Methods and means for diagnosis and risk stratification of juvenile granulocytic leukemia

Abstract

The present invention relates to the diagnosis and assessment of juvenile granulocytic leukemia (JMML). In particular, it relates to a method of diagnosing JMML in a subject, said method comprising: a) determining the amount of at least one biomarker present on or in hematopoietic stem and progenitor cells (HSPC) in a biological sample, said at least one biomarker being selected from the group consisting 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, RABI1A, SELL, VAMPS, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, b) comparing the quantity determined in step a) with a reference; and c) diagnosing the JMML based on the comparison of step b). In addition, the invention also relates to a method for classifying subjects suffering from JMML as a low-risk or high-risk group of JMML. Furthermore, the invention relates to the use of at least one biomarker present on or in HSPC in a biological sample for diagnosing JMML in a subject suffering from or at risk of developing into JMML as a low-risk or high-risk group of JMML. Furthermore, the present invention relates to a kit for diagnosing JMML in a subject or classifying a subject suffering from JMML as a low-risk group or a high-risk group of JMML. Furthermore, the present invention relates to the use of an inhibitor for the treatment and / or prevention of JMML, said inhibitor specifically inhibiting 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, RABI1A, SELL, VAMPS, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, CD34, IGLL1, B, present on or in hematopoietic The invention also relates to a pharmaceutical composition for the treatment and / or prevention of JMML, said pharmaceutical composition comprising at least two inhibitors according to the invention. Finally, the present invention envisages a method of treating and / or preventing JMML.

Description

[0001] The present invention relates to the diagnosis and evaluation of juvenile myelomonocytic leukemia (JMML). In particular, it relates to a method for diagnosing JMML in a subject, the method comprising: a) determining the amount of at least one biomarker present on or in hematopoietic stem and progenitor cells (HSPCs) in a biological sample, the at least one biomarker being 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, SELK, PRRG3, NINJ1, MGST1, and HLA-G, b) comparing the amount determined in step a) with a reference, c) diagnosing JMML based on the comparison of step b). Furthermore, the present invention also relates to a method for classifying a subject with JMML into a low-risk or high-risk group of JMML. In addition, the present invention also relates to the use of at least one biomarker present on or in HSPCs in a biological sample for diagnosing JMML in a subject having or at risk of developing JMML into a low-risk or high-risk group of JMML. Moreover, the present invention relates to a kit for diagnosing JMML in a subject or classifying a subject with JMML into a low-risk group or a high-risk group of JMML. In addition, the present invention also relates to the use of an inhibitor for the treatment and / or prevention of JMML, the inhibitor specifically inhibiting 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 and progenitor cells (HSPCs). The present invention also relates to a pharmaceutical composition for the treatment and / or prevention of JMML, the pharmaceutical composition comprising at least two inhibitors according to the present invention.Finally, the present invention contemplates a method for treating and / or preventing JMML.

[0002] Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive blood cancer that typically presents in early childhood, with a median age at diagnosis of less than 2 years (Niemeyer et al. 1997, Blood). According to the latest WHO classification, JMML is defined as a myeloproliferative neoplasm (MPN) (Khoury et al. 2022 Leukemia), sharing some characteristics with adult chronic myelomonocytic leukemia. This disorder originates from pluripotent hematopoietic stem / progenitor cells and is characterized by the overproduction of mature and immature myeloid cells. Hyperactive RAS signaling is presumed to be the major driver of JMML. Somatic mutations in the KRAS, NRAS, PTPN11, NF1, and / or CBL genes are present in the majority of patients (approximately 90%), which genetically and clinically define distinct subtypes ( et al., Clin Cancer Res. 2021 Jan 1; 27(1):158-168).

[0003] Another key feature of JMML is its heterogeneity. For example, the clinical course of the disease is highly heterogeneous, and only partial prediction can be made using clinical features. This clinical heterogeneity is reflected in the treatment considerations, ranging from watchful waiting to early allogeneic hematopoietic stem cell transplantation (HSCT) (Niemeyer & Flotho 2019, Blood). Although approximately 15% of cases will ultimately resolve spontaneously, more than 50% of patients will relapse after HSCT (Locatelli et al. 2005 Blood).

[0004] This clinical heterogeneity highlights the urgent need for a diagnostic method that allows for the early identification of high-risk patients. In addition, there is a need to develop and test novel risk-adapted molecular targeted therapy regimens. On the one hand, clinical studies of JMML have identified gene subsets defined by driver mutations that lead to overactivation of the RAS pathway (Neubauer et al. 1991 Blood; Flotho et al. 1999 Leukemia). On the other hand, gene expression-related signatures have revealed non-genetic features that vary in JMML (Bresolin et al. 2010 Journal of Clinical Oncology; Helsmoortel et al. 2016 Blood). Although both genetic and non-genetic features have been shown to have a certain degree of prognostic ability, these biomarkers have failed to capture the full spectrum of clinical heterogeneity observed in JMML. Therefore, clinical studies of JMML have long lacked meaningful and robust prognostic biomarkers.

[0005] It is thought that gene regulatory or epigenetic programs are the missing link between the genetic and non-genetic features that underlie the clinical heterogeneity of JMML. Indeed, stratifying JMML patients by DNA methylation patterns has revealed a significant correlation between the epigenetics of JMML and prognosis (Olk-Batz et al. 2011, Blood). Using genome-wide array-based DNA methylation analysis, epigenetic subgroups of JMML have been established (Lipka et al. 2017, Nature Communications; Murakami et al. 2018, Blood; Stieglitz et al. 2017, Nature Communications), which have revealed DNA methylation as the only important factor predicting overall survival in JMML. In addition, DNA methylation appears to predict treatment response. Treatment with hypomethylating agents seems to be particularly effective in the low-risk and intermediate-risk DNA methylation subgroups (Niemeyer et al. 2021 Blood Advances). However, given the poor response to hypomethylating agents and the high relapse rate after HSCT, there is currently a lack of treatment options for high-risk JMML (Loh 2011, British Journal of Haematology).

[0006] Preclinical studies using xenograft mouse models derived from patients have revealed that transplanted JMML cells are able to 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). In addition, the DNA methylation patterns specific to the JMML epigenetic subsets are also re-established in xenograft mice. This suggests that hematopoietic stem cells (HSCs) can be regarded as the leukemic initiating cells of JMML, which has slowly increased the interest in this cell population in the field (Louka et al. 2021, Journal of Experimental Medicine). Currently, the interpretation of clinical outcomes through clinical or genetic characteristics is insufficient, and the dysregulation of the DNA methylation landscape has been shown to be a non-random subset-specific prognostic feature. Currently, there is a lack of systematic and subset-stratified characterization of HSPCs in JMML.

[0007] In principle, JMML is a rare aggressive clonal neoplasm (DeVos 2022, Mayerhofer 2021). It is estimated that in children aged 0 to 14 years, the incidence of JMML is 1.3 cases per million person-years (according to the WHO classification of tumors). Therefore, different from other tumors, only very limited patient samples can be obtained. In addition, the biological samples stored in the European JMML biobank are usually DNA samples rather than viable cryopreserved cells. However, pediatric cancers, especially aggressive cancers occurring in early childhood such as JMML, have limited treatment options to date, so there has always been a need for treatment methods (Laetsch 2021).

[0008] Therefore, there is a need for prognostic and diagnostic means for risk stratification of JMML and the development of new subset-specific therapies.

[0009] The fundamental technical problem of the present invention can be regarded as providing means and methods to meet the above needs. This technical problem is solved by the claims and the embodiments described below.

[0010] The present invention relates to a method for diagnosing juvenile myelomonocytic leukemia (JMML) in a subject, the method comprising:

[0011] a) determining the amount of at least one biomarker on hematopoietic stem and progenitor cells (HSPCs) in a biological sample, the at least one biomarker being selected from each of the following:

[0012] 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

[0013] ii) Group II consisting of: IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1 and HLA-G;

[0014] b) Comparing the quantity determined in step a) with a reference; and

[0015] c) Diagnosing JMML based on the comparison in step b).

[0016] It should be understood that in the specification and claims, "a" or "an" may refer to one or more of the items mentioned below, depending on the context in which it is used. Thus, for example, reference to "an" item may mean that at least one item is available.

[0017] The terms "having", "comprise" or "include" as used hereinafter are not exclusive. Thus, these terms can refer both to a situation where no other features exist in the entity described in the present context in addition to the features introduced by these terms, and to a situation where one or more other features exist. For example, the expressions "A has B", "A comprises B" and "A includes B" can refer both to a situation where there are no other elements in A except B (i.e., the situation where A consists only and exclusively of B), and to a situation where there are one or more other elements in entity A in addition to B, such as element C, elements C and D, or more elements.

[0018] The terms "especially", "more especially", "typically", "more typically" or similar terms are used in conjunction with additional and / or alternative features, but do not limit the possibility of alternatives. Thus, the features introduced by these terms are additional features and / or alternative features and are not intended to limit the scope of the claims in any way. As will be recognized by the person skilled in the art, the present invention can be achieved by using more alternative features. Similarly, features introduced with expressions such as "in an embodiment of the present invention" are intended as additional and / or alternative features, without any limitation on alternative embodiments of the present invention, without any limitation on the scope of the present invention, and without any limitation on the possibility of combining the features introduced in this way with other additional and / or alternative or non-additional and / or alternative features of the present invention.

[0019] Furthermore, hereinafter, the terms "preferred", "more preferred", "most preferred", "especially", "more especially", "typically" and "more typically" are used in conjunction with features to indicate that these features are preferred features, i.e., these terms should indicate that alternative features can also be envisaged in accordance with the present invention.

[0020] Furthermore, it should be understood that the term "at least one" as used herein means that one or more of the items mentioned after this term can be used in the present invention. For example, if the term indicates that at least one item should be used, this can be understood as one item or more than one item, i.e., two, three, four, five or any other number. Depending on the items mentioned by the term, the person skilled in the art understands the upper limit (if any) that the term can refer to.

[0021] The term "about" as used herein means that for any number recited after this term, there is an exact interval within which the technical effect can be achieved. Thus, in the context of the present invention, the term "about" means a deviation of ±20%, ±10%, ±5%, ±2% or ±1% from the indicated parameter or value. This also takes into account the deviations typically caused by measurement techniques and the like.

[0022] The method of the present invention may consist of the above steps or may include other steps, such as steps for further evaluating the evaluation results obtained in step (c), steps for suggesting treatment measures such as treatment. Furthermore, it may also include steps before step (a), such as steps related to sample pretreatment. However, preferably, it is contemplated that the above method is an in vitro method that does not require any steps to be performed on a human or animal body. In addition, the method can also be automated. Typically, the determination of biomarkers can be supported by robotic instruments, while the comparison and evaluation can be supported by data processing instruments such as computers.

[0023] As used herein, the term "diagnosis" refers to the assessment of the health status of a subject. Thus, "diagnosis" as used herein refers to determining whether a subject has juvenile myelomonocytic leukemia (JMML), predicting the risk of developing JMML, and / or predicting any deterioration in the health status of the subject, particularly with respect to the signs and symptoms associated with JMML. The most common symptoms and signs of JMML include hepatosplenomegaly, lymphadenopathy, and anemia, as well as thrombocytopenia, which result in pallor, fatigue, weakness, bleeding, and / or bruising, respectively. Patients may also experience bone and joint pain, abdominal pain, recurrent fevers, infections, and / or dry cough and dyspnea.

[0024] As is known to those skilled in the art, although such a diagnosis is preferred, it may not generally be 100% correct for all subjects under study. However, the term requires that a significant portion of the subjects be correctly diagnosed in a statistical sense. Using various well-known statistical evaluation tools, such as determination of confidence intervals, determination of P-values, Student's t-test, Mann-Whitney test, etc., those skilled in the art can readily determine whether a portion is statistically significant without much ado. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Typically contemplated 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, 0.05. It is understood that the diagnostic method of the present invention should assist medical personnel in making a final diagnosis. To improve the accuracy of the diagnosis, medical examinations may also consider more factors, such as the presence of JMML symptoms described elsewhere herein.

[0025] As used herein, a "subject" can be an animal, and preferably a mammal. More preferably, the subject is a human. The subject under study by the method of the present invention is preferably an adolescent, i.e., a child or young adult. More particularly, the subject is up to 16 years old, up to 15 years old, up to 14 years old, up to 13 years old, up to 12 years old, up to 11 years old, up to 10 years old, up to 9 years old, up to 8 years old, up to 7 years old, or up to 6 years old, up to 5.5 years old, up to 5 years old, up to 4.5 years old, up to 4 years old, up to 3.5 years old, up to 3 years old, up to 2.5 years old, up to 2 years old, up to 1.5 years old, up to 1 year old, up to 6 months, or less than 6 months.

