to have a low risk of developing JMML with an exacerbation of JMML or any associated signs or symptoms and / or fatality due to JMML, without treatment within a period of up to 8 years, 7 years, 6.5 years, 6 years, 5.5 years, 5 years, 4.5 years, 4 years, 3.5 years, 3 years, 2.5 years, 2 years, 1.5 years, 1 year, 6 months, or less than 6 months after diagnosis. This term also refers to a group of subjects known to have JMML that often exhibit spontaneous remission. Subjects known to have JMML may also be classified as JMML low-risk group if certain biomarkers on the HSPC are detected.

[0163] Thus, in one embodiment of the method of the present invention, if at least one biomarker selected from group II is determined in step a), the subject is classified into the JMML low-risk group. In such cases, it is understood that, preferably, a reference for at least one group II biomarker can be obtained from at least one subject known to originate from the JMML low-risk group. A determined amount of at least one biomarker from group I that is identical or increased compared to the reference indicates a low-risk subject. Furthermore, it is understood that, preferably, a reference for at least one group II biomarker can be obtained from at least one subject known not to originate from the low-risk JMML group. A determined amount of at least one biomarker from group II that is identical or decreased compared to the reference indicates that the subject is not a low-risk subject. The present invention further provides a method for identifying whether a subject belongs to the JMML high-risk group,

[0164] a) A step of determining the amount of at least one biomarker present on or in HSPC in a biological sample, selected from group I consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34; b) A step of comparing the determined amount in step a) with a reference; and c) A step to identify whether the subject belongs to the JMML high-risk group based on the comparison in step b). This includes methods.

[0165] The reference can preferably be obtained from at least one subject known to belong to the JMML high-risk group. If at least one determined biomarker is identical to or increased compared to the reference, the subject under investigation is understood to also belong to the JMML high-risk group. Furthermore, the reference can preferably be obtained from at least one subject known not to belong to the JMML high-risk group. If at least one determined biomarker is identical to or decreased compared to the reference, the subject under investigation is understood to also not belong to the JMML high-risk group. The present invention further provides a method for identifying whether a subject belongs to the low-risk group for JMML,

[0166] a) A step of determining the amount of at least one biomarker present on or in the HSPC in a biological sample, which is selected from group II consisting of IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G; b) A step of comparing the determined amount in step a) with a reference; and c) A step to identify whether the subject belongs to the low-risk group for JMML based on the comparison in step b). This includes methods.

[0167] The reference can preferably be obtained from at least one subject known to belong to the JMML low-risk group. If at least one determined biomarker is identical to or increased compared to the reference, the subject under investigation is understood to also belong to the JMML low-risk group. Furthermore, the reference can preferably be obtained from at least one subject known not to belong to the JMML low-risk group. If at least one determined biomarker is identical to or decreased compared to the reference, the subject under investigation is understood to also not belong to the JMML low-risk group.

[0168] The method of the present invention preferably further includes selecting a therapy for a subject suffering from JMML based on the identified JMML risk group in step c).

[0169] As used herein, the term “therapy” means any means aimed at improving and / or treating JMML or any associated signs or symptoms. Such means may include the administration of drugs, the administration of disease-modulating radiotherapy, surgery or transplantation, scheduling of further appointments with a physician, or a combination thereof.

[0170] Preferably, the preferred therapy is selected depending on whether the subject with JMML is identified as belonging to a low or high-risk group for JMML. Typically, one of the following JMML treatment regimens is considered: i) allogeneic stem cell transplantation or ii) drug therapy. Drug therapy typically involves administering azacitidine (Vidaza®), a chemical analog of nucleoside cytidine. Preferably, the drug therapy involves administering an inhibitor that specifically inhibits at least one group I or II biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G, which are present on or in hematopoietic stem cells and progenitor cells (HSPCs) as specified elsewhere in this specification.

[0171] The present invention relates to the following groups for diagnosing juvenile myelomonocytic leukemia in subjects, or for classifying subjects with JMML or those at risk of developing JMML into low- or high-risk groups: a) Group I consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34, and b) Group II consisting of IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G Further consideration is the use of at least one biomarker present on or within hematopoietic stem cells and progenitor cells (HSPCs) in a biological sample, selected from one of the following.

[0172] The present invention further relates to a kit for diagnosing JMML in a subject or for classifying a subject suffering from JMML into a low or high-risk group for JMML, comprising at least one detection agent and instructions for carrying out the method of the present invention, wherein the at least one detection agent is present on or in HSPC as CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, This invention relates to a kit capable of specifically detecting a group I or II biomarker selected from the group consisting of CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G.

[0173] As used herein, the term “kit” typically refers to a collection of the above-mentioned components, provided separately or in a single container. The container also typically includes instructions for performing the methods of the present invention. These instructions may be in the form of a manual, or they may be provided as computer program code that, when implemented on a computer or data processing device, can perform or assist in determining the biomarkers referred to in the methods of the present invention. The computer program code may be provided on a data storage medium or device such as an optical storage medium (e.g., a compact disk), or directly on a computer or data processing device, or in a downloadable format such as a link to an accessible server or cloud. Furthermore, the kit may typically include standards for reference amounts of biomarkers for calibration purposes, as described in detail elsewhere herein. The kit according to the present invention may also include further components necessary for performing the methods of the present invention, such as solvents, buffers, washing solutions and / or reagents necessary for the detection of the released second molecule. Furthermore, it may include the devices of the present invention, separately or as a whole.

[0174] As used herein, the term “detector” means a detector as referred to in accordance with the methods of the present invention as described elsewhere herein. In particular, the detector may depend on the nature of the biomarker to be detected. Preferably, for the detection of transcribed nucleic acid molecules, nucleic acid molecules that can specifically hybridize to said transcribed nucleic acid, such as antisense nucleic acid probes or oligonucleotide primers, may be used as the detector. For protein biomarkers, aptamers, antibodies, adnectin, ankyrin, antibody mimetics and other protein scaffolds, small molecules, nucleic acids, lectins, aphibodies, nanobodies, avimers and peptidomimetics may be used.

[0175] The present invention further envisions an inhibitor for use in the treatment and / or prevention of juvenile myelomonocytic leukemia (JMML) that specifically inhibits at least one group I or II biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G.

[0176] The term "inhibitor" refers to a substance that affects one or more biological or chemical reactions induced by at least one of the biomarkers mentioned above, i.e., affects their biological activity in such a way that they are slowed, inhibited, or prevented. Depending on the type of substance, the inhibition may be reversible or irreversible. It is understood that the one or more biological or chemical reactions are associated with JMML, i.e., they may directly or indirectly contribute to the onset or progression of the disease.

[0177] As used herein, the term “to treat” refers to improving and / or curing JMML as described herein, preventing disease progression, or significantly improving at least one symptom associated with the disease. Where used herein, “to treat” also encompasses complete recovery of health with respect to JMML. It is understood that, in most cases, the treatments referred herein will not be successful in all subjects treated. However, it is assumed that the treatments will be effective in at least a statistically significant portion of the subjects treated. For example, whether a statistically significant portion of a cohort of subjects can be successfully treated can preferably be determined by statistical tests using various well-known statistical evaluation methods, such as determining confidence intervals, determining p-values, Student's t-test, Mann-Whitney U tests, etc. Preferably, the treatment should be effective in at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the subjects in a given cohort or population.