[0026] Subjects studied by the methods of the present invention should be subjects suffering from or at risk of developing JMML. As used herein, suffering from JMML means that the subject should exhibit clinical parameters, signs, and / or symptoms of JMML. Thus, according to the present invention, the subject is generally a subject suffering from or suspected of suffering from JMML. As used herein, at risk of developing JMML refers to a subject who appears healthy, has not yet exhibited clinical signs or symptoms of JMML, but has an increased risk of developing JMML; or a subject who exhibits faint signs or symptoms of disease, but has a risk of deterioration of the associated disease or associated signs or symptoms.

[0027] According to the present invention, the term "biomarker" refers to a biomolecule, the presence, absence, or abundance of which can indicate a health condition. According to the present invention, the health condition can be JMML or no JMML, low-risk JMML, and / or high-risk JMML. According to the present invention, the biomarker can be a protein or a fragment thereof more specifically mentioned in other parts of this text. However, the biomarker can be a transcribed nucleic acid molecule, the presence, absence, or abundance of which can be used as a surrogate for the protein. Preferably, such a transcribed nucleic acid molecule is a messenger RNA molecule (mRNA) or any precursor or variant thereof, including pre-mRNA or mRNA for splice variants. According to the present invention, these RNA nucleic acid molecules can also be determined as biomarkers. Thus, it should be understood that if, for example, CD52 is to be determined as a biomarker according to the present invention, then both the CD52 protein and the transcribed nucleic acid molecule encoding the CD52 protein, such as CD52 mRNA, can be determined. This also applies to all other biomarkers mentioned herein, unless otherwise stated. Proteins are mentioned in the specification, however, those skilled in the art are well aware of the transcribed nucleic acid molecules belonging to the said proteins and the genes encoding them.

[0028] Biomarkers used according to the present invention cover biomarkers associated with high-risk JMML or low-risk JMML.

[0029] High-risk JMML biomarkers are those biomarkers of Group I as mentioned in 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, high-risk JMML biomarkers are those biomarkers of Group I as mentioned in the present invention and are 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, high-risk JMML biomarkers are those biomarkers of Group I as mentioned in the present invention and are 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 biomarkers of Group I are selected from the group consisting of: CD52, CD69, CD164, IGHM, RALA, and HLA-DRA.

[0030] Low-risk JMML biomarkers refer to the biomarkers of Group II as mentioned in the present invention and are selected from the group consisting of: IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G. More preferably, the low-risk biomarkers of Group II are selected from the group consisting of: IGLL1 and HLA-G.

[0031] Thus, in order to diagnose JMML in a subject, at least one biomarker must be determined from the aforementioned high-risk JMML biomarkers (Group I) and at least one biomarker must be determined from the aforementioned low-risk JMML biomarkers (Group II). More preferably, all of the aforementioned biomarkers in either of the aforementioned Groups I and II can be determined. Thus, the diagnosis of JMML (including all disease stages from low risk to high risk) can be improved.

[0032] As used herein, the term "CD52" refers to the cluster of differentiation 52 glycoprotein encoded by the CD52 gene in humans, which is 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 polypeptide consisting of 61 amino acids, anchored to glycosylphosphatidylinositol (GPI). It is speculated that it acts as an anti-adhesion protein, allowing cells to move freely because it is highly negatively charged and present on sperm cells and lymphocytes. Several orthologs of CD52 in various animal species have been reported.

[0033] The CD52 protein referred to in the present invention is preferably human CD52 having the amino acid sequence deposited under UniProt accession number P31358. It should be understood that the term "CD52" also relates to variants of the aforementioned protein. These variants have at least the same basic biological and immunological properties as the aforementioned CD52 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence that differs by at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the human CD52 protein, preferably over the entire length of the CD52 protein.

[0034] As used herein, the term "RAMP1" refers to receptor activity-modifying protein 1 encoded by the RAMP1 gene in humans. It belongs to the RAMP family of single transmembrane domain proteins called receptor (calcitonin) activity-modifying proteins, including three members, RAMP1, RAMP2, and RAMP3. RAMPs are considered type I transmembrane proteins with an extracellular N-terminus and a cytoplasmic C-terminus. An important function of RAMP1 is to control the glycosylation of the calcitonin receptor (CRL) and thus transport it to the cell membrane. RAMP1 is widely expressed in the brain, spinal cord, gastrointestinal tract, adrenal glands, perivascular nerves, and arterial smooth muscle. Several orthologs of RAMP1 in various animal species have been reported.

[0035] The RAMP1 protein referred to in the present invention is preferably human RAMP1 having the amino acid sequence deposited under UniProt accession number O60894. It should be understood that the term "RAMP1" also refers to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned RAMP1 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence that is different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the human RAMP1 protein, preferably over the entire length of the RAMP1 protein.

[0036] The term "LTB" (LTβ) as used herein refers to the lymphotoxin-β protein encoded by the LTB gene in humans, also known as tumor necrosis factor C (TNF-C). It is a type II membrane protein of the TNF superfamily and is the main ligand of the lymphotoxin-β receptor. LTB interacts with two ligands: the membrane heterotrimeric lymphotoxin α (LTα) and the homotrimeric LIGHT. Generally, it is expressed by epithelial cells, stromal cells, dendritic cells (DCs), and macrophages, but not on lymphocytes. It is well known that LTB is a key regulator of lymphoid organogenesis and inflammation. In addition, it has been determined that LTB has a tumor-promoting function. For example, in several cancer models, mice overexpressing LTα or LTβ showed increased tumor growth and metastasis. For LTB, two human isoforms (UniProt accession numbers Q06643-1 and Q06643-2) are known. Several orthologs of RAMP1 have been reported in various animal species.

[0037] The LTB protein mentioned according to the present invention is preferably human LTB having the amino acid sequence deposited under UniProt accession number Q06643. It should be understood that the term "LTB" also refers to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned LTB protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the human LTB protein, preferably over the entire length of the LTB protein.

[0038] The term "LST1" used herein 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 regulating the immune response. To date, 13 isoforms have been described, which are generated by alternative splicing. For example, isoform 1 and isoform 2 have an inhibitory effect on lymphocyte proliferation. Several orthologs of LST1 have been reported in various animal species.

[0039] The LST1 protein mentioned according to the present invention is preferably human LST1 having the amino acid sequence deposited under UniProt accession number O00453. It should be understood that the term "LST1" also refers to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned LST1 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the human LST1 protein, preferably over the entire length of the LST1 protein.

[0040] As used herein, the term "JAML" refers to the junctional adhesion molecule-like protein encoded by the JAML gene in humans or AMICA1. JAML is a transmembrane protein on the leukocyte plasma membrane that controls the migration and activation of adjacent epithelial and endothelial cells by interacting with the plasma membrane receptor CXADR found on these cells. The interaction between these two receptors mediates the activation of γ-δ T cells, a subset of T cells resident in epithelial tissues that are involved in tissue homeostasis and repair. After binding to epithelial CXADR, JAML induces downstream cell signaling events in γ-δ T cells via PI3 kinase and MAP kinase. It leads to the proliferation of T cells and the production of cytokines and growth factors, which in turn stimulate the repair of epithelial tissues. It also controls the migration of leukocytes within epithelial and endothelial tissues through adhesive interactions with epithelial and endothelial CXADR. Typically, JAML is located in bicellular tight junctions, the nuclear cytoplasm, and the plasma membrane. Four isoforms of JAML have been described, and several orthologs of JAML in different animal species have been reported.

[0041] The JAML protein referred to in the present invention is preferably human JAML having the amino acid sequence deposited under UniProt accession number Q86YT9. It should be understood that the term "JAML" also relates to variants of the above protein. These variants have at least the same basic biological and immunological properties as the above JAML protein. In particular, they have the same basic biological and immunological properties if they can be detected by the same specific detection methods mentioned in this specification. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence that differs by at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the human JAML protein, preferably over the entire length of the JAML protein.

[0042] As used herein, the term "IFITM3" refers to interferon-induced transmembrane protein 3 encoded by the IFITM3 gene in humans. The IFITM3 protein is a family of interferon-induced antiviral proteins. This family contains five members, including IFITM1, IFITM2, and IFITM3, and belongs to the CD225 superfamily. This protein restricts the entry of a variety of viral pathogens into cells, such as influenza A virus, Ebola virus, and Sars-CoV-2. Several orthologs of IFTIM3 in various animal species have been reported.

[0043] The IFITM3 protein referred to in the present invention is preferably human IFITM3 having the amino acid sequence deposited under UniProt accession number Q01628. It should be understood that the term "IFITM3" also relates to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned IFITM3 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the specific amino acid sequence of the IFITM3 protein, preferably over the entire length of the IFITM3 protein.

[0044] The term "CD7" as used herein refers to the cluster of differentiation 7 protein encoded by the CD7 gene in humans. CD7 is a transmembrane protein on thymocytes and mature T cells. It belongs to the immunoglobulin superfamily and plays an important role in T cell interactions and T cell / B cell interactions during early lymphoid development. Five potential isoforms are currently known, and several orthologs of CD7 in different animal species have been reported.

[0045] The CD7 protein referred to in the present invention is preferably human CD7 having the amino acid sequence deposited under UniProt accession number P09564. It should be understood that the term "CD7" also relates to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned CD7 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the specific amino acid sequence of the CD7 protein, preferably over the entire length of the CD7 protein.

[0046] As used herein, the term "CD69" refers to the cluster of differentiation 69 protein encoded by the CD69 gene in humans. It is a disulfide-linked homodimeric protein with two distinct subunits. Each subunit consists of: an extracellular C-type lectin domain (CTLD), linked to a single transmembrane region, followed by a short cytoplasmic tail. It is an early activation marker and is 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 the expression of CD69. It is involved in lymphocyte proliferation and functions as a signaling receptor in lymphocytes. It is also associated with T cell differentiation and the retention of lymphocytes in lymphoid organs. Currently, one potential isoform is known, and several orthologs of CD69 have been reported in various animal species.

[0047] The CD69 protein referred to in the present invention is preferably human CD69 having the amino acid sequence deposited under UniProt accession number Q07108. It should be understood that the term "CD69" also encompasses variants of the above protein. These variants have at least the same basic biological and immunological properties as the above CD69 protein. In particular, they have the same basic biological and immunological properties if they can be detected by the same specific detection methods mentioned in this specification. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence that differs by at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the CD69 protein, preferably over the entire length of the CD69 protein.

[0048] As used herein, the term "CD164" refers to the cluster of differentiation 164 protein encoded by the CD164 gene in humans or sialomucin core protein 24 (also known as endolyn). This gene encodes a transmembrane sialomucin and cell adhesion molecule that regulates the proliferation, adhesion, and migration of hematopoietic progenitor cells. The encoded protein also interacts with C-X-C chemokine receptor type 4 (CXCR4) and can regulate muscle development. Elevated expression of this gene has been observed in human patients with Sezary syndrome, a type of blood cancer, and mutations in this gene may be associated with hearing impairment. Currently, five isoforms are known, and several orthologs of CD164 have been reported in various animal species.

[0049] The CD164 protein referred to in the present invention is preferably human CD164 having the amino acid sequence deposited under UniProt accession number Q04900. It should be understood that the term "CD164" also relates to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned CD164 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the CD164 protein, preferably over the entire length of the CD164 protein.

[0050] The term "CD74" as used herein refers to the cluster of differentiation 74 protein or HLA class II histocompatibility antigen gamma chain (also known as HLA-DR antigen-associated invariant chain) encoded by the CD74 gene in humans. The protein encoded by this gene is associated with the class II major histocompatibility complex (MHC) and is an important molecular chaperone that regulates antigen presentation in the immune response. It also serves as a cell surface receptor for the cytokine macrophage migration inhibitory factor (MIF), which can initiate survival pathways and cell proliferation after binding to the encoded protein. A number of alternatively spliced transcript variants encoding five different isoforms have been identified, and several orthologs of CD74 in various animal species have been reported.

[0051] The CD74 protein referred to in the present invention is preferably human CD74 having the amino acid sequence deposited under UniProt accession number P04233. It should be understood that the term "CD74" also relates to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned CD74 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the CD74 protein, preferably over the entire length of the CD74 protein.

[0052] As used herein, the term "TNF" refers to the tumor necrosis factor protein encoded by the TNF gene in humans. TNF belongs to the TNF superfamily, which consists of various transmembrane proteins with a homologous TNF domain. It is an adipokine and cytokine, produced by a variety of cell types, including lymphocytes, 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 regulating cell signaling in a broad spectrum of biological processes, including cell proliferation, differentiation, apoptosis, lipid metabolism, and blood coagulation. This cytokine is associated with 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. Mouse gene knockout studies have also shown that this cytokine has neuroprotective functions.

[0053] The TNF protein referred to in the present invention is preferably human TNF having the amino acid sequence deposited under UniProt accession number P01375. It should be understood that the term "TNF" also refers to variants of the above protein. These variants have at least the same basic biological and immunological properties as the above TNF protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence that differs by at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the TNF protein, preferably over the entire length of the TNF protein. Several orthologs of TNF have been reported in various animal species.

[0054] 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 regulates the tissue factor (TF)-dependent blood coagulation pathway. In particular, TFPI is a single-chain polypeptide that can reversibly inhibit factor Xa in the coagulation cascade. Two different isoforms have been identified, and several orthologs of TFPI have been reported in different animal species.

[0055] The TFPI protein mentioned according to the present invention is preferably human TFPI having the amino acid sequence deposited under UniProt accession number P10646. It should be understood that the term "TFPI" also relates to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned TFPI protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the TFPI protein, preferably over the entire length of the TFPI protein.

[0056] The term "DLK1" as used herein refers to the protein delta homolog 1 encoded by the DLK1 gene in humans. This gene encodes a transmembrane protein that contains multiple epidermal growth factor repeats and has a function of regulating cell growth. Soluble DLK1 cleaved by ADAM17 is involved in inhibiting adipogenesis, that is, the differentiation of preadipocytes into adipocytes. DKL1 is a member of the EGF-like family of homologous proteins. Two different isoforms have been identified so far, and several orthologs of DKL1 in different animal species have been reported.

[0057] The DLK1 protein mentioned according to the present invention is preferably human DLK1 having the amino acid sequence deposited under UniProt accession number P80370. It should be understood that the term "DLK1" also relates to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned DLK1 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the DLK1 protein, preferably over the entire length of the DLK1 protein.

[0058] As used herein, the term "CD82" refers to the cluster of differentiation 82 protein encoded by the CD82 gene in humans. CD82 belongs to the tetraspanin / transmembrane 4 superfamily. This protein functions as a metastasis suppressor. Studies have shown that during tumor progression in human cancers, the expression of this gene is downregulated and can be activated by p53 through consensus binding sequences in the promoter. Its expression is closely related to that of p53, and the loss of expression of both proteins is associated with poor survival in prostate cancer patients. Two alternatively spliced transcript variants encoding different isoforms have been identified, and several orthologs of CD82 in various animal species have been reported.

[0059] The CD82 protein referred to in the present invention is preferably human CD82 having the amino acid sequence deposited under UniProt accession number P22701. It should be understood that the term "CD82" also relates to variants of the above protein. These variants have at least the same basic biological and immunological properties as the above CD82 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence that differs by at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the CD82 protein, preferably over the entire length of the CD82 protein.

[0060] As used herein, the term "IGHM" refers to the Igμ chain C-region protein encoded by the IGHM gene in humans. The IGHM gene encodes the C-region of the μ heavy chain, which defines the isotype of IgM. Naive B cells express transmembrane forms of IgM and IgD on their surface. During an antibody response, activated B cells can switch to the expression of a single downstream heavy chain C-region gene through the process of somatic recombination (i.e., isotype switching). IGHM is associated with agammaglobulinemia-1. Two alternatively spliced transcript variants encoding two different isoforms have been identified, and several orthologs of IGHM in various animal species have been reported.

[0061] The IGHM protein mentioned according to the present invention is preferably human IGHM having the amino acid sequence deposited under UniProt accession number P01871. It should be understood that the term "IGHM" also refers to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned IGHM protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the specific amino acid sequence of the IGHM protein, preferably over the entire length of the IGHM protein.

[0062] As used herein, the term "CALCRL" refers to the calcitonin gene-related peptide type 1 receptor protein encoded by the CALCRL gene in humans. It is a G protein-coupled receptor related to the calcitonin receptor. Under normal circumstances, CALCRL is located in the endoplasmic reticulum, endosomes, and lysosomes and is presumably active in the plasma membrane. Studies have shown that this protein can regulate various physical functions of all major systems (such as respiratory, endocrine, gastrointestinal, immune, and cardiovascular). More particularly, it is thought to be involved in several processes, including the G protein-coupled receptor signaling pathway, the cell's response to sucrose stimulation, and the endocytosis of receptors. Several orthologs of CALCRL have been reported in various animal species.

[0063] The CALCRL protein mentioned according to the present invention is preferably human CALCRL having the amino acid sequence deposited under UniProt accession number Q16602. It should be understood that the term "CALCRL" also refers to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned CALCRL protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the specific amino acid sequence of the CALCRL protein, preferably over the entire length of the CALCRL protein.

[0064] As used herein, the term "RALA" refers to 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 (GTPase) superfamily, the Ras protein family. As a Ras GTPase, RalA functions as a molecular switch that becomes active when bound to GTP and inactive when bound to GDP. RalA can be activated by RalGEF and in turn activates effectors in signal transduction pathways, leading to biological outcomes. Other downstream functions include exocytosis, receptor-mediated endocytosis, tight junction biogenesis, filopodia formation, mitochondrial fission, and cytokinesis. Several orthologs of RALA have been reported in various animal species.

[0065] The RALA protein referred to in the present invention is preferably human RALA having the amino acid sequence deposited under UniProt accession number P11233. It should be understood that the term "RALA" also relates to variants of the above protein. These variants have at least the same basic biological and immunological properties as the above RALA protein. In particular, they have the same basic biological and immunological properties if they can be detected by the same specific detection methods mentioned in this specification. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence that differs by at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the RALA protein, preferably over the entire length of the RALA protein.

[0066] The term "SLC2A5" (also known as GLUT5) as used herein refers to solute carrier family 2 facilitated glucose transporter member 5 protein encoded by the SLC2A5 gene in humans. Generally, SCL2A5 is expressed at the apical border of enterocytes in the small intestine, in skeletal muscle, testis, kidney, adipose tissue, and brain. The protein encoded by this gene is a fructose transporter responsible for the absorption of fructose in the small intestine. SLC2A5 is also required for high-fructose diet-induced elevation of blood pressure. Two alternatively spliced transcript variants encoding two different isoforms have been identified, and several orthologs of SLC2A5 have been reported in various animal species.

[0067] The SLC2A5 protein referred to in the present invention is preferably human SLC2A5 having the amino acid sequence deposited under UniProt accession number P22732. It should be understood that the term "SLC2A5" also relates to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned SLC2A5 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the SLC2A5 protein, preferably over the entire length of the SLC2A5 protein.

[0068] The term "HSPA5" as used herein refers to the 70 kDa heat shock protein 5 encoded by the HSPA5 gene in humans. It is also known as binding immunoglobulin protein (BiP) or 78 kDa glucose-regulated protein (GRP-78). Under normal circumstances, it is located in the lumen of the endoplasmic reticulum (ER), where it functions as an HSP70 molecular chaperone, participating in protein folding and assembly and being a major regulator of ER homeostasis. It has been reported that the expression of this protein is elevated and atypically translocated to the cell surface in viral infections and certain types of cancer cells. Several orthologs of HSPA5 in various animal species have been reported.

[0069] The HSPA5 protein referred to in the present invention is preferably human HSPA5 having the amino acid sequence deposited under UniProt accession number P11021. It should be understood that the term "HSPA5" also relates to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned HSPA5 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the HSPA5 protein, preferably over the entire length of the HSPA5 protein.

[0070] As used herein, the term "HLA-DRA" refers to the HLA class II histocompatibility antigen DRα chain protein encoded by the HLA-DRA gene in humans. This protein is a heterodimer composed of an α chain and a β chain, both of which are anchored to the membrane. 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 response by presenting peptides from extracellular proteins, particularly pathogen-derived peptides, to T cells. Several orthologs of HLA-DRA have been reported in different animal species.

[0071] The HLA-DRA protein referred to in the present invention is preferably human HLA-DRA having the amino acid sequence deposited under UniProt accession number P01903. It should be understood that the term "HLA-DRA" also relates to variants of the above protein. These variants have at least the same basic biological and immunological properties as the above HLA-DRA protein. In particular, they have the same basic biological and immunological properties if they can be detected by the same specific detection methods mentioned in this specification. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different by at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the HLA-DRA protein, preferably over the entire length of the HLA-DRA protein.

[0072] 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 of the small GTPase superfamily. It is involved in the constitutive and regulated secretory pathways and may be involved in protein trafficking. Rab-11a controls the intracellular trafficking of the innate immune receptor TLR4 and thus also controls the transmission of receptor signals. Two isoforms are known, and several orthologs of RAB11A have been reported in various animal species.

[0073] The RAB11A protein mentioned according to the present invention is preferably human RAB11A having the amino acid sequence deposited under UniProt accession number P62491. It should be understood that the term "RAB11A" also relates to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned RAB11A protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the RAB11A protein, preferably over the entire length of the RAB11A protein.

[0074] As used herein, the term "SELL" refers to the L-selectin protein encoded by the SELL gene in humans, also known as CD62L. This gene encodes a cell surface adhesion molecule that belongs to the adhesion / homing receptor family. The encoded protein contains a C-type lectin-like domain, a calcium-binding epidermal growth factor-like domain, and two short complement-like repeats. The gene product is required for the binding of leukocytes to endothelial cells and subsequent rolling, and it promotes the migration of leukocytes to secondary lymphoid organs and sites of inflammation. Single nucleotide polymorphisms of this gene are associated with various diseases including immunoglobulin A nephropathy. Two isoforms are known, and several orthologs of SELL have been reported in various animal species.

[0075] The SELL protein mentioned according to the present invention is preferably human SELL having the amino acid sequence deposited under UniProt accession number P14151. It should be understood that the term "SELL" also relates to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned SELL protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the SELL protein, preferably over the entire length of the SELL protein.

[0076] As used herein, the term "VAMP5" refers to vesicle-associated membrane protein 5 encoded by the VAMP5 gene in humans. Synaptobrevin / VAMP, syntaxin, and 25-kD synaptosomal-associated protein are the main components of a protein complex involved in vesicle docking and / or fusion with the cell membrane. The VAMP5 gene is a member of the vesicle-associated membrane protein (VAMP) / synaptobrevin family and the SNARE superfamily. Members of this VAMP family can be involved in vesicle trafficking events related to myogenesis. Several orthologs of VAMP5 have been reported in various animal species.

[0077] The VAMP5 protein referred to in the present invention is preferably human VAMP5 having the amino acid sequence deposited under UniProt accession number O95183. It should be understood that the term "VAMP5" also relates to variants of the above protein. These variants have at least the same basic biological and immunological properties as the above VAMP5 protein. In particular, they have the same basic biological and immunological properties if they can be detected by the same specific detection methods mentioned in this specification. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different by at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the VAMP5 protein, preferably over the entire length of the VAMP5 protein.

[0078] 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 can play a role in the immune system process. It can prevent apoptosis induced by FAS, TNFα, and FADD, but does not increase the expression of apoptosis inhibitors BCL2 and BCLXL, and seems to activate an inhibitory pathway to prevent the activation of CASP8 after FAS stimulation, rather than blocking downstream apoptotic signals. FCMR also shows that it plays a role in inhibiting FAS-induced apoptosis by upregulating CFLAR to prevent the processing of CASP8. Three isoforms are known, and several orthologs of FCMR have been reported in various animal species.

[0079] The FCMR protein mentioned according to the present invention is preferably human FCMR having the amino acid sequence deposited under UniProt accession number O60667. It should be understood that the term "FCMR" also relates to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned FCMR protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the specific amino acid sequence of the FCMR protein, preferably over the entire length of the FCMR protein.

[0080] 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 fold and a cytoplasmic domain with an immunoreceptor tyrosine-based activation motif. It acts as a pattern recognition receptor, recognizing β-1,3-linked glucan and β-1,6-linked glucan from fungi and plants, and in this way plays a role in the innate immune response. Ten isomers are currently known, and several orthologs of CLEC7A have been reported in various animal species.

[0081] The CLEC7A protein mentioned according to the present invention is preferably human CLEC7A having the amino acid sequence deposited under UniProt accession number Q9BXN2. It should be understood that the term "CLEC7A" also relates to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned CLEC7A protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the specific amino acid sequence of the CLEC7A protein, preferably over the entire length of the CLEC7A protein.

[0082] 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 with three transmembrane domains. It is a potential target for ubiquitination by the Nedd4 protein family. NDFIP1 is considered to be part of the integral Golgi membrane protein family. Eight isoforms are currently known, and several orthologs of NDFIP1 have been reported in various animal species.

[0083] The NDFIP1 protein referred to in the present invention is preferably human NDFIP1 having the amino acid sequence deposited under UniProt accession number Q96PU5. It should be understood that the term "NDFIP1" also relates to variants of the above protein. These variants have at least the same basic biological and immunological properties as the above NDFIP1 protein. In particular, they have the same basic biological and immunological properties if they can be detected by the same specific detection methods mentioned in this specification. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the specific amino acid sequence of the NDFIP1 protein, preferably over the entire length of the NDFIP1 protein.

[0084] As used herein, the term "CLEC9A" refers to C-type lectin domain family 9 member A protein encoded by the CLEC9A gene in humans. This protein is typically expressed by myeloid lineage cells. CLEC9A is a group V C-type lectin-like receptor (CTLR) that acts as an endocytic receptor on a subset of myeloid cells, specifically internalizing and processing material from dying cells. It recognizes filamentous actin associated with specific actin-binding domains of cytoskeletal proteins, including spectrin, which are exposed upon cell membrane damage, and mediates cross-presentation of dead cell-associated antigens in a Syk-dependent manner.