[0178] The term “prevention” means maintaining health with respect to JMML in a subject over a specific period of time. It is understood that the said period may depend on the therapy used or the amount of the drug compound administered. It should be understood that prevention may not be effective in all subjects administered with the binder described herein. However, this term preferably requires that a statistically significant portion of a cohort or population of subjects is effectively prevented from developing the disease or disorder or associated symptoms described herein. Preferably, a cohort or population of subjects that would normally, i.e., develop JMML without the preventive measures according to the present invention, is assumed in this context. Whether a portion is statistically significant can be determined by those skilled in the art without further work using various well-known statistical evaluation methods discussed elsewhere in this specification.

[0179] In particular, the present invention envisions a group I or II biomarker that is a protein selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G. Preferably, the inhibitor is thus a peptide, protein, small molecule, lipid, or aptamer.

[0180] As used herein, “peptides” or “proteins” refer to molecules consisting of amino acid residues linked by peptide bonds. Molecules with short amino acid chains, for example, up to about 100 amino acids, are referred to as peptides, while molecules with longer amino acid chains are referred to as proteins. Preferably, the peptide or protein directly or indirectly inhibits the biological activity of at least one biomarker. More preferably, the inhibitor specifically binds to at least one biomarker, thereby inhibiting its biological activity.

[0181] As used herein, the term "low molecular weight" refers to molecules having a low molecular weight. Typically, low molecular weight molecules are organic compounds having a molecular weight of less than 900 daltons. Examples of low molecular weight molecules include alkaloids, lipids, glycosides, terpenes, tetrapyrroles, phenazines, oligonucleotides, or small secondary metabolites such as peptide-like low molecular weight molecules. Preferably, the low molecular weight directly or indirectly inhibits the biological activity of at least one biomarker. More preferably, the inhibitor specifically binds to at least one biomarker, thereby inhibiting its biological activity.

[0182] As used herein, “lipids” refer to hydrophobic or amphiphilic small molecules. Examples of lipids include fatty acids and their derivatives, such as triglycerides, diglycerides, monoglycerides, phospholipids, lysophospholipids, such as lysophosphatidylcholine (LPC), glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, prenolipids, and sterollipids. Preferably, the lipid directly or indirectly inhibits the biological activity of at least one biomarker. More preferably, the inhibitor specifically binds to at least one biomarker, thereby inhibiting its biological activity.

[0183] As used herein, the term "aptamer" refers to a polynucleotide or polypeptide that specifically binds to a target molecule due to its three-dimensional structure. Peptide aptamers are preferably peptides containing 8 to 80 amino acids, more preferably 10 to 50 amino acids, and most preferably 15 to 30 amino acids. They can be isolated from randomized peptide expression libraries in a suitable host system, such as baker's yeast (see, for example, Klevenz et al., Cell Mol Life Sci. 2002, 59: 1993-1998). Peptide aptamers are preferably free peptides; however, peptide aptamers are also intended to be fused to a polypeptide that functions as a “scaffold,” meaning that covalent linkage to the polypeptide functions to fix the three-dimensional structure of the peptide aptamer to a specific conformation. Preferably, the aptamer specifically binds to at least one biomarker and inhibits its biological activity.

[0184] In another embodiment, the inhibitor is an antibody or its antigen-binding fragment, as defined in detail elsewhere herein. In a more preferred embodiment, the antibody or its antigen-binding fragment is alemtuzumab. Alemtuzumab refers to a humanized monoclonal antibody commercially available under the trademark names Campath and Lemtrada. The antibody specifically binds to CD52 and has been used in the treatment of multiple sclerosis and chronic lymphocytic leukemia (CLL) (Havrdova E. et al., Ther Adv Neurol Discord. 2015 Jan; 8(1):31-45; Fraser G. et al., Curr Oncol. 2007 Jun; 14(3):96-109).

[0185] The present invention also envisions a biomarker which is a transcribed nucleic acid, preferably mRNA. Preferably, the inhibitor is thus a ribozyme, an inhibitory RNA molecule, an antisense oligonucleotide, or a morpholino.

[0186] As used herein, the term “ribozyme” refers to a catalytic RNA molecule having a clearly defined tertiary structure capable of catalyzing either the hydrolysis of one of its own phosphodiester bonds (self-cleaving ribozymes) or the hydrolysis of a bond in another RNA, which has also been found to catalyze the aminotransferase activity of ribosomes. The ribozymes envisioned by the present invention are preferably those that specifically hydrolyze a target transcript. Hammerhead ribozymes are particularly preferred according to the present invention. Methods for generating and using such ribozymes are well known in the art (see, for example, Hean J, Weinberg MS (2008). “The Hammerhead Ribozyme Revisited: New Biological Insights for the Development of Therapeutic Agents and for Reverse Genomics Applications.” In Morris KL. RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Norfolk, England: Caister Academic Press).

[0187] As used herein, the term “inhibitory RNA molecule” refers to an RNA molecule that inhibits gene expression in a sequence-specific manner. Examples of inhibitory RNA molecules include small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), and microRNAs (miRNAs). Inhibitory RNA molecules typically induce a process known as RNA interference (RNAi), resulting in cleavage and / or translation inhibition of a target mRNA having a complementary sequence. It is known to those skilled in the art that inhibitory RNA molecules may exhibit complete or incomplete base pairing with a complementary target sequence. siRNAs and shRNAs typically exhibit complete base pairing, inducing mRNA cleavage only at a single specific target. Conversely, miRNAs usually exhibit incomplete base pairing with their target and often inhibit the translation of many different mRNAs having similar sequences. Inhibitory RNA molecules can be chemically synthesized or expressed in cells, for example, by introducing their respective recombinant DNA constructs. It is understood that such DNA constructs may contain additional regulatory elements such as enhancers, constitutive or inductive promoters, or terminators. Inhibitory RNA molecules are direct or indirect inhibitors of at least one group I or II biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G. Preferably, an inhibitory RNA molecule for at least one biomarker weakens the translation of each mRNA into a functional protein, thus inhibiting the activity of each protein, or it may degrade the mRNA or facilitate its degradation.

[0188] As used herein, “antisense oligonucleotide” means a single-stranded DNA and / or RNA molecule capable of inhibiting DNA and / or RNA processing. Antisense oligonucleotides contain nucleic acid sequences complementary to specific RNA or DNA sequences. Typically, antisense oligonucleotides bind to their respective complementary oligonucleotides, DNA, or RNA in a sequence-specific manner, thereby inhibiting DNA and / or RNA processing. It is known to those skilled in the art that antisense oligonucleotides may inhibit mRNA processing by RNase H-mediated degradation, translational arrest, or splicing modulation, or they may act by steric hindrance of proteins. Means and methods for designing and synthesizing antisense oligonucleotides are well known in the art, including, for example, rational design, chemical modification, and design and solid-phase chemical synthesis of antisense oligonucleotides containing locked nucleic acids (LNAs). Antisense oligonucleotides can be chemically synthesized or expressed in cells, for example, by introducing their respective recombinant DNA constructs. It is understood to those skilled in the art that such DNA constructs may contain additional regulatory elements such as enhancers, constitutive or inductive promoters, or terminators. Preferably, the antisense oligonucleotide has a length of at least 8, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, or at least 50 nucleotides. The antisense oligonucleotide may include deoxyribonucleotides, ribonucleotides, or a combination of both. Preferably, the antisense oligonucleotide is a DNA and / or RNA molecule that inhibits the expression and / or translation of one of the biomarkers so that it cannot produce a functional protein.