[0085] The CLEC9A protein referred to in the present invention is preferably human CLEC9A having the amino acid sequence deposited under UniProt accession number Q6UXN8. It should be understood that the term "CLEC9A" also relates to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned CLEC9A protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the specific amino acid sequence of the CLEC9A protein, preferably over the entire length of the CLEC9A protein. A number of orthologs of CLEC9A have been reported in various animal species.

[0086] As used herein, the term "HCST" refers to the hematopoietic cell signal transduction protein encoded by the HCST gene in humans. This gene encodes a transmembrane signal transduction adaptor that contains a YxxM motif in its cytoplasmic domain. The encoded protein may form part of an immune recognition receptor complex with the C-type lectin-like receptor NKG2D. As part of such a receptor complex, this protein can activate the phosphatidylinositol 3-kinase-dependent signaling pathway through its cytoplasmic YxxM motif. Such a receptor complex can play a role in cell survival and proliferation by activating NK and T cell responses. Two isoforms are known, and a number of orthologs of HCST have been reported in various animal species.

[0087] The HCST protein referred to in the present invention is preferably human HCST having the amino acid sequence deposited under UniProt accession number Q9UBK5. It should be understood that the term "HCST" also relates to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned HCST protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the specific amino acid sequence of the HCST protein, preferably over the entire length of the HCST protein.

[0088] As used herein, the term "LPAR6" refers to lysophosphatidic acid receptor 6 encoded by the LPAR6 gene in humans. LPAR6 is also known as LPA 6 , P2RY5, and GPR87. The protein encoded by this gene belongs to the G protein-coupled receptor family and is preferentially activated by adenosine and uridine nucleotides. Mutations in this gene can lead to rare hereditary hair loss (monilethrix). Several orthologs of LPAR6 have been reported in various animal species.

[0089] The LPAR6 protein referred to in the present invention is preferably human LPAR6 having the amino acid sequence deposited under UniProt accession number P43657. It should be understood that the term "LPAR6" also refers to variants of the above protein. These variants have at least the same basic biological and immunological properties as the above LPAR6 protein. In particular, they have the same basic biological and immunological properties if they can be detected by the same specific detection methods mentioned in this specification. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence that differs by at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the LPAR6 protein, preferably over the entire length of the LPAR6 protein.

[0090] As used herein, the term "HLA-DQA1" refers to the major histocompatibility complex class II, DQα1 protein encoded by the HLA-DQA1 gene on chromosome 6 in humans. HLA-DQA1 is a heterodimer composed of an α chain (DQA) and a β chain (DQB), both of which are anchored to the membrane. 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 from extracellular proteins. Several orthologs of HLA-DQA1 have been reported in various animal species.

[0091] The HLA-DQA1 protein mentioned according to the present invention is preferably the human HLA-DQA1 having the amino acid sequence deposited under UniProt accession number P01909. It should be understood that the term "HLA-DQA1" also refers to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned HLA-DQA1 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the HLA-DQA1 protein, preferably over the entire length of the HLA-DQA1 protein.

[0092] The term "HLA-DRB5" as used herein 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 composed of an alpha (DRA) chain and a beta (DRB) chain, both of which are anchored to the membrane. It plays a central role in the immune system by presenting peptides from extracellular proteins. Diseases associated with HLA-DRB5 include pityriasis rosea and multiple epiphyseal dysplasia caused by abnormal collagen 9. Several orthologs of HLA-DRB5 have been reported in various animal species.

[0093] The HLA-DRB5 protein mentioned according to the present invention is preferably the human HLA-DRB5 having the amino acid sequence deposited under UniProt accession number Q30154. It should be understood that the term "HLA-DRB5" also refers to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned HLA-DRB5 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the HLA-DRB5 protein, preferably over the entire length of the HLA-DRB5 protein.

[0094] As used herein, the term "CD34" refers to cluster of differentiation 34 protein encoded by the CD34 gene in humans. This transmembrane phosphoglycoprotein may function as an adhesion molecule during early hematopoiesis by mediating the attachment of stem cells to the bone marrow extracellular matrix or directly to stromal cells. Alternative spliced transcript variants encoding two different isoforms have been identified, and several orthologs of CD34 in various animal species have been reported.

[0095] The CD34 protein referred to in the present invention is preferably human CD34 having the amino acid sequence deposited under UniProt accession number P28906. It should be understood that the term "CD34" also relates to variants of the above protein. These variants have at least the same basic biological and immunological properties as the above CD34 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different by at least one amino acid substitution, deletion and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the specific amino acid sequence of the CD34 protein, preferably over the entire length of the CD34 protein.

[0096] As used herein, the term "IGLL1" refers to immunoglobulin lambda-like polypeptide 1 protein encoded by the IGLL1 gene in humans. This protein is involved in the transduction of cell proliferation signals, the differentiation from proB cells to preB cell stage, allelic exclusion of the Ig heavy chain locus and the promotion of Ig light chain gene rearrangement. Mutations in this gene can lead to B cell deficiency and agammaglobulinemia, an autosomal recessive disease in which patients produce little or no gamma globulin or antibodies. Two different isoforms have been identified, and several orthologs of IGLL1 in different animal species have been reported.

[0097] The IGLL1 protein referred to in the present invention is preferably human IGLL1 having the amino acid sequence deposited under UniProt accession number P15814. It should be understood that the term "IGLL1" also relates to variants of the above protein. These variants have at least the same basic biological and immunological properties as the above IGLL1 protein. In particular, they have the same basic biological and immunological properties if they can be detected by the same specific detection methods mentioned in this specification. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence that differs by at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the IGLL1 protein, preferably over the entire length of the IGLL1 protein.

[0098] As used herein, the term "BEST1" refers to the bestrophin1 protein encoded by the BEST1 gene in humans. It belongs to the bestrophin family, which includes four related genes that encode integral membrane proteins. BEST1 is characterized by a highly conserved N-terminus and has four to six transmembrane domains. Bestrophin can form chloride channels or can regulate voltage-gated L-type calcium channels. It is generally believed that Bestrophin can form calcium-activated chloride channels in epithelial cells, but there are also studies showing that they are highly permeable to bicarbonate ion transport in retinal tissue. Mutations in this gene are the cause of juvenile vitelliform macular dystrophy (VMD2), also known as Best macular dystrophy, in addition to adult vitelliform macular dystrophy (AVMD) and other retinal pathologies. 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 orthologs of BEST1 in different animal species have been reported.

[0099] The BEST1 protein mentioned according to the present invention is preferably human BEST1 having the amino acid sequence deposited under UniProt accession number O76090. It should be understood that the term "BEST1" also refers to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned BEST1 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the specific amino acid sequence of the BEST1 protein, preferably over the entire length of the BEST1 protein.

[0100] As used herein, the term "EREG" refers to the epiregulin protein encoded by the EREG gene in humans. This gene encodes a secreted peptide hormone and belongs to the 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 can be involved in a wide range of biological processes, including inflammation, wound healing, oocyte maturation, and cell proliferation. In addition, this encoded protein can promote the development of cancers in various human tissues. Several orthologs of EREG have been reported in various animal species.

[0101] The EREG protein mentioned according to the present invention is preferably human EREG having the amino acid sequence deposited under UniProt accession number O14944. It should be understood that the term "EREG" also refers to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned EREG protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the specific amino acid sequence of the EREG protein, preferably over the entire length of the EREG protein.

[0102] As used herein, the term "SLC5A3" refers to the sodium / inositol cotransporter encoded by the SLC5A3 gene in humans. Generally, it is located in the plasma membrane. SLC5A3 is a sodium / inositol cotransporter. It is also said to act upstream or within multiple processes, including the development of the peripheral nervous system, the positive regulation of the reactive oxygen species biosynthetic process, and the regulation of respiratory gas exchange. Several orthologs of SLC5A3 have been reported in various animal species.

[0103] The SLC5A3 protein referred to in the present invention is preferably the human SLC5A3 having the amino acid sequence deposited under UniProt accession number P53794. It should be understood that the term "SLC5A3" also relates to variants of the above protein. These variants have at least the same basic biological and immunological properties as the above SLC5A3 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the SLC5A3 protein, preferably over the entire length of the SLC5A3 protein.

[0104] 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 glycoproteins. It also plays a role in protecting cells from ER stress-induced apoptosis. Gene knockout studies conducted in mice have shown that this gene is important in promoting Ca 2+ flux in immune cells and triggering an effective immune response. SELK also plays a role in T cell proliferation and T cell and neutrophil migration. Several orthologs of SELK have been reported in various animal species.

[0105] The SELK protein mentioned according to the present invention is preferably human SELK having the amino acid sequence deposited under UniProt accession number Q9Y6D0. It should be understood that the term "SELK" also relates to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned SELK protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the specific amino acid sequence of the SELK protein, preferably over the entire length of the SELK protein.

[0106] The term "PRRG3" as used herein refers to 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 vitamin K-dependent transmembrane protein family, and such proteins contain an extracellular domain rich in glutamate. Diseases associated with PRRG3 include hereditary vitamin K-dependent coagulation factor complex deficiency and vitamin K deficiency bleeding. Several orthologs of PRRG3 have been reported in various animal species.

[0107] The PRRG3 protein mentioned according to the present invention is preferably human PRRG3 having the amino acid sequence deposited under UniProt accession number Q9BZD7. It should be understood that the term "PRRG3" also relates to variants of the above-mentioned protein. These variants have at least the same basic biological and immunological properties as the above-mentioned PRRG3 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the specific amino acid sequence of the PRRG3 protein, preferably over the entire length of the PRRG3 protein.

[0108] As used herein, the term "NINJ1" refers to the nerve injury-induced protein 1 (ninjurin-1) encoded by the NINJ1 gene in humans. It is upregulated in both dorsal root ganglion neurons and Schwann cells after nerve injury. NINJ1 is a homophilic transmembrane adhesion molecule involved in various processes such as inflammation, cell death, axon growth, cell chemotaxis, and angiogenesis. Several orthologs of NINJ1 have been reported in various animal species.

[0109] The NINJ1 protein referred to in the present invention is preferably human NINJ1 having the amino acid sequence deposited under UniProt accession number Q92982. It should be understood that the term "NINJ1" also relates to variants of the above protein. These variants have at least the same basic biological and immunological properties as the above NINJ1 protein. In particular, they have the same basic biological and immunological properties if they can be detected by the same specific detection methods mentioned in this specification. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to the specific amino acid sequence of the NINJ1 protein, preferably over the entire length of the NINJ1 protein.

[0110] 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 with electrophiles and the reduction of lipid hydroperoxides. Typically, MGST1 is localized to the endoplasmic reticulum and the outer mitochondrial membrane, where it is thought to protect these membranes from oxidative stress. It is involved in the cellular defense against toxic, carcinogenic, and pharmacologically active electrophilic compounds. Two isoforms are known, and several orthologs of MGST1 have been reported in various animal species.

[0111] The MGST1 protein mentioned according to the present invention is preferably human MGST1 having the amino acid sequence deposited under UniProt accession number P10620. It should be understood that the term "MGST1" also relates to variants of the above protein. These variants have at least the same basic biological and immunological properties as the above-mentioned MGST1 protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the specific amino acid sequence of the MGST1 protein, preferably over the entire length of the MGST1 protein.

[0112] The term "HLA-G" used herein refers to HLA-G histocompatibility antigen class I G, also known as human leukocyte antigen G, a protein encoded by the HLA-G gene in humans. HLA-G belongs to the HLA class I heavy chain paralogues. This class I molecule is a heterodimer composed of a heavy chain and a light chain (β-2 microglobulin). The heavy chain is anchored to the membrane. HLA-G is expressed on placental cells of fetal origin. It acts as a major immune checkpoint, for example, it downregulates the response of the immune system. HLA-G also plays a role in immune tolerance during pregnancy and the regulation of the immune response to parasitic diseases, and has been shown to be associated with tumor escape in cancer and allergic responses. Seven isotypes are known, and several orthologues of HLA-G have been reported in various animal species.

[0113] The HLA-G protein mentioned according to the present invention is preferably human HLA-G having the amino acid sequence deposited under UniProt accession number P17693. It should be understood that the term "HLA-G" also relates to variants of the above protein. Such variants have at least the same basic biological and immunological properties as the above-mentioned HLA-G protein. In particular, if they can be detected by the same specific detection methods mentioned in this specification, they have the same basic biological and immunological properties. In addition, it should be understood that the variants according to the present invention should have an amino acid sequence different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the specific amino acid sequence of the HLA-G protein, preferably over the entire length of the HLA-G protein.

[0114] According to the present invention, the degree of identity between two amino acid sequences can be determined by algorithms well known in the art. Preferably, the degree of identity is determined by comparing two optimally aligned sequences within a comparison window, wherein the fragment of the amino acid sequence within the comparison window may include additions or deletions (e.g., gaps or overhang portions) compared to the reference sequence used for the optimal alignment (which does not include additions or deletions). The percentage is calculated by determining the number of positions at which the same amino acid residue occurs in both sequences to yield 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 derive the percentage of sequence identity. The optimal alignment of the sequences to be compared can be carried out by: the local homology algorithm disclosed by Smith, the homology alignment algorithm of Needleman, 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 visual inspection. Assuming that two sequences have been identified for comparison, GAP and BESTFIT are preferably used to determine their optimal alignment and thus the degree of identity. Preferably, the default values of a gap weight of 5.00 and a gap weight length of 0.30 are used. The variants mentioned above can be allelic variants or any other species-specific homologs, paralogs, or orthologs. The variants mentioned above can be allelic variants or any other species-specific homologs, paralogs, or orthologs.

[0115] The term "determining the amount of at least one biomarker" as used according to the present invention refers to the qualitative and quantitative determination of the biomarker, i.e., the term includes determining the presence or absence of the biomarker or determining its absolute or relative amount. The term also encompasses measuring amounts or concentrations, preferably semi-quantitative or quantitative.

[0116] As used herein, the term "quantity" refers to the absolute quantity of a biomarker, the relative quantity or concentration of a biomarker, and any value or parameter related to or derivable from the same. These values or parameters include intensity signal values of all specific physical or chemical properties obtained from the biomarker or detection molecule and / or detectable label. These values or parameters can be obtained by direct or indirect measurement. Direct measurement involves measuring the quantity or concentration of a biomarker based on a signal obtained from the biomarker molecule itself, and the intensity thereof is directly related to the number of biomarker molecules present in the sample. For example, such a signal can be obtained by measuring the intensity value of a specific physical or chemical property of the biomarker molecule, which is sometimes referred to as an intensity signal herein. Indirect measurement includes measuring a signal obtained from a secondary component, i.e., a component that is not the biomarker molecule itself. It should be understood that values related to the aforementioned quantity or parameter can also be obtained and / or modified by all standard mathematical operations.

[0117] In the method of the present invention, determining the quantity can be carried out by any technique that allows detection of the presence, absence, or quantity of the biomarker. Suitable techniques depend on the molecular nature and characteristics of the biomarker. For example, except in the case of a nucleic acid molecule biomarker of transcription, a protein biomarker can be determined by measuring characteristics. The differences in these measurable characteristics are well known to those skilled in the art. In addition, it should be understood that a protein biomarker can be detected by using detection reagents and / or techniques different from those used for detecting nucleic acid molecule biomarkers of transcription. However, those skilled in the art are also well aware of the different detection reagents and / or techniques.

[0118] According to the present invention, determining the quantity of a biomarker can be achieved by all known means for determining such quantity in a sample. Such means include immunoassay devices and methods, which can utilize various sandwich, competitive, or other assay formats of labeled molecules. The assay will generate a signal that indicates the presence or absence of the protein. In addition, the signal intensity can preferably be directly or indirectly related (e.g., inversely proportional) to the quantity of the biomarker present in the sample. Other suitable methods include measuring the physical or chemical properties unique to the biomarker. The methods preferably include biosensors, optical devices coupled to immunoassays, biochips, or other analytical devices such as chromatographic devices or single-cell analysis devices such as FACS analyzers or devices for PCR analysis such as devices for single-cell PCR, qPCR, or bulk PCR, or sequencing devices.

[0119] 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. Protein biomarkers can be quantified proportionally in a cell population by counting positive and negative sorting events. Thus, biomarker data for a clinical sample is not a single value representing overall staining intensity, but a value reflecting the proportion of individual cells exceeding the intensity threshold for a particular biomarker. The gating criteria for positive sorting events can be set to a combination of desired signal intensities for the protein biomarker used.

[0120] Preferably, one or more biomarkers to be determined according to the present invention can be determined as proteins. For this purpose, a binding molecule that specifically binds to the biomarker protein is typically applied, and the binding molecule can be detected by a detectable label present in the binding molecule or by a second binding molecule that specifically binds to the first binding molecule and contains a detectable label. The binding molecule can be exposed to the biomarker in solution or when the binding molecule is immobilized on a solid support.

[0121] A binding molecule in this context refers to any molecule capable of specifically binding to the biomarker to be detected. The binding molecule is selected based on the type of analysis to be performed. Binding molecules include, but are not limited to, aptamers, antibodies, adnectins, ankyrins, antibody mimetics and other protein scaffolds, small molecules, nucleic acids, lectins, affybodies, nanobodies, avimers, and peptide mimetics. Preferably, such a binding molecule can be an antibody or an antigen-binding fragment thereof.

[0122] An "antibody" according to the present invention can encompass all types of antibodies that specifically bind to the biomarker protein. Preferably, the antibodies of the present invention are monoclonal antibodies, polyclonal antibodies, single-chain antibodies, chimeric antibodies, or any fragment or derivative of these antibodies that is still capable of specifically binding to the biomarker protein.

[0123] The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed by the term "antigen-binding fragment" include fragment antigen-binding (Fab) fragments, Fab' fragments, F(ab') 2 fragments, heavy chain antibodies, single-domain antibodies (sdAb), single-chain variable fragments (scFv), variable fragments (Fv), V H domains, V LDomains, single-domain antibodies, nanobodies, IgNAR (immunoglobulin new antigen receptor), di-scFv, bispecific T cell engagers (BITE), dual-affinity re-targeting (DART) molecules, triple bodies, diabodies, single-chain diabodies, alternative scaffold proteins, and fusion proteins thereof.

[0124] Specific binding as used in the context of the antibodies of the present invention refers to the antibody not cross-reacting with other molecules present in the sample under investigation. Specific binding can be tested by various well-known techniques. Generally, antibodies or fragments thereof can be obtained by using methods described in standard textbooks, such as in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies can be prepared by techniques that include fusing mouse myeloma cells with spleen cells from an immunized mammal, preferably an immunized mouse. Preferably, an immunogenic peptide is applied to the mammal. The peptide is preferably conjugated to a carrier protein 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 encompass Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface-active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). 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 high specific binding affinity of the antibody for a specific antigen, i.e., the biomarker protein. The binding event results in a physicochemical change, and such a change can be detected as described elsewhere herein.

[0125] As used herein, "adnectin" refers to a synthetic binding protein, also known as a monomer, which is based on the 10th fibronectin type III ( 10The Fn3 domain. It is a member of the immunoglobulin superfamily and contains a "β-sandwich" protein fold that is strikingly similar to antibody domains. Thus, adnectin represents a simple and robust alternative to antibodies in terms of generating target-binding proteins. The main advantage of adnectin over conventional antibodies is that adnectin can be readily used as a genetically encoded intracellular inhibitor, i.e., one can express an adnectin inhibitor in selected cells by simply transfecting the cells with an adnectin expression vector. Preferably, the adnectin used herein specifically binds to the following: 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.

[0126] The "ankyrin" used herein refers to a family of proteins that includes binding sites for a wide range of membrane proteins. Ankyrin contains four functional domains: (i) an N-terminal domain with 24 tandem ankyrin repeats, which is responsible for recognizing a variety of 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 between different ankyrins. Ankyrin is encoded by three genes in humans, which in turn generate multiple proteins through alternative splicing. Preferably, the ankyrin used herein should specifically bind to at least one biomarker described elsewhere herein.

[0127] As used herein, an "antibody mimetic" refers to a compound that can specifically bind to an antigen, similar to an antibody but structurally unrelated to an antibody. Generally, antibody mimetics are artificial peptides or proteins with a molecular weight of about 3 to 20 kDa, which include one, two or more exposed domains that specifically bind to an antigen. Examples particularly include LACI-D1 (lipoprotein-associated coagulation inhibitor); affilin, such as human γB-crystallin or human ubiquitin; cystatin; Sac7D from Sulfolobus acidocaldarius; lipocalin and anticalin derived from lipocalin; DARPin (designed ankyrin repeat domain); the SH3 domain of Fyn; the Kunits domain of protease inhibitors; monobody, such as the 10th type III domain of fibronectin; adnectin; knottin (cysteine knot miniprotein); atrimer; evibody, such as a CTLA4-based binder; affibody, such as a three-helix bundle derived from the Z domain of Staphylococcus aureus protein A; Trans-body, such as human transferrin; tetrameric protein, such as monomeric or trimeric human C-type lectin domain; microbody, such as trypsin inhibitor II; affilin; armadillo repeat protein. Nucleic acids and small molecules are sometimes also considered antibody mimetics (aptamers), but not artificial antibodies, antibody fragments and fusion proteins composed of them. The common advantages over antibodies are better solubility, tissue permeability, stability to heat and enzymes, and relatively low production costs. Preferably, the antibody mimetics used herein should specifically bind to at least one biomarker described elsewhere herein.

[0128] As used herein, a "scaffold protein" refers to a specific protein whose main function is to mediate a protein complex. Scaffold proteins generally have multiple protein domains that mediate binding to other proteins. Examples of scaffold proteins include, but are not limited to, the protein inaD from the rhabdomere of Drosophila melanogaster or the protein titin found in muscle.

[0129] As used herein, the term "lectin" refers to a carbohydrate-binding protein that is highly specific for sugar groups. They are ubiquitous in nature and can bind to soluble carbohydrates or carbohydrate moieties that are part of glycoproteins or glycolipids. Lectins typically agglutinate certain cells and / or precipitate glycoconjugates. Thus, they can be used in medicine, particularly for blood typing. Lectins are also used in neuroscience for anterograde labeling to trace the path of efferent axons. Preferably, the lectins used herein should specifically bind to at least one biomarker described elsewhere herein.

[0130] As used herein, an "affibody" is a small and robust protein with high affinity for a target protein. Compared to antibodies, affibodies consist of α-helices and have no disulfide bonds. In particular, they are based on a three-helix bundle domain with 58 amino acids and have a molar mass of approximately 6 kDa. They can be expressed alone or as a fusion with other protein partners in various host cells in a soluble and proteolytically stable form. 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 should specifically bind to at least one biomarker described elsewhere herein.

[0131] As used herein, the term "nanobody" refers to a small recombinant antigen-binding fragment, typically consisting of a single monomeric variable antibody domain. Although nanobodies lack the light and heavy chain constant regions, their antigen-binding ability is still similar to that of conventional antibodies. Typically, the complementarity-determining region 3 (CDR3) of nanobodies is similar to or even longer than that of the human variable domain of the heavy immunoglobulin chain (VH). They can form finger-like structures to recognize cavities or hidden epitopes that cannot be recognized by monoclonal antibodies, a feature that enhances the binding affinity and specificity of nanobodies. Preferably, the nanobodies used herein should specifically bind to at least one biomarker described elsewhere herein.

[0132] As used herein, "avimer" (short for avidity multimer) refers to an artificial protein having multiple binding sites that specifically bind to certain antigens. They are structurally unrelated to antibodies and are thus classified as antibody mimetics. Typically, they consist of two or more peptide sequences of 30 to 35 amino acids, linked by a linker peptide. The individual sequences are derived from the A domains of various membrane receptors and have a rigid structure, stabilized by disulfide bonds and calcium. Each A domain can bind to a certain epitope of the target protein. The combination of domains that bind to different epitopes of the same protein increases the affinity for that protein, an effect known as avidity. Avimers are widely used in tissue imaging, therapy, and early detection in carcinogenesis studies. Preferably, the avimers used herein should specifically bind to at least one biomarker described elsewhere herein.

[0133] As used herein, the term "peptide mimetic" refers to a compound that mimics one or more structural aspects or biological activities of a naturally occurring polypeptide, but which includes one or more non-peptide or non-naturally occurring chemical structures or bonds. Peptide mimetics are often used to mimic the biological actions of peptides, and thus they can be small protein-like chains designed to mimic one or more peptides. Peptide mimetics are typically synthesized based on an existing target peptide, with one or more modifications made to alter the structure or properties of the molecule. The modifications can alter the stability, half-life, biological activity, absorption, or side effects (e.g., toxicity, solubility, hydrophobicity, side chain charge, or flexibility) of the peptide molecule. Peptide mimetics can be used as rationally developed pharmaceuticals or drug-like compounds, or obtained by modifying existing peptides with known or putative biological activity. Preferably, the peptide mimetics used herein should specifically bind to at least one biomarker described elsewhere herein.

[0134] "Detectable labels" that can be used in accordance with the present invention as referred to herein include gold particles, latex beads, acridinium esters, luminol, ruthenium, enzyme activity labels, radioactive labels, magnetic labels such as magnetic beads, including paramagnetic and superparamagnetic labels, and fluorescent labels. Enzyme activity labels include, for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, luciferase, and their derivatives. Suitable substrates for detection include diaminobenzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-nitroblue tetrazolium chloride), and 5-bromo-4-chloro-3-indolyl-phosphate. Suitable enzyme-substrate combinations can produce colored reaction products, fluorescence, or chemiluminescence, which can be measured according to methods known in the art (such as using a light-sensitive film or a suitable photographic system). As for measuring enzymatic reactions, the same criteria given above apply. Typical fluorescent labels include, for example, fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red, fluorescein, Alexa dyes, Brilliant Violet, or Brilliant Ultraviolet. In addition, the use of quantum dots as fluorescent labels is also contemplated. Typical radioactive labels include 35S, 125I, 32P, 33P, etc. Radioactive labels can be detected by any known and suitable method, such as a light-sensitive film or a phosphor imager. Suitable labels can also be or include tags such as biotin, digoxin, His-tag, GST-tag, FLAG-tag, GFP-tag, MYC-tag, influenza A virus hemagglutinin (HA), maltose-binding protein, etc.