[0189] As used herein, the term "morpholino" refers to a molecule that blocks the access of other molecules to a small, specific sequence on the base-pairing surface of RNA. Typically, the small, specific sequence is about 25 nucleotides long. Generally, morpholinos consist of a methylenemorpholine ring backbone and a phosphorodiamidate bond. Morpholinos are also commonly known as morpholino oligomers (MO nucleic acid analogs) and phosphorodiamidate morpholino oligomers (PMOs). Morpholinos typically do not result in the degradation of their target RNA molecule, but rather act by steric blockage, i.e., by binding to the target sequence within the RNA, thereby preventing molecules that would otherwise interact with the RNA. Preferably, morpholino directly binds to the premRNA and / or mRNA of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G, thereby inhibiting their translation and / or splicing, resulting in the production of low-function or non-functional proteins. Thus, the activity of any of the respective proteins is inhibited.

[0190] The present invention further envisions a pharmaceutical composition for use in treating and / or preventing JMML, comprising at least two inhibitors as defined elsewhere herein, wherein each inhibitor specifically inhibits a different biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G, which is present on or within hematopoietic stem cells and progenitor cells (HSPCs).

[0191] Thus, the pharmaceutical composition of the present invention comprises two or more different inhibitors that inhibit two or more previously described different biomarkers.

[0192] As used herein, the term “pharmaceutical composition” refers to a composition comprising the compound of the present invention and, preferably, one or more pharmaceutically acceptable carriers. The compound of the present invention can be formulated as a pharmaceutically acceptable salt. Preferred acceptable salts include acetates, HCl, sulfates, and chlorides. The pharmaceutical composition is preferably administered systemically. Preferred routes of administration commonly used for drug administration are oral, intravenous, subcutaneous, parenteral, and inhalation. However, depending on the properties and mechanism of action of the compound, the pharmaceutical composition may also be administered by other routes. Furthermore, the compound may be administered together with other drugs in a general pharmaceutical composition or as another pharmaceutical composition which may be provided in the form of a kit.

[0193] The compound is preferably administered in a conventional dosage form prepared by combining the drug with a standard pharmaceutical carrier according to conventional procedures. These procedures may include mixing, granulating, and compressing or dissolving components suitable for the desired preparation. It is understood that the form and properties of a pharmaceutically acceptable carrier or diluent are determined by the amount of the active ingredient combined with it, the route of administration, and other well-known variables.

[0194] The carrier must be acceptable in the sense that it is compatible with the other components of the formulation and is not harmful to its recipient. The pharmaceutical carrier used may be, for example, a solid, gel, or liquid. Examples of solid carriers include lactose, clay, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, and biodegradable polymers such as PLGA (DeYoung et al. (2011), DIABETES TECHNOLOGY & THERAPEUTICS 13:1145; Ramazani et al. (2016), Int J Pharm. 499(1-2): 358-367). Examples of liquid carriers include phosphate-buffered saline solution, syrup, oils such as peanut oil and olive oil, water, emulsions, various types of wetting agents, and sterile solutions. Similarly, the carrier or diluent may include time-delaying materials well known in the art, such as glyceryl monostearate or glyceryl distearate, either alone or with wax. The aforementioned suitable carriers include those described above and others well known in the industry; see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

[0195] The diluent is selected so as not to affect the biological activity of the compound or multiple compounds. Examples of such diluents include distilled water, physiological saline, Ringer's solution, dextrose solution, and Hanks' solution. Furthermore, the pharmaceutical composition or formulation may also contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, reactive oxygen scavengers, etc.

[0196] The pharmaceutical composition is preferably administered in a conventional dosage form prepared by combining an active compound with a standard pharmaceutical carrier according to conventional procedures. These procedures may, if necessary, include mixing the components to obtain the desired preparation. It is understood that the form and properties of a pharmaceutically acceptable carrier or diluent are determined by the amount of the active ingredient it is combined with, the route of administration, and other well-known variables. Similarly, the carrier or diluent may contain a time-delaying material well known in the art, such as glyceryl monostearate or glyceryl distearate, either alone or with a wax. The therapeutically effective amount refers to the amount of the active compound used in the pharmaceutical composition of the present invention that produces the effect referred to herein. The therapeutic efficacy and toxicity of such compounds are determined by standard pharmaceutical procedures in cell culture or experimental animals, e.g., ED 50 (Therapeutic dose effective in 50% of the population) and LD 50 (The lethal dose for 50% of the population) can be determined by the dose ratio between therapeutic and toxic effects, which is the therapeutic index, and is the LD50. 50 / ED 50It can be expressed as a ratio. The dosage regimen is determined by the attending physician and other clinical factors. As is well known in the medical field, the dosage for any given patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, sex, time and route of administration, general health status, and other drugs administered concurrently. Progress can be monitored by periodic evaluations. Typical doses can be in the range of, for example, 1 μg to 1000 mg; however, doses below or above this exemplary range are also conceivable, especially considering the factors mentioned above. In general, a regimen as a regular administration of a pharmaceutical composition should be in the range of 1 μg to 100 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 1 mg units per kilogram of body weight per minute, respectively. Preferably, the pharmaceutical composition is administered to the subject once, i.e., preferably used as a single treatment. Depending on the subject and mode of administration, the amount of substance administered may vary widely to provide a dose ranging from approximately 0.01 mg / kg body weight to approximately 100 mg / kg body weight. The pharmaceutical compositions and formulations described herein are administered at least once to treat, improve, or prevent the diseases or conditions described herein. However, the pharmaceutical compositions may be administered two or more times, for example, 2 to 50 times, more preferably 5 to 50 times. Preferably, the administration is adjusted to maintain an effective concentration in the subject's body over the intended period. Progress can be monitored by periodic evaluations.

[0197] The present invention further relates to a method for treating and / or preventing JMML, comprising administering to a subject in need thereof a therapeutically effective dose of at least one inhibitor as defined elsewhere herein.

[0198] "Therapeutally effective dose" means the amount of at least one inhibitor of the present invention that prevents, improves, or cures JMML or the symptoms associated with the disease described herein. The therapeutic efficacy and toxicity of such compounds are determined by standard pharmaceutical procedures in cell culture or experimental animals, e.g., ED 50(Therapeutic dose effective in 50% of the population) and LD 50 (The lethal dose for 50% of the population) can be determined by the dose ratio between therapeutic and toxic effects, which is the therapeutic index, and is the LD50. 50 / ED 50 It can be expressed as a ratio. The dosage regimen is determined by the attending physician and other clinical factors, preferably according to one of the methods described above.