[0135] A biomarker / binding molecule complex can be used to detect the amount of a biomarker. The amount can also be detected indirectly from the biomarker / binding molecule complex, for example, as a result of a reaction after biomarker / binding molecule interaction, but depending on the formation of the biomarker / binding molecule complex. In some instances, the amount of a biomarker can be detected directly from the biomarker in a biological sample. The amount of a biomarker can also be detected using a multiplex format that allows the simultaneous detection of two or more biomarkers in a biological sample. In the multiplex format, binding molecules are immobilized at discrete positions on a solid support, either directly or indirectly, covalently or non-covalently.

[0136] Also preferably, one or more biomarkers to be determined in accordance with the present invention can be determined as transcribed nucleic acid molecules. For this purpose, binding molecules, such as nucleic acid molecules that specifically hybridize, for example, through a reverse transcription reaction, to a transcribed nucleic acid molecule or any molecule derived therefrom, can be used for detection. The hybridizing binding molecule itself can include a detectable label, or it can specifically bind to a secondary molecule that includes such a detectable label.

[0137] Equally preferably, the amount of the 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 for detecting and quantifying 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 during each amplification cycle. The increase in the fluorescent signal is proportional to the amount of exponentially increasing PCR product molecules (amplicons) generated during the repetitive phase of the reaction.

[0138] More preferably, the amount of the biomarker can be detected by transcriptome sequencing, also known as single-cell sequencing (scRNA-seq). Transcriptome sequencing technology is a well-known method that refers to sequencing the genome or transcriptome of a single cell to obtain genomic, transcriptomic, or other multi-omics information. Thus, "transcriptome sequencing" as used herein refers to a method of analyzing RNA expression in a large population of cells, preferably hematopoietic stem and progenitor cell populations. Generally, the method includes isolating single cells and their RNA, followed by reverse transcription, amplification, library generation, and sequencing. These techniques encompass but are not limited to droplet-based and plate-based scRNA-seq techniques. Plate-based methods require isolation of single cells, for example, by FACS. However, droplet-based methods use the formation of lipid droplets to isolate single cells by phase separation in a large sample of single cells in suspension. The latter technique allows analysis of thousands of cells, while plate-based techniques are typically applicable to hundreds of cells.

[0139] When a biomarker indicates an abnormal process or disease in a subject, the biomarker is typically described as overexpressed compared to the expression level or amount of a biomarker indicating a normal process or the absence of disease or other condition in the subject. "Overexpression" refers to a level or amount of a 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.

[0140] The biomarkers of the present invention should be present on or in hematopoietic stem and progenitor cells (HSPCs). According to the present invention, biomarkers presented on the surface of HSPCs, such as in the form of surface proteins, receptors, lipids, etc., are present on HSPCs. Biomarkers generated within HSPCs and intended to remain intracellular, such as transcribed nucleic acid molecules, are thus present in HSPCs. Hematopoietic stem and progenitor cells (HSPCs) are a heterogeneous cell population responsible for generating almost all types of mature blood and immune cells. HSPCs encompass a range of undifferentiated cells in a lowly primed initiation state, including hematopoietic stem cells (HSCs) (Velten et al. 2017 Nature Cell Biology), which are considered the initiating cells of postnatal hematopoietic differentiation. HSCs are capable of self-renewal and gradually acquire lineage biases in multiple directions within the HSPC compartment, which respectively give rise to committed or unilineage-restricted progenitors. For example, myeloid progenitors ultimately give rise to differentiated cells such as monocytes, macrophages, neutrophils, or dendritic cells, while lymphoid progenitors ultimately give rise to, for example, T cells, B cells, or natural killer cells. The term "HSPC" as used herein refers to CD34 + - cells, preferably CD34 + CD38 - - cells, more preferably Lin - CD34 + CD38 - - cells. The cells can preferably be isolated from the samples mentioned herein, preferably from tissue samples or body fluid samples, for example, by 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 even 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 - cells correspond to CD4 - CD8 - CD11b - CD14 - CD19 - CD20 - CD56 - CD235a - CD34 +CD38 - cells.

[0141] The term "sample" refers to any sample containing HSPCs that can be obtained from a subject to be studied. Preferably, the sample can be a tissue or body fluid sample. Preferably, the tissue sample is a connective tissue sample, preferably a bone marrow sample or a spleen sample. Preferably, the body fluid sample is a peripheral blood sample or a cord blood sample. A blood sample also includes its fraction. For example, a blood sample can be separated into serum, plasma, or a fraction containing a specific type of blood cell, such as red blood cells or white blood cells (leukocytes). The term "sample" also includes materials containing homogenized solid materials, such as from a tissue sample or a tissue biopsy. 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 by biopsy, for example. Isolated cells can be obtained from body fluids or tissues or organs by separation techniques such as centrifugation or cell sorting. Thus, the term refers to the biological sample itself, which is used directly in the process of determining the amount of a biomarker. However, the term can also refer to a sample that must undergo various steps (e.g., separating cells from biological material) before the amount of the biomarker is determined.

[0142] In the method according to the invention, the amount of the determined biomarker is compared to a reference. As used herein, the term "reference" refers to an amount or value that allows assignment of a subject to a group of subjects having a disease or disorder or at risk of developing said disease or disorder, or to a group of subjects not having said disease or disorder or not at risk of developing said disease or disorder. Such a reference can be a threshold amount that separates these groups from each other. Thus, the reference should be an amount that allows assignment of a subject to a group of subjects having a disease or disorder or at risk of developing said disease or disorder, or not. A suitable threshold amount that separates these two groups can be calculated without much ado based on the amount of the biomarker from subjects or groups of subjects known to have a certain disease or disorder, or at risk of developing said disease or disorder, and subjects or groups of subjects known not to have said disease or disorder, or not at risk of developing said disease or disorder, according to the statistical test methods mentioned elsewhere herein. The reference amount applicable to an individual subject can vary according to various physiological parameters such as age, sex or subpopulation. Preferably, the reference can be a reference for each biomarker from at least one subject known to have JMML disease. However, preferably, it is a reference for each biomarker from at least one subject known not to have JMML disease. More preferably, the reference for each biomarker is from at least one subject diagnosed with JMML or belonging to a high-risk or low-risk JMML group by DNA methylome analysis (preferably as performed in the attached examples below).

[0143] In principle, a reference amount can be calculated for a cohort of subjects based on a given parameter such as the mean or median of the amount of a biomarker by applying standard statistical methods. In particular, the accuracy of a test, such as a method designed to diagnose or rule out an event, is preferably described by its receiver operating characteristic (ROC) (see especially Zweig 1993, Clin. Chem. 39:561-577). An ROC plot is a plot of all sensitivity / specificity pairs generated by continuously varying the decision threshold over the entire range of observed data. The clinical performance of a diagnostic method depends on its accuracy, i.e., its ability to correctly assign subjects to a particular prognosis or diagnosis. The ROC plot shows the overlap between two distributions by plotting sensitivity against 1 - specificity over the full range of thresholds applicable for discrimination. On the y-axis is sensitivity, or true positive fraction, 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 is also known as positive in the presence of disease or disorder. It is calculated only from the affected subpopulation. On the x-axis is the false positive fraction, 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. This is the specificity index and is calculated entirely from the unaffected subpopulation. Since the true positive and false positive fractions are calculated completely separately, by using test results from two different subpopulations, the ROC plot is independent of the incidence of events in the cohort. Each point on the ROC plot represents a sensitivity / -specificity pair corresponding to a particular decision threshold. A test with perfect discrimination (no overlap in the two outcome distributions) has an ROC plot passing through the upper left corner, where the true positive fraction is 1.0 or 100% (perfect sensitivity) and the false positive fraction is 0 (perfect specificity). The theoretical plot for a non-discriminating test (identical distributions of the two sets of results) is a 45° diagonal line from the lower left to the upper right. Most plots fall between these two extremes. If the ROC plot is entirely below the 45° diagonal, this can easily be remedied by changing the "positive" criterion from "greater than" to "less than", and 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, a threshold can be derived from the ROC curve, allowing diagnosis or prediction of a given event with an appropriate balance of sensitivity and specificity, respectively. Thus, a reference for the above-described method of the present invention, i.e., a threshold that allows discrimination between subjects with a coagulation defect and subjects without a coagulation defect, can generally be generated by establishing the ROC of the cohort as described above and deriving a threshold amount therefrom. Depending on the sensitivity and specificity required for the diagnostic method, the ROC plot allows derivation of a suitable threshold. It should be understood that when excluding subjects at high risk (i.e., exclusion), optimal sensitivity is desired; while when assessing subjects at high risk (i.e., confirmation), optimal specificity is desired.

[0144] As used herein, the term "comparison" encompasses comparing a determined amount of a biomarker mentioned herein with a reference. It should be understood that the comparison used herein refers to any kind of comparison between the value of that amount and the reference. However, it should be understood that preferably, values of the same type are compared with each other. For example, if absolute amounts are determined and compared in the methods of the present invention, the reference should also be an absolute amount; if relative amounts are determined and compared in the methods of the present invention, the reference should also be a relative amount, and so on. The comparison can be performed manually or with computer assistance. The value of the amount and the reference can be compared with each other, for example, and the comparison can be automatically performed by a computer program that executes a comparison algorithm. The computer program that performs the evaluation will provide the desired evaluation result in a suitable output format.

[0145] As used in the present invention, the term "JMML" encompasses JMML, JMML-like tumors, and myeloproliferative disorders associated with neurofibromatosis or Noonan syndrome (CBL syndrome)-associated JMML. The typical symptoms and signs of diagnosing JMML are splenomegaly, hepatomegaly, lymphadenopathy, pallor, and fever. Less common are infections, bleeding, cough, and rash. Occasionally, café-au-lait spots, abdominal pain, xanthomas, bone pain, diarrhea, and CNS infiltration (Arber et al. 2022 Blood) occur. Usually, if certain diagnostic criteria for JMML can be verified by specific tests such as blood tests, bone marrow aspiration, cytogenetics, and molecular tests, the subject is known to have JMML.

[0146] According to the present invention, the diagnostic criteria for JMML can preferably be the International Consensus Classification (ICC) of myeloid neoplasms and acute leukemias. The ICC classifies JMML and related disorders as MDS / MPN and groups them with pediatric and / or germline mutation-related diseases (Arber, ibid.). JMML is a childhood clonal disorder characterized by constitutive activation of the RAS signal transduction pathway. More than 95% of patients carry mutations in the RAS pathway. Typical mutations affect the PTPN11, NRAS, KRAS, NF1, CBL genes, and rarely affect the RRAS gene. Clonal diseases that phenotypically mimic JMML but do not carry one of these mutations are classified as JMML-like tumors. Polyclonal Noonan syndrome-related myeloproliferative disorders can be clinically similar to JMML. All these cases, namely JMML, JMML-like tumors, Noonan syndrome-related myeloproliferative disorders, have typical characteristics.

[0147] The diagnostic criteria for JMML listed by the ICC include genetic, clinical, and hematological features. The percentage of blasts in peripheral blood and bone marrow needs to be <20%, and there is no BCR::ABL1 fusion (or Philadelphia chromosome). Most cases have splenomegaly (absent in ~3% of cases), and the monocyte count ≥ 1×10 9 / L (not achieved in ~7% of cases). Genetically, one of the following findings is usually observed:

[0148] - Somatic mutations in PTPN11, KRAS, NRAS, or RRAS;

[0149] - Or germline mutations in NF1 and loss of heterozygosity or clinical diagnosis of neurofibromatosis type 1;

[0150] - Or the presence of CBL mutations and CBL loss of heterozygosity.

[0151] In addition, monosomy of chromosome 7 or any other chromosomal abnormality, hemoglobin F level higher than the normal level for the patient's age, myeloid precursors in the blood, hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) in colony assays, and hyperphosphorylation of STAT5 may indicate JMML, while KMT2A rearrangements are excluded (Niemeyer & Flotho 2019 Blood). Although GM-CSF hypersensitivity and hematological parameters are most important in the early stage, the current WHO classification emphasizes RAS pathway mutations as the most important criterion. JMML-like tumors lack mutations in the RAS pathway but phenotypically resemble JMML. This group includes rearrangements such as ALK, ROS1, FIP1L1::RARA, or CCDC88C::FLT3 fusions (Arber, ibid.).

[0152] According to the present invention, the diagnostic criteria for JMML can also preferably be the diagnostic criteria in Chapter 5 of the WHO on myelodysplastic / myeloproliferative neoplasms (Orazi et al. 2016). Therefore, all 4 are necessary according to the following clinical and hematological criteria:

[0153] - Peripheral blood monocyte count ≥ 1×10 9 cells / L;

[0154] - Percentage of blasts in peripheral blood and bone marrow <20%;

[0155] - Splenomegaly; and

[0156] - Absence of Philadelphia (Ph) chromosome or BCR-ABL1 fusion.