[0199] All explanations and definitions of the above terms apply to the following embodiments, with modifications as necessary. [Modes for carrying out the invention]

[0200] The following embodiments are specific preferred embodiments of the present invention. Embodiment 1: A method for diagnosing juvenile myelomonocytic leukemia (JMML) in a subject, a) At least one biomarker present on or in hematopoietic stem cells and progenitor cells (HSPCs) in a biological sample, i) Group I consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34, and ii) Group II consisting of IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G A step of determining the amount of at least one biomarker selected from each of the following; b) A step of comparing the determined amount in step a) with a reference; and c) Step to diagnose JMML based on the comparison in step b) Methods that include...

[0201] Embodiment 2: The method according to Embodiment 1, wherein the biological sample is a tissue sample or a body fluid sample.

[0202] Embodiment 3: The method according to any one of Embodiments 1 and 2, wherein the tissue sample is a connective tissue sample, preferably bone marrow.

[0203] Embodiment 4: The method according to any one of Embodiments 1 to 3, wherein the body fluid sample is a peripheral blood sample or an umbilical cord blood sample.

[0204] Embodiment 5: The method according to any one of Embodiments 1 to 4, wherein the subject is a human.

[0205] Embodiment 6: The method according to Embodiment 5, wherein the subject is at most 16 years old, at most 15 years old, at most 14 years old, at most 13 years old, at most 12 years old, at most 11 years old, at most 10 years old, at most 9 years old, at most 8 years old, at most 7 years old, at most 6 years old, at most 5.5 years old, at most 5 years old, at most 2.5 years old, at most 2 years old, at most 1.5 years old, at most 1 year old, at most 6 months old, or less than 6 months old.

[0206] Embodiment 7: The method according to any one of Embodiments 1 to 6, wherein the reference is derived from at least one subject known to be affected by JMML.

[0207] Embodiment 8: The method of Embodiment 7, wherein the amount determined in step a) is identical to or greater than the reference, indicating that the subject is infected with JMML, or the amount determined in step a) is lower than the reference, indicating that the subject is not infected with JMML.

[0208] Embodiment 9: The method according to any one of Embodiments 1 to 8, wherein the reference is derived from at least one subject known not to have JMML.

[0209] Embodiment 10: The method according to Embodiment 9, wherein the amount determined in step a) is identical to or smaller than the reference, indicating that the subject is not infected with JMML, or the amount determined in step a) is greater than the reference, indicating that the subject is infected with JMML.

[0210] Embodiment 11: The method according to any one of Embodiments 1 to 10, wherein at least one biomarker is determined by flow cytometry, quantitative PCR (qPCR), or transcriptome sequencing, preferably bulk RNA-seq or scRNA-seq.

[0211] Embodiment 12: A method for classifying subjects suffering from JMML into low-risk or high-risk groups for JMML, a) At least one biomarker present on or in hematopoietic stem cells and progenitor cells (HSPCs) in a biological sample, i) Group I consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34, and ii) Group II consisting of IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G A step of determining the amount of at least one biomarker selected from each of the following; b) A step of comparing the determined amount in step a) with a reference; and c) A step in which subjects are classified into the JMML low-risk group or the JMML high-risk group based on the comparison in step b). Methods that include...

[0212] Embodiment 13: When at least one biomarker selected from group I is determined in step a), - A subject is classified as high-risk for JMML if the reference is derived from at least one subject known to have high-risk JMML, and the determined amount of at least one biomarker is the same as or increased compared to the reference; or - If the reference is derived from at least one subject known not to have high-risk JMML, and the determined amount of at least one biomarker is the same as or decreased compared to the reference, the subject is not classified as high-risk for JMML. The method according to Embodiment 12.

[0213] Embodiment 14: When at least one biomarker selected from group II is determined in step a), - A subject is classified as low-risk for JMML if the reference is derived from at least one subject known to have low-risk JMML, and the determined amount of at least one biomarker is the same as or increased compared to the reference; or - If the reference is derived from at least one subject known not to have low-risk JMML, and the determined amount of at least one biomarker is the same as or decreased compared to the reference, the subject is not classified as a low-risk JMML subject. The method according to any one of embodiments 12 to 13.

[0214] Embodiment 15: A method for identifying whether a subject belongs to the JMML high-risk group, a) A step of determining the amount of at least one biomarker present on or in hematopoietic stem cells and progenitor cells (HSPCs) in a biological sample, selected from group I consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34; b) A step of comparing the determined amount in step a) with a reference; and c) A step to identify whether the subject belongs to the JMML high-risk group based on the comparison in step b). Methods that include...

[0215] Embodiment 16: - A subject is classified as high-risk for JMML if the reference is derived from at least one subject known to have high-risk JMML, and the determined amount of at least one biomarker is the same as or increased compared to the reference; or - If the reference is derived from at least one subject known not to have high-risk JMML, and the determined amount of at least one biomarker is the same as or decreased compared to the reference, the subject is not classified as high-risk for JMML. The method according to Embodiment 15.

[0216] Embodiment 17: A method for identifying whether a subject belongs to the low-risk group for JMML, a) A step of determining the amount of at least one biomarker present on or in hematopoietic stem cells and progenitor cells (HSPCs) in a biological sample, selected from group II consisting of IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G; b) the step of comparing the amount determined in step a) with a reference derived from at least one subject known not to have JMML; and c) If the amount determined in step a) is the same as or lower than the reference, and based on the comparison in step b), indicates that the subject does not have JMML, then identify whether the subject belongs to the low-risk group for JMML. Methods that include...

[0217] Embodiment 18: - A subject is classified as low-risk for JMML if the reference is derived from at least one subject known to have low-risk JMML, and the determined amount of at least one biomarker is the same as or increased compared to the reference; or - If the reference is derived from at least one subject known not to have low-risk JMML, and the determined amount of at least one biomarker is the same as or decreased compared to the reference, the subject is not classified as a low-risk JMML subject. The method described in Embodiment 17.

[0218] Embodiment 19: The method according to any one of Embodiments 12 to 18, further comprising selecting a therapy for a subject suffering from JMML based on the identified JMML risk group in step c).

[0219] Embodiment 20: For diagnosing juvenile myelomonocytic leukemia in a subject, or for classifying subjects with JMML or at risk of developing it into low- or high-risk JMML groups, the following groups are used: a) Group I consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34, and b) Group II consisting of IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G The use of at least one biomarker present on or within hematopoietic stem cells and progenitor cells (HSPCs) in a biological sample, selected from one of the following.

[0220] Embodiment 21: A kit for diagnosing JMML in a subject or for classifying a subject suffering from JMML into a low or high-risk group for JMML, comprising at least one detection agent and instructions for performing the method described in any one of Embodiments 1 to 18, wherein the at least one detection agent is CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFP present on or in hematopoietic stem cells and progenitor cells (HSPCs). A kit capable of specifically detecting biomarkers selected from the group consisting of I, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G.

[0221] Embodiment 22: An inhibitor for use in the treatment and / or prevention of juvenile myelomonocytic leukemia (JMML) that specifically inhibits at least one biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G, present on or in hematopoietic stem cells and progenitor cells (HSPCs).

[0222] Embodiment 23: The inhibitor for use according to Embodiment 22, wherein the inhibitor specifically binds to and inhibits at least one biomarker.

[0223] Embodiment 24: The inhibitor for use according to Embodiment 22 or 23, wherein the inhibitor is a peptide, protein, small molecule, lipid, or aptamer.

[0224] Embodiment 25: The inhibitor for use according to Embodiment 22 or 23, wherein the inhibitor is an antibody or an antigen-binding fragment thereof.

[0225] Embodiment 26: The inhibitor for use according to Embodiment 25, wherein the antibody or its antigen-binding fragment is alemtuzumab.

[0226] Embodiment 27: The inhibitor according to Embodiment 22, wherein the inhibitor specifically binds to at least one biomarker and inhibits its translation, and the at least one biomarker is expressed in nucleic acid, preferably mRNA.

[0227] Embodiment 28: The inhibitor for use according to Embodiment 27, wherein the inhibitor is a ribozyme, an inhibitory RNA molecule, an antisense oligonucleotide, or a morpholino.

[0228] Embodiment 29: A pharmaceutical composition for use in treating and / or preventing juvenile myelomonocytic leukemia (JMML), comprising at least two inhibitors as defined in any one of Embodiments 22 to 28, wherein each of the inhibitors is present on or in hematopoietic stem cells and progenitor cells (HSPCs), such as CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, I A pharmaceutical composition that specifically inhibits different biomarkers selected from the group consisting of GHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G.

[0229] Embodiment 30: A pharmaceutical composition for use according to Embodiment 29, further comprising a pharmaceutically acceptable carrier.

[0230] Embodiment 31: A method for treating and / or preventing juvenile myelomonocytic leukemia (JMML), comprising administering a therapeutically effective dose of at least one inhibitor as defined in any one of Embodiments 22 to 28 to a subject in need thereof.

[0231] All references cited throughout this specification are incorporated herein by reference, both in terms of the specific disclosures they contain and in their entirety. [Brief explanation of the drawing]

[0232] [Figure 1]Patient selection for deep multimode analysis to detect novel biomarkers and therapeutic targets for JMML stem cells. Total hematopoietic cell DNA methylation array analysis (EPIC array) from eight different patients diagnosed with JMML. The heatmap shows the mean DNA methylation values ​​(β values) of 124 classifier CpGs derived from previously published DNA methylation classifiers for JMML (Schonung et al., 2021 Clinical Cancer Research). Epigenotype refers to the consensus definition of DNA methylation subgroups in JMML. High-risk group refers to HM patients, and low-risk group refers to LM and IM patients. [Figure 2] Biomarker identification and validation. (A) Dot plot summarizing scRNA-seq expression data for identified high and low-risk HSC biomarkers. Biomarkers represent genes that encode putative cell surface markers and are differentially expressed in HSCs between high-risk and low-risk JMML patients. High and low-risk groups are defined as described in Figure 1 above. The normal group represents umbilical cord blood-derived HSCs from healthy donors. The dot color represents the normalized mean expression value of single-cell data per group. The dot size represents the percentage of cells detected to express the marker gene per group. (B) Histograms of FACS analysis of high-risk JMML HSC surface markers CD52, CD69, and CD164 on JMML Lin-CD34+ HSPCs from low-to-high-risk patients determined in Figure 1, confirming the expected increase in cell surface protein expression in high-risk patients. [Figure 3] Functional validation of anti-CD52 treatment in JMML patient-derived xenograft (PDX) mice. (A) Representative FACS plots of control and anti-CD52 treated (aremtuzumab) JMML PDX mice. Extracellular CD52 levels on human hematopoietic cells were detected using anti-CD52-FITC. FSC-A is the lateral scattering region. (B) Quantification of live human hematopoietic CD45+ cells in the bone marrow of control and anti-CD52 treated (aremtuzumab) PDX mice (n=10 mice per group). [Figure 4] Total human hematopoietic depletion in JMML PDX mice treated with anti-CD52. Panels (A)–(G) represent the number of living human cells across different hematopoietic lineages on a logarithmic scale, quantified by FACS analysis of total bone marrow from anti-CD52 treated (aremtuzumab) or control JMML PDX mice. Total hematopoietic depletion, including CD52-negative cells such as erythrocytes, confirms the JMML stem cell markers identified here as disease-transmitting cells and therapeutic targets in JMML. [Figure 5] Disease transmission of JMML is interrupted in serial transplantation experiments with anti-CD52 treatment. 2° recipient mice received total bone marrow from control mice (PBS) or mice treated with anti-CD52 (aremtuzumab). (A) Engraftment of human CD45+ cells in 2° recipients (n=7-8 mice per group), demonstrating the interruption of disease transmission with anti-CD52 treatment. (B) Kaplan-Meier curves summarizing leukemia-free survival in 2° recipients (n=3 mice per group), confirming the effectiveness of targeting JMML HSC surface markers. [Figure 6]Conservation of JMML epigenotypes in stem cells and total hematopoiesis. (A) Ultra-low input whole-genome bisulfite sequencing (WGBS) of Lin-CD34+CD38-JMML HSPCs demonstrates conservation of JMML epigenotypes in the immature JMML stem cell compartment. (B) Integration of ultra-low WGBS and DNA methylation array analysis shows that the DNA methylation pattern of JMML stem cells is conserved across the DNA methylomes of 147 patients (LM=62, IM=45, HM=40). 450k = Illumina 450k DNA methylation array data, summarized for low-risk (LM+IM) and high-risk (HM) patients. Values ​​between 0 and 1 refer to DNA methylation beta values. Conservation in 147 patients suggests that molecular programs in JMML stem cells are generally prognostically and therapeutically valuable in JMML. (C) Integration of DNA methylation data (WGBS) derived from JMML stem cells and DNA methylation array data (Illumina 450k and EPIC) derived from JMML bulk material containing mature immune cells. Risk-related DNA methylation changes in JMML stem cells were used to examine preservation in bulk patient material from 331 patients. Principal component 1 (PC1) accurately summarizes the epigenotypes of all patients as determined by the use of DNA methylation array analysis, confirming the transfer of epigenotypes from immature JMML cells to mature JMML cells. [Figure 7] Relationship between DNA methylation and surface marker gene expression. (A) Very low input WGBS data show differential methylation of JMML stem cell surface markers. (B) scRNA-seq shows methylation-related differential gene expression of JMML stem cell surface markers. Heatmaps align differentially expressed gene expression with associated differentially methylated regions (DMRs) in a 200 Mb region around each transcription start site (TSS). Identification of methylation-related gene expression changes across JMML epitypes was performed using a generalized linear model with stepwise feature removal using AIC to identify DEGs associated with DMRs.

[0233] (Examples) The examples provided are merely illustrative of the present invention and should not be construed as limiting its scope. [Example 1]

[0234] Enrichment of hematopoietic stem cells and progenitor cells To classify the epigenotypes and thus the risk groups of JMML patients, DNA methylation array analysis of primary hematopoietic cells (Infinium Human Methylation EPIC Bead Chip (EPIC) array) was performed as previously described (Schonung et al., 2021, Clinical Cancer Research). Briefly, 100–250 ng of genomic DNA (gDNA) was submitted to the Genomics and Proteomics Core Facility at the German Cancer Research Center (Heidelberg, Germany), and the data were analyzed using the previously described "RnBeads" Bioconductor package (Lipka et al., 2018, Nature Communications, Schonung et al., 2021 Clinical Cancer Research). Based on 124 previously published CpG DNA methylation classifiers for JMML (Schonung et al., 2021 Clinical Cancer Research), patients were classified into one of three epigenetic subgroups: hypomethylated (LM), moderately methylated (IM), or hypermethylated (HM) .