[0157] According to the following genetic criteria, at least one criterion is sufficient:

[0158] - Somatic mutations in PTPN11, KRAS, or NRAS, with germline mutations (indicating Noonan syndrome) excluded;

[0159] - Clinical diagnosis of neurofibromatosis type 1 or NF1 mutations; and

[0160] - Germline CBL mutations and LOH of CBL, with occasional cases having heterozygous splice site mutations.

[0161] For cases that do not meet any of the above genetic criteria, in addition to the above clinical and hematological criteria, the following criteria must also be met:

[0162] - Monosomy of chromosome 7 or any other chromosomal abnormality,

[0163] Or

[0164] - At least two of the following criteria:

[0165] - Hemoglobin F increases with age;

[0166] - Myeloid or erythroid precursors on peripheral blood smear;

[0167] - Hypersensitivity to granulocyte-macrophage colony-stimulating factor (also known as CSF2) in colony assays; and

[0168] - STAT5 hyperphosphorylation.

[0169] More preferably, subjects with JMML are identified by, for example, DNA methylome analysis as described in the attached examples below.

[0170] In one embodiment, the reference is derived from at least one subject known to have JMML.

[0171] Preferably, a subject with JMML is identified by determining the amount of at least one biomarker on or in HSPCs in a biological sample, wherein the at least one biomarker is 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, SELK, PRRG3, NINJ1, MGST1, and HLA-G, and comparing the determined amount with a reference.

[0172] In an embodiment of the method of the invention, wherein the reference is derived from at least one subject known to have JMML, a determined amount of at least one of the above biomarkers being the same as or greater than the reference indicates that the subject has JMML. A determined amount of at least one of the above biomarkers being lower than the reference may indicate that the subject does not have JMML.

[0173] In another embodiment, the reference is derived from at least one subject known not to have JMML.

[0174] Generally, if the above diagnostic criteria for JMML are not met, the subject is known not to have JMML. Preferably, a subject not having JMML is identified by DNA methylome analysis and as described, for example, in the attached examples below.

[0175] Preferably, a subject known not to have JMML may also have a physiological abundance profile of biomarkers on or in HSPCs in the biological sample of the subject 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] Preferably, a subject not suffering from JMML is identified by determining the amount of at least one biomarker on or in HSPCs in a biological sample, said at least one biomarker being 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, SELK, PRRG3, NINJ1, MGST1 and HLA-G, and comparing the determined amount with a reference.

[0177] In an embodiment, wherein the reference is derived from at least one subject known not to suffer from JMML, a determined amount of at least one of the above markers being the same as or lower than the reference indicates that the subject does not suffer from JMML. A determined amount of at least one of the above markers being higher than the reference may indicate that the subject suffers from JMML.

[0178] It should be understood that, according to the method of the present invention, advantageously, JMML can be diagnosed based on the presence or abundance of one or more biomarkers of Group I, i.e., high-risk JMML biomarkers on or in HSPCs, or one or more biomarkers of Group II, i.e., low-risk JMML biomarkers on or in HSPCs. The method of the present invention will also provide an auxiliary means for diagnosing the absence of JMML, i.e., if it is determined that there are no biomarkers of Group I and no biomarkers of Group II on or in HSPCs. Therefore, by studying the biomarkers of Group I and Group II, it is possible to diagnose whether a subject has JMML. Due to the findings in the basic research of the present invention, it is possible to effectively screen for JMML in the adolescent subject population or more reliably diagnose individual subjects. Therefore, the treatment effect can be improved. As mentioned above, JMML is a rare and aggressive childhood cancer. Considering the low incidence and prevalence of the disease, the availability of patient samples for research is limited, which makes the diagnosis and treatment development difficult and troublesome. In the basic research of the present invention, it was possible to isolate live cells from a limited number of patient samples representing all known JMML subtypes. In live cells, the number of HSPCs (Lin-CD34+CD38-HSPCs) is usually low. However, by applying single-cell technologies, including scRNA-seq or FACS technologies, it was possible to obtain rich datasets from the existing materials.

[0179] Furthermore, by identifying the specific biomarkers of high-risk JMML subjects and the biomarkers of low-risk JMML subjects, the present invention allows for the classification of subjects and the direct identification of high-risk or low-risk JMML subjects.

[0180] Therefore, in accordance with the foregoing, the present invention also contemplates a method for classifying subjects suffering from JMML into a low-risk or high-risk group of JMML, the method comprising:

[0181] a) determining the amount of at least one biomarker present on or in hematopoietic stem and progenitor cells (HSPCs) in a biological sample, the at least one biomarker being selected from each of the following:

[0182] 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

[0183] ii) Group II, consisting of: IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G;

[0184] b) comparing the determined amount in step a) with a reference; and

[0185] c) classifying the subject into a JMML low or high-risk group based on the comparison in step b).

[0186] As used herein, the term "classifying" refers to assigning a subject to a group of subjects exhibiting a similar or identical disease state. The disease states into which a subject can be classified by applying the method of the present invention are the JMML high-risk group or the JMML low-risk group.

[0187] As used herein, the term "JMML high-risk group" refers to a group of subjects known to have JMML who, 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 3.5 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, have a high risk of JMML deterioration or any signs or symptoms associated therewith and / or death due to JMML without treatment. The term also refers to a group of subjects known to have JMML with a high risk of recurrence. A subject known to have JMML can also be classified into the JMML high-risk group if a specific biomarker on HSPC is detected.

[0188] 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 this case, it should be understood that preferably, the reference for at least one biomarker in Group I can be obtained from at least one subject known to be from the high-risk JMML group. If the determined amount of at least one biomarker from Group I is the same as or increased compared to the reference, it will indicate a high-risk subject. However, it should be understood that preferably, the reference for at least one biomarker in Group I can be obtained from at least one subject known not to be from the JMML high-risk group. If the determined amount of at least one biomarker from Group I is the same as or decreased compared to the reference, it will indicate that the subject is not a high-risk subject.

[0189] As used herein, the term "low-risk group of JMML" refers to a group of subjects known to have JMML who, 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 3.5 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, have a low risk of JMML progression or any signs or symptoms associated therewith and / or death due to JMML without treatment. This term also refers to a group of subjects known to have JMML who typically exhibit spontaneous remission. Subjects known to have JMML can also be classified into the low-risk group of JMML if specific biomarkers on HSPCs are detected.

[0190] Thus, in one embodiment of the above 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 low-risk group of JMML. In this case, it should be understood that, preferably, the reference for at least one biomarker in Group II can be obtained from at least one subject known to be from the low-risk group of JMML. If the determined amount of at least one biomarker from Group I is the same as or increased compared to the reference, the subject will be indicated as a low-risk subject. However, it should be understood that, preferably, the reference for at least one biomarker in Group II can be obtained from at least one subject known not to be from the low-risk JMML group. If the determined amount of at least one biomarker from Group II is the same as or decreased compared to the reference, the subject will be indicated not to be a low-risk subject.

[0191] The present invention also relates to a method for identifying whether a subject belongs to the high-risk group of JMML, the method comprising:

[0192] a) determining the amount of at least one biomarker present on or in HSPCs in a biological sample, the at least one biomarker being 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;

[0193] b) comparing the determined amount in step a) with a reference; and

[0194] c) identifying whether the subject belongs to the high-risk group of JMML based on the comparison in step b).

[0195] Preferably, the reference can be obtained from at least one subject known to belong to the high-risk group of JMML. If at least one identified biomarker is the same as or increased compared to the reference, it can be understood that the subject under study also belongs to the high-risk group of JMML. However, preferably, the reference can be obtained from at least one subject known not to belong to the high-risk group of JMML. If at least one identified biomarker is the same as or decreased compared to the reference, it can be understood that the subject under study also does not belong to the high-risk group of JMML.

[0196] The present invention also relates to a method for identifying whether a subject belongs to the low-risk group of JMML, the method comprising:

[0197] a) determining the amount of at least one biomarker present on or in HSPCs in a biological sample, said at least one biomarker being selected from Group II consisting of: IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G;

[0198] b) comparing the determined amount in step a) with a reference; and

[0199] c) identifying whether the subject belongs to the low-risk group of JMML based on the comparison in step b).

[0200] Preferably, the reference can be obtained from at least one subject known to belong to the low-risk group of JMML. If at least one identified biomarker is the same as or increased compared to the reference, it can be understood that the subject under study also belongs to the low-risk group of JMML. However, preferably, the reference can be obtained from at least one subject known not to belong to the low-risk group of JMML. If at least one identified biomarker is the same as or decreased compared to the reference, it can be understood that the subject under study also does not belong to the low-risk group of JMML.

[0201] Preferably, the method of the present invention further comprises selecting a therapy for a subject suffering from JMML based on the JMML risk group identified in step c).

[0202] As used herein, the term "therapy" refers to any measure intended to improve and / or cure JMML or any signs or symptoms associated therewith. Such measures can be selected from administering drugs, administering radiotherapy for modulating the disease, surgery or transplantation, scheduling further appointments with medical staff, or any combination thereof.

[0203] Preferably, an appropriate therapy is selected based on whether a subject with JMML is identified as belonging to a JMML low-risk group or a high-risk group. Generally, one of the following JMML treatment regimens is considered: i) allogeneic stem cell transplantation or ii) drug therapy. Drug therapy generally includes administration of approved azacitidin (Vidaza ), a chemical analogue of the nucleoside cytidine. Preferably, the drug therapy includes administration of an inhibitor that specifically inhibits at least one biomarker selected from Group I or Group II present on or in hematopoietic stem and progenitor cells (HSPCs) as described elsewhere herein: 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.

[0204] The present invention further contemplates at least one biomarker present on or in hematopoietic stem and progenitor cells (HSPCs) in a biological sample for use in diagnosing juvenile myelomonocytic leukemia in a subject with JMML or at risk of developing JMML or classifying a subject with JMML into a JMML low-risk group or a high-risk group, wherein the biomarker is selected from one of the following groups:

[0205] 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

[0206] b) Group II consisting of: IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G.

[0207] The present invention further relates to a kit for diagnosing JMML in a subject or classifying a subject having JMML into a low-risk or high-risk group of JMML, which comprises at least one detection agent and instructions for carrying out the method of the present invention, wherein the at least one detection agent is capable of specifically detecting a Group I or Group 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 present on or in HSPCs.

[0208] As used herein, the term "kit" refers to a collection of the above components, usually provided separately or in a single container. The container usually also includes instructions for carrying out the method of the present invention. These instructions may be in the form of a manual, or may be provided by computer program code which, when implemented on a computer or data processing device, is capable of performing or supporting the determination of the biomarker(s) referred to in the method 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 CD), or directly on a computer or data processing device, or may be provided in a downloadable format such as a link to an accessible server or cloud. In addition, the kit may generally include a standard of the reference amount of the biomarker for calibration purposes, as described in detail elsewhere herein. The kit according to the present invention may also include other components necessary for carrying out the method of the present invention, such as solvents, buffers, wash solutions and / or reagents required for detecting the released second molecule. In addition, it may include, in whole or in part, the device of the present invention.

[0209] As used herein, the term "detection agent" refers to the detection agent referred to in the method of the present invention as described elsewhere herein. In particular, the detection agent may depend on the nature of the biomarker to be detected. Preferably, for detecting a transcribed nucleic acid molecule, a nucleic acid molecule capable of specifically hybridizing to the transcribed nucleic acid, such as an antisense nucleic acid probe or oligonucleotide primer, may be used as the detection agent, while for a protein biomarker, an aptamer, an antibody, an adnectin, an ankyrin, an antibody mimetic and other protein scaffolds, a small molecule, a nucleic acid, a lectin, an affibody, a nanobody, an avimer and a peptide mimetic may be used.

[0210] The present invention also contemplates the use of an inhibitor for the treatment and / or prevention of juvenile myelomonocytic leukemia (JMML), said inhibitor specifically inhibiting at least one Group I or Group 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 is present on or in hematopoietic stem and progenitor cells (HSPC).

[0211] The term "inhibitor" refers to a substance that affects one or more biological or chemical reactions (i.e., its biological activity) triggered by at least one of the above-mentioned biomarkers, in a manner that slows down, hinders, or prevents these reactions. Depending on the type of substance, reversible or irreversible inhibition may occur. It should be understood that the one or more biological or chemical reactions are associated with JMML, i.e., they can directly or indirectly lead to the occurrence or development of the disease.

[0212] As used herein, the term "treatment" relates to improving and / or curing JMML as described herein, preventing the progression of the disease or at least significantly improving at least one symptom associated with the disease. The treatment as used herein also encompasses a complete restoration of health with respect to JMML. It should be understood that the treatment mentioned herein is likely not to be successful in all subjects treated. However, it is expected that the treatment will be effective in at least a statistically significant portion of the subjects treated. Preferably, it can be determined whether successful treatment can be achieved, for example, in a statistically significant portion of a cohort of subjects, by statistical tests using various statistical evaluation tools, such as confidence interval determination, p-value determination, Student's t-test, Mann-Whitney test, etc. Preferably, the treatment will 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.