[0235] To obtain material for single-cell RNA sequencing (scRNA-seq) of JMML hematopoietic stem cells and progenitor cells (HSPCs), primary cells derived from JMML patients across epigenetic risk groups were isolated from biopsies using fluorescence-activated cell sorting (FACS). Table 1 summarizes the antibodies used to enrich Lin-CD34+CD38-HSPCs.

[0236] [Table 1] [Example 2]

[0237] Single-cell sequencing Approximately 10,000 JMML HSPCs were subjected to single-cell RNA sequencing (scRNA-seq) using a 10X Genomics chromium platform, following the manufacturer's instructions. Single-cell sequencing libraries were generated using the Chromium Single Cell 3' Library & Gel Bead Kit v2 and Single Cell A Chip Kit. All libraries were sequenced on an Illumina HiSeq 4000 in 26+74bp paired-end mode at the Genomics and Proteomics Core Facility of the German Cancer Research Center (Heidelberg, Germany). [Example 3]

[0238] Data evaluation from single-cell sequencing Approximately 10,000 JMML HSPCs were subjected to single-cell RNA sequencing (scRNA-seq) using a 10X Genomics chromium platform, following the manufacturer's instructions. Single-cell sequencing libraries were generated using the Chromium Single Cell 3' Library & Gel Bead Kit v2 and Single Cell A Chip Kit. All libraries were sequenced on an Illumina HiSeq 4000 in 26+74bp paired-end mode at the Genomics and Proteomics Core Facility of the German Cancer Research Center (Heidelberg, Germany).

[0239] scRNA-seq data were aligned and quantified using Cell Ranger Single-Cell Software Suite (10X Genomics) and GRCh38 as the human reference genome. As a quality control, only cells with the following characteristics were retained for further analysis: more than 200 UMIs per cell, more than 100 genes per cell, and less than 5% mitochondrial reads per cell. The median absolute deviation (MAD) was used for outlier or doublet removal, respectively.

[0240] Downstream analyses were performed using Seurat V3 or V4 (Stuart and Butler et al., 2019 Cell; Hao and Hao et al., 2021 Cell) with default parameters for NormalizeData (LogNormalize), ScaleData, and FindVariableGenes. Data dimensionality reduction was performed by applying RunPCA to variable genes with a number of PCs selected based on elbow plot analysis.

[0241] As a healthy, normal control, we used scRNA-seq data derived from human umbilical cord blood, which is publicly available from the Human Cell Atlas (HCA) Data Portal (https: / / data.humancellatlas.org / explore / projects / cc95ff89-2e68-4a08-a234-480eca21ce79).

[0242] To generate consensus cell type definitions across the integrated datasets, we applied Seurat's label transfer function using publicly available human reference scRNA-seq data of hematopoiesis from healthy adults from the HCA project (Hay et al., 2018, Experimental Hematology). As a result, hematopoietic stem cells (HSCs) could be identified in both JMML and healthy reference data. [Example 4]

[0243] Identification of biomarkers specific to risk groups To identify risk-specific biomarkers, patients from the HM subgroup were considered high-risk, while those from the IM and LM subgroups were considered low-risk. To call differentially expressed genes (DEGs) between JMML high- and low-risk HSCs, Seurat's FindMarkers function was applied with default settings: genes expressed in at least 10% of cells, expression differences on a natural logarithmic scale of at least 0.25, a one-sided Wilcoxon rank-sum test, and adjusted p-values ​​for multiple tests using Bonferroni correction. To identify DEGs encoding cell surface markers, the entire list of DEGs was used as input for SurfaceGenie to calculate Surface Protein Consensus (SPC) scores (Waas et al., 2020, Bioinformatics). This resulted in a list of 61 genes with SPC scores greater than 0. This list was further reduced through manual curation to exclude genes that were most highly expressed within the JMML IM subgroup, or genes that were more highly expressed in normal control HSCs than in the JMML risk group with the highest expression of the corresponding gene. In this way, statistically significant, risk-group-specific DEGs were identified, presumably encoding cell surface factors. These genes are (1) predictive intracellular and extracellular biomarkers for high and low risk groups in JMML, and (2) potential drug targets on the cell surface of HMML HSCs. [Example 5]

[0244] Evaluation of the functional role of biomarkers To evaluate the functional role of such surface markers in the molecular pathogenesis of JMML, patient-derived xenograft mice were generated as previously described (Krombholz et al., 2016 Haematologica; Krombholz et al., 2019 Leukemia). One to four days after birth, primary JMML cells were transplanted into immunodeficient Rag2− / −γc− / − mice. Seven weeks after transplantation, anti-CD52 treatment was performed by application of alemtuzumab (Campath, Sanofi) at 100 μg / kg (i.v.) once a week for 4 rounds. Human CD45 + tissue infiltration of cells and human CD52 + cell amounts were quantified by flow cytometry using FACS. To evaluate the clinical impact of anti-CD52 treatment, total bone marrow from treated and untreated mice was applied to secondary transplantation and the overall survival of secondary recipients was monitored. Table 2 contains the antibodies used to analyze PDX mice.

[0245]

Table 2

[0246] The heatmap of the DNA methylation array analysis (EPIC array) in Figure 1 shows the average methylation values of 124 classifier CpGs in total hematopoietic cells from 8 different JMML patients. Based on these values, patients were classified into 2 LM patients (P1 - 2), 2 IM patients (P3 - 4), and 4 HM patients (P5 - 8). The subgroup of HM patients was considered high-risk JMML, while LM and IM patients were grouped together as low-risk JMML.

[0247] Hematopoietic stem and progenitor cells (HSPC) were Lin - CD34 + CD38 -Hematopoietic stem cells (HSPCs) were defined as cells and enriched by flow cytometry from each of eight patients. These HSPCs were analyzed using droplet-based single-cell RNA sequencing (scRNA-seq). In total, 13,594 hematopoietic stem cells (HSCs) were identified using reference-based cell type annotation. These JMML HSCs were used to call genes (DEGs) that are differentially expressed across the above-defined risk groups (high-risk vs. low-risk). From this transcriptome-wide list of DEGs, genes encoding putative cell surface markers were selected. To determine genes abnormally upregulated in JMML, the mean gene expression levels of JMML HSCs were compared to 1,069 HSCs isolated from human umbilical cord blood. Figure 2A summarizes the mean expression levels of 41 determined surface marker genes, showing (1) risk-group-specific differential expression patterns and (2) disease-specific overexpression. These genes are both prognostic biomarkers and therapeutic targets for JMML HSCs. Figure 2B illustrates the corresponding protein expression for the high-risk JMML biomarkers CD52, CD69, and CD164.

[0248] To functionally evaluate the roles of these surface marker genes and assess their therapeutic potential, we applied alemtuzumab, a monoclonal therapeutic anti-CD52 antibody, to an established preclinical patient-derived xenograft (PDX) mouse model of JMML. Figure 3A shows that anti-CD52 treatment was superior to human CD52 compared to control mice that did not receive alemtuzumab. + This demonstrates that we were able to specifically deplete the cells. This treatment resulted in overall depletion of human CD45+ cells in the treated mice, but the number of engrafted leukemia cells was clearly higher in the control mice (Figure 3B).