[0213] The term "prevention" refers to maintaining the health of a subject with respect to JMML over a period of time. It should be understood that the period of time can depend on the therapy used or the amount of the pharmaceutical compound that has been administered. It should be understood that prevention may not be effective for all subjects to whom the binder according to the invention has been administered. However, the term requires that, preferably, a statistically significant portion of a cohort or population of subjects be effectively prevented from suffering from the diseases or disorders or their attendant symptoms mentioned herein. Preferably, in this context, a cohort or population of subjects is envisioned who, typically, i.e., without the preventive measures according to the invention, would develop into JMML. A person skilled in the art can readily determine whether a portion is statistically significant using the various well-known statistical evaluation tools discussed elsewhere in this specification.

[0214] In particular, the present invention contemplates that the Group I or Group II biomarker 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. Thus, preferably, the inhibitor is a peptide, protein, small molecule, lipid, or aptamer.

[0215] As used herein, the term "peptide" or "protein" refers to a molecule composed of amino acid residues linked by peptide bonds. A molecule composed of a short amino acid chain (e.g., up to about 100 amino acids) is called a peptide, while a molecule with a larger amino acid chain is called a protein. Preferably, the peptide or protein will directly or indirectly inhibit the biological activity of at least one biomarker. More preferably, the inhibitor will specifically bind to at least one biomarker and thereby inhibit the biological activity of at least one biomarker.

[0216] The term "small molecule" as used herein refers to a molecule of low molecular weight. Typically, a small molecule is an organic compound with a molecular weight less than 900 daltons. Small molecules include, for example, small secondary metabolites such as alkaloids, lipids, glycosides, terpenes, tetrapyrroles, phenazines, oligonucleotides, or small peptide-like molecules. Preferably, the small molecule will directly or indirectly inhibit the biological activity of at least one biomarker. More preferably, the inhibitor will specifically bind to at least one biomarker and thereby inhibit the biological activity of at least one biomarker.

[0217] As used herein, "lipid" refers to hydrophobic or amphiphilic small molecules. Lipids include fatty acids and their derivatives such as triglycerides, diglycerides, monoglycerides, phospholipids, lysophospholipids such as lysophosphatidylcholine (LPC), glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides, prenol lipids, and sterol lipids. Preferably, the lipid will directly or indirectly inhibit the biological activity of at least one biomarker. More preferably, the inhibitor will specifically bind to at least one biomarker and thereby inhibit the biological activity of at least one biomarker.

[0218] As used herein, the term "aptamer" refers to a polynucleotide or polypeptide that specifically binds to a target molecule by virtue of its three-dimensional structure. Peptide aptamers are preferably peptides comprising 8 - 80 amino acids, more preferably 10 - 50 amino acids, and most preferably 15 - 30 amino acids. They can be isolated, for example, from a random peptide expression library in a suitable host system such as Saccharomyces cerevisiae (see, e.g., Klevenz et al., Cell Mol Life Sci. 2002, 59:1993 - 1998). Peptide aptamers are preferably free peptides; however, it is also contemplated that peptide aptamers are fused to a polypeptide that acts as a "scaffold", meaning a covalent linkage to the polypeptide is used to fix the three-dimensional structure of the peptide aptamer into a specific conformation. Preferably, the aptamer specifically binds to at least one biomarker and inhibits the biological activity of at least one biomarker.

[0219] In another embodiment, the inhibitor is an antibody or an antigen-binding fragment thereof as defined in detail elsewhere herein. In a more preferred embodiment, the antibody or an antigen-binding fragment thereof is alemtuzumab. Alemtuzumab refers to a humanized monoclonal antibody commercially available under the trade 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).

[0220] The present invention also contemplates that the biomarker is a transcribed nucleic acid, preferably mRNA. Thus, preferably, the inhibitor is a ribozyme, an inhibitory RNA molecule, an antisense oligonucleotide, or a morpholino oligonucleotide.

[0221] As used herein, the term "ribozyme" refers to a catalytic RNA molecule with a defined tertiary structure that permits catalysis of the hydrolysis of one of its own phosphodiester bonds (self-cleaving ribozymes), or of bonds in other RNAs, but they have also been found to catalyze the transaminase activity of ribosomes. Ribozymes contemplated by the present invention are preferably those that specifically hydrolyze a target transcript. In particular, according to the present invention, hammerhead ribozymes are preferred. How to generate and use such ribozymes is 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).

[0222] As used herein, the term "inhibitory RNA molecule" refers to an RNA molecule that inhibits gene expression in a sequence-specific manner. Inhibitory RNA molecules include, for example, small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), and microRNAs (miRNAs). Inhibitory RNA molecules typically induce a process called RNA interference (RNAi), resulting in cleavage and / or translational inhibition of target mRNAs with complementary sequences. As is known to those skilled in the art, inhibitory RNA molecules can exhibit complete or incomplete base pairing with complementary target sequences. siRNAs and shRNAs typically have complete base pairing and induce mRNA cleavage only in a single, specific target. In contrast, miRNAs typically have incomplete base pairing with targets and often inhibit the translation of many different mRNAs with similar sequences. Inhibitory RNA molecules can be chemically synthesized or expressed intracellularly, for example, by introducing the respective recombinant DNA constructs. It should be understood that such DNA constructs can contain additional regulatory elements such as enhancers, constitutive or inducible promoters, or terminators. The inhibitory RNA molecule should be a direct or indirect inhibitor of a Group I or Group 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. Preferably, the inhibitory RNA molecule of at least one biomarker will attenuate the translation of the corresponding mRNA into a functional protein, thereby inhibiting the activity of the corresponding protein, or it can degrade or promote the degradation of the said mRNA.

[0223] As used herein, "antisense oligonucleotide" refers to single-stranded DNA and / or RNA molecules that can interfere with DNA and / or RNA processing. Antisense oligonucleotides include nucleic acid sequences complementary to specific RNA or DNA sequences. Generally, antisense oligonucleotides will bind to their respective complementary oligonucleotides, DNA, or RNA in a sequence-specific manner, thereby interfering with DNA and / or RNA processing. As is known to those skilled in the art, antisense oligonucleotides can interfere with mRNA processing through RNase H-mediated degradation, translational arrest, splicing regulation, or they can act through 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 the design of antisense oligonucleotides containing locked nucleic acids (LNAs), as well as solid-phase chemical synthesis. Antisense oligonucleotides can be chemically synthesized or expressed intracellularly, for example, by introducing the respective recombinant DNA constructs. Those skilled in the art will understand that such DNA constructs can contain additional regulatory elements such as enhancers, constitutive or inducible promoters, or terminators. Preferably, the antisense oligonucleotides have 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, at least 50, or more nucleotides. Antisense oligonucleotides can include deoxyribonucleotides, ribonucleotides, or a combination of both. Preferably, the antisense oligonucleotides are DNA and / or RNA molecules that interfere with the expression and / or translation of one of the aforementioned biomarkers, thereby preventing the production of a functional protein.

[0224] The term "morpholino" as used herein refers to molecules of small, specific sequences that block access of other molecules to the base-pairing surfaces of RNA. Typically, the small, specific sequences have a length of about 25 nucleotides. Generally, morpholino oligonucleotides comprise a backbone of methylene morpholine rings and phosphorodiamidate bonds. Morpholino oligonucleotides are also commonly referred to as morpholino oligomers (MO nucleic acid analogs) and phosphorodiamidate morpholino oligomers (PMO). Morpholino oligonucleotides generally do not cause degradation of their target RNA molecules, but rather act by steric hindrance, i.e., binding to the target sequence in the RNA, thereby preventing molecules that might interact with the RNA. Preferably, the morpholino oligonucleotides bind directly to the following pre-mRNA and / or mRNA: 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, and thus, it interferes with their translation and / or splicing processes, resulting in the production of proteins with reduced or no function. Accordingly, the activity of any corresponding protein is inhibited.

[0225] The present invention further contemplates a pharmaceutical composition for use in treating and / or preventing JMML, said pharmaceutical composition comprising at least two inhibitors as defined elsewhere, wherein each inhibitor specifically inhibits a different biomarker selected from the group consisting of those present on or in hematopoietic stem and progenitor cells (HSPCs): 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.

[0226] Thus, the above-mentioned pharmaceutical composition of the present invention comprises two or more different inhibitors that inhibit two or more different biomarkers, as described above.

[0227] As used herein, the term "pharmaceutical composition" refers to a composition comprising a compound of the present invention and preferably one or more pharmaceutically acceptable carriers. The compounds of the present invention can be formulated into pharmaceutically acceptable salts. Preferred acceptable salts are acetate, HCl, sulfate, chloride, etc. The pharmaceutical composition is preferably administered systemically. Suitable routes of administration conventionally used for drug administration are oral, intravenous, subcutaneous or parenteral administration, and inhalation. However, depending on the nature and mode of action of the compound, the pharmaceutical composition can also be administered by other routes. In addition, the compound can be administered in a conventional pharmaceutical composition or in combination with other drugs as a separate pharmaceutical composition, wherein the separate pharmaceutical composition can be provided in the form of a kit.

[0228] Preferably, the compound is administered in a conventional dosage form, which is prepared by combining the drug with a standard pharmaceutical carrier according to conventional procedures. These procedures can include mixing, granulating, pressing or dissolving the ingredients suitable for the desired formulation. It should be understood that the form and characteristics of the pharmaceutically acceptable carrier or diluent depend on the amount of the active ingredient combined therewith, the route of administration, and other well-known variables.

[0229] The carrier must be acceptable in the sense that it is compatible with the other components of the formulation and harmless to the recipient. The pharmaceutical carriers used can be, for example, solids, gels or liquids. Examples of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, biodegradable polymers such as PLGA (DeYoung at al. (2011), DIABETES TECHNOLOGY & THERAPEUTICS 13:1145; Ramazani etal., (2016), Int J Pharm. 499(1-2):358-367 etc.). Exemplary liquid carriers are phosphate buffered saline solutions, syrups, oils such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions, etc. Similarly, the carrier or diluent may include retardation materials well known in the art, such as glyceryl monostearate or glyceryl distearate alone or together with waxes. The suitable carriers include those mentioned above and other carriers well known in the art, see, for example, Remington s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

[0230] One or more diluents are selected so as not to affect the biological activity of one or more compounds. Examples of such diluents are distilled water, physiological saline, Ringer's solution, glucose solution and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants or non-toxic, non-therapeutic, non-immunogenic stabilizers, reactive oxygen species scavengers, etc.

[0231] The pharmaceutical composition is preferably administered in conventional dosage forms, which are prepared by combining the active compound with standard pharmaceutical carriers according to conventional procedures. These procedures may include mixing or dissolving the appropriate ingredients to obtain the desired formulation. It should be understood that the form and characteristics of the pharmaceutically acceptable carrier or diluent depend on the amount of the active ingredient combined therewith, the route of administration and other well-known variables. Similarly, the carrier or diluent may include retardation materials well known in the art, such as glyceryl monostearate or glyceryl distearate alone or together with waxes. A therapeutically effective dose refers to the amount of the active compound used in the pharmaceutical composition of the present invention, which provides the effects mentioned in this specification. The therapeutic effects and toxicities of such compounds can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as ED 50 (the dose that has a therapeutic effect on 50% of the population) and LD 50 (the dose that is fatal to 50% of the population). The dose ratio between the therapeutic effect and the toxic effect is the therapeutic index, which can be expressed as the ratio LD 50 / ED50 The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical art, the dosage for any given patient depends on many factors, which can include the patient's body size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic evaluation. For example, typical dosages can range from 1 μg to 1000 mg; however, dosages below or above this exemplary range can be envisioned, taking into account the above factors. Generally, a conventional dosage regimen for the 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 be in the range of 1 μg to 1 mg units per kilogram body weight per minute. Preferably, the pharmaceutical composition is administered to the subject once, i.e., preferably used as a single-dose treatment. Depending on the subject and the mode of administration, the amount of the substance administered can vary widely to provide from about 0.01 mg / kg body mass to about 100 mg / kg body mass. The pharmaceutical compositions and formulations described herein are administered at least once to treat or ameliorate or prevent the diseases or disorders described in this specification. However, the pharmaceutical composition can be administered more than once, for example, two to 50 times, more preferably five to 50 times. Preferably, the administration is adjusted to maintain an effective concentration in the subject's body over the desired period of time. Progress can be monitored by periodic evaluation.

[0232] The present invention also relates to a method of treating and / or preventing JMML, comprising administering to a subject in need thereof a therapeutically effective amount of at least one inhibitor as defined elsewhere herein.

[0233] "Therapeutically effective amount" means an amount of at least one inhibitor of the present invention that prevents, ameliorates, or cures JMML or the symptoms associated with said disease as mentioned in this specification. The therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED 50 (the dose that has a therapeutic effect on 50% of