[0249] To further evaluate the therapeutic potential of anti-CD52 targeted therapy, alemtuzumab-treated and untreated JMML PDX mice were analyzed using flow cytometry. FACS was used to analyze human CD52 + Not only was efficient depletion of human CD34 confirmed, but human CD34 +CD38 - CD52 including cells - Efficient depletion of mature hematopoietic cells across the entire hematopoietic lineage, including individual cells, was also observed (Figure 4). Thus, alemtuzumab-treated targeted human HSPCs resulted in virtually complete depletion of the hematopoietic system, demonstrating that JMML HSPCs contain therapeutically vulnerable disease-transmitting cells.

[0250] Second transplantation of total bone marrow derived from alemtuzumab-treated animals and control animals confirmed that anti-CD52 treatment prevented leukemia engraftment in 2° recipients. FACS analysis showed a significant reduction in the number of engrafted human (leukemia) cells in the blood, bone marrow, spleen, liver, and lungs (Figure 5A). Furthermore, leukemia-free survival in mice receiving bone marrow from first-transplant-treated mice was strongly improved compared to mice receiving bone marrow from untreated PDX mice in the second transplantation experiment (Figure 5B). This result not only confirms the functional validity of such surface markers, but also illustrates the therapeutic potential of the biomarkers identified here. In conclusion, CD52 targeting results in efficient depletion of leukemia-transmitting stem cells in vivo and provides a preclinical rationale for further evaluation of anti-CD52 treatment for JMML patients. Moreover, this embodiment demonstrates the power of such high-precision molecular techniques.

[0251] In summary, the inventors identified novel intracellular and extracellular biomarkers for rapid, reliable, and economical risk stratification using FACS or expression analysis. Furthermore, the functional validity and therapeutic potential of such surface markers were demonstrated by anti-CD52 treatment of patient-derived xenografts. Thus, CD52 is a novel therapeutic target, as well as a prognostic and predictive biomarker for high-risk JMML. In conclusion, the inventors identified a first drug target specific to high-risk JMML. [Example 6]

[0252] The methylation patterns of patients exhibiting prognostic biomarker expression are conserved across large patient cohorts. To evaluate the preservation of molecular programs in JMML stem cells, we performed the first-ever analysis of the entire methylome of JMML stem cells by applying ultra-low input whole-genome bisulfite sequencing (WGBS) to JMML HSPCs enriched by flow cytometry. We modified a single-cell bisulfite protocol (Clark et al., 2017 Nature Protocols) to analyze up to 100 highly purified JMML HSPCs to which the above selection strategy was applied.

[0253] The DNA methylome of JMML stem cells, when determined by bulk DNA methylation array analysis in Figure 1, showed conservation of JMML epigenotypes (Figure 6A). Integration of ultra-low input WGBS and DNA methylation array data from 147 JMML patients showed conservation of disease-specific DNA methylation signatures in bulk JMML samples, indicating epigenotype transfer from immature JMML cells to mature JMML cells (Figure 6B). To evaluate JMML stem cells as the source of disease-specific epigenotypes, we showed that JMML stem cell-specific DNA methylation signatures can summarize the epigenotypes of 331 JMML patients (Figure 6C).

[0254] Furthermore, the inventors demonstrate that changes in DNA methylation are associated with the expression of JMML stem cell surface markers such as CD52 by integrating ultra-low WGBS and scRNA-seq data of JMML stem cells (Figure 7). In conclusion, the preservation of disease-specific abnormalities in more than 300 patients confirms the functional value of the JMML stem cell signature as an relevant prognostic biomarker and therapeutic target in JMML.

[0255] Cited literature Arber DA, Orazi A, Hasserjian RP et al. Blood 2022 Sep 15;140(11):1200-1228 Orazi A et al., Chapter 5: Myelodysplastic / myeloproliferative neoplasms, Swerdlow SH, editor. WHO classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th edn. Lyon, France: International Agency for Research on Cancer; 2017 Schoenung M, Meyer J, Noellke P et al. “International Consensus Definition of DNA Methylation Subgroups in Juvenile Myelomonocytic Leukemia”; Clin Cancer Res. 2021 Jan 1; 27(1):158-168 Lipka DB, Witte T, Toth R et al. “RAS-pathway mutation patterns define epigenetic subclasses in juvenile myelomonocytic leukemia”. Nat Commun 8, 2126 (2017) Krombholz CF, Gallego-Villar L, Sahoo SS et al. “Azacitidine is effective for targeting leukemia-initiating cells in juvenile myelomonocytic leukemia.” Leukemia; 2019 Jul;33(7):1805-1810 Louka E, Povinelli B, Rodriguez-Meira A et al. “Heterogeneous disease-propagating stem cells in juvenile myelomonocytic leukemia”; J Exp Med. 2021 Feb 1;218(2):e20180853 DeVos N., Hofmans, M., Lammens, T., DeWilde, B., VanRoy N., DeMoerloose, B. “Targeted therapy in juvenilemyelomonocytic leukemia:Where arewe now?” Pediatr Blood Cancer. 2022; e29930. Mayerhofer, C., Niemeyer, C.M., Flotho, C. “Current Treatment of Juvenile Myelomonocytic Leukemia” J. Clin. Med. 2021, 10, 3084. Laetsch TW., DuBois, SG., Glade Bender, J., Macy, M.E., Moreno, L. “Opportunities and Challenges in Drug Development for Pediatric Cancers” Cancer Discov (2021) 11 (3): 545-559. Niemeyer CM and Flotho C. “Juvenile myleomonocytic leukemia: who’s the driver at the wheel?”; Blood 2019 133(10): 1060-1070 Niemeyer CM, Arico M, Basso G et al. “Chronic myelomonocytic leukemia in childhood: a retrospective analysis of 110 cases”; Blood 1997 May 15;89(10):3534-43 Locatelli F, Noellke P, Zecca M, et al. “Hematopoietic stem cell transplantation (HSCT) in children with juvenile myelomonocytic leukemia (JMML): results of the EWOG-MDS / EBMT trial”. Blood 2005 Jan 1;105(1):410-9 Neubauer A, Shannon K, Liu E. “Mutations of the ras proto-oncogenes in childhood monosomy 7”. Blood. 1991;77(3):594-598. Flotho C, Valcamonica S, Mach-Pascual S et al. “RAS mutations and clonality analysis in children with juvenile myelomonocytic leukemia (JMML)”. Leukemia 1999 Jan;13(1):32-7 Bresolin S, Zecca M, Flotho C et al. “Gene expression-based classification as an independent predictor of clinical outcome in juvenile myelomonocytic leukemia”. J Clin Oncol. 2010 Apr 10;28(11):1919-27 Helsmoortel HH, Bresolin S, Lammens T et al. “LIN28B overexpression defines a novel fetal-like subgroup of juvenile myelomonocytic leukemia”. Blood 2016; 127(9): 1663-1172 Olk-Batz C, Poetsch AR, Noellke P et al. "Aberrant DNA methylation characterizes juvenile myelomonocytic leukemia with poor outcome”; Blood 2011 May 5;117(18):4871-80 Murakami N, Okuno Y, Yoshida K et al. “Integrated molecular profiling of juvenile myelomonocytic leukemia”; Blood 2018 Apr 5;131(14):1576-1586 Stieglitz E, Mazor T, Olshen AB et al. "Genome-wide DNA methylation is predictive of outcome in juvenile myelomonocytic leukemia”; Nat Commun 8, 2127 (2017) Loh ML, “Recent advances in the pathogenesis and treatment of juvenile myelomonocytic leukaemia”; British Journal of Haematology, vol. 152, issue 6, p. 677-687 Lapidot T, Grunberger T, Vormoor J, et al. ”Identification of human juvenile chronic myelogenous leukemia stem cells capable of initiating the disease in primary and secondary SCID mice”; Blood. 1996;88(7):2655-2664. Iversen PO, Lewis ID, Turczynowicz S et al. “Inhibition of granulocyte-macrophage colony-stimulating factor prevents dissemination and induces remission of juvenile myelomonocytic leukemia in engrafted immunodeficient mice”; Blood 1997; 90:4910-7 Krombholz CF, Aumann K, Kollek M et al. “Long-term serial xenotransplantation of juvenile myelomonocytic leukemia recapitulates human disease in Rag2- / -γc- / - mice”; Haematologica 2016 May, vol. 101, no. 5 Velten L, Haas SF, Raffel S, et al. “Human haematopoietic stem cell lineage commitment is a continuous process”; Nature Cell Biology 2017 Apr;19(4):271-281 Hay SB, Ferchen K, Chetal K et al. “The Human Cell Atlas bone marrow single-cell interactive web portal”; Exp Hematol. 2018 Dec;68:51-61 Waas M, Snarrenberg ST, Littrell J et al. “SurfaceGenie: a web-based application for prioritizing cell-type-specific marker candidates”; Bioinformatics. 2020 Jun 1;36(11):3447-3456 Orazi et al. 2016 WHO, Chapter 5, Myelodysplastic / myeloproliferative neoplasms

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Claims

1. A method for diagnosing juvenile myelomonocytic leukemia (JMML) in the subject, a) At least one biomarker present on or in hematopoietic stem cells and progenitor cells (HSPCs) in a biological sample, i) Group I consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34, and ii) Group II consisting of IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G A step of determining the amount of at least one biomarker selected from each of the following; b) A step of comparing the determined amount in step a) with a reference; and c) Step to diagnose JMML based on the comparison in step b) Methods that include...

2. The method according to claim 1, wherein the biological sample is a tissue sample or a body fluid sample.

3. The method according to claim 1 or 2, wherein the subject is a human being and is at most 16 years old, at most 15 years old, at most 14 years old, at most 13 years old, at most 12 years old, at most 11 years old, at most 10 years old, at most 9 years old, at most 8 years old, at most 7 years old, at most 6 years old, at most 5.5 years old, at most 5 years old, at most 2.5 years old, at most 2 years old, at most 1.5 years old, at most 1 year old, at most 6 months old, or less than 6 months old.

4. The method according to any one of claims 1 to 3, wherein the reference is derived from at least one subject known to have JMML, and the amount determined in step a) which is identical to or greater than the reference indicates that the subject has JMML, or the amount determined in step a) which is lower than the reference indicates that the subject does not have JMML.

5. The method according to any one of claims 1 to 3, wherein the reference is derived from a subject known not to have JMML, and the amount determined in step a) which is identical to or smaller than the reference indicates that the subject is not affected by JMML, or the amount determined in step a) which is greater than the reference indicates that the subject is affected by JMML.

6. A method for classifying individuals suffering from JMML into low-risk or high-risk groups for JMML, a) At least one biomarker present on or in hematopoietic stem cells and progenitor cells (HSPCs) in a biological sample, i) Group I consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34, and ii) Group II consisting of IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G A step of determining the amount of at least one biomarker selected from each of the following; b) A step of comparing the determined amount in step a) with a reference; and c) A step to classify subjects into low or high-risk groups for JMML based on the comparison in step b). Methods that include...

7. The method according to claim 6, wherein if at least one biomarker selected from group I is determined in step a), the subject is classified into the high-risk group for JMML, or if at least one biomarker selected from group II is determined in step a), the subject is classified into the low-risk group for JMML.

8. A method for identifying whether a subject belongs to the JMML high-risk group, a) A step of determining the amount of at least one biomarker present on or in hematopoietic stem cells and progenitor cells (HSPCs) in a biological sample, selected from group I consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34; b) A step of comparing the determined amount in step a) with a reference; and c) A step to identify whether the subject belongs to the JMML high-risk group based on the comparison in step b). Methods that include...

9. A method for identifying whether a subject belongs to the low-risk group for JMML, a) A step of determining the amount of at least one biomarker present on or in hematopoietic stem cells and progenitor cells (HSPCs) in a biological sample, selected from group II consisting of IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G; b) A step of comparing the determined amount in step a) with a reference; and c) A step to identify whether the subject belongs to the JMML low-risk group based on the comparison in step b). Methods that include...

10. The method according to claim 8 or 9, further comprising selecting a therapy for subjects suffering from JMML based on the identified JMML risk group in step c).

11. To diagnose juvenile myelomonocytic leukemia (JMML) in the subjects, or to classify subjects with JMML or those at risk of developing it into low- or high-risk JMML groups, the following groups are used: a) Group I consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34, and b) Group II consisting of IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G The use of at least one biomarker present on or within hematopoietic stem cells and progenitor cells (HSPCs) in a biological sample, selected from one of the following.

12. A kit for diagnosing juvenile myelomonocytic leukemia (JMML) in a subject, or for classifying a subject with JMML into a low- or high-risk group for JMML, comprising at least one detection agent and instructions for performing the method according to any one of claims 1 to 10, wherein the at least one detection agent contains CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, present on or in hematopoietic stem cells and progenitor cells (HSPCs). A kit capable of specifically detecting biomarkers selected from the group consisting of TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G.

13. An inhibitor for use in the treatment and / or prevention of juvenile myelomonocytic leukemia (JMML) that specifically inhibits at least one biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G, present on or within hematopoietic stem cells and progenitor cells (HSPCs).

14. The inhibitor for use according to claim 13, wherein the inhibitor specifically binds to and inhibits at least one biomarker.

15. The inhibitor for use according to claim 14, wherein the inhibitor is a peptide, protein, small molecule, lipid, aptamer, or antibody or antigen-binding fragment thereof, preferably alemtuzumab.

16. The inhibitor according to claim 13, wherein the inhibitor specifically binds to at least one biomarker and inhibits its translation, and the at least one biomarker is expressed in nucleic acid, preferably mRNA, and preferably the inhibitor is a ribozyme, an inhibitory RNA molecule, an antisense oligonucleotide, or a morpholino.

17. A pharmaceutical composition for use in treating and / or preventing juvenile myelomonocytic leukemia (JMML), comprising at least two inhibitors as defined in any one of claims 20 to 26, wherein each of the inhibitors is present on or in hematopoietic stem cells and progenitor cells (HSPCs), such as CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, A pharmaceutical composition that specifically inhibits different biomarkers selected from the group consisting of CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RAB11A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, BEST1, EREG, SLC5A3, SERK, PRRG3, NINJ1, MGST1, and HLA-G.

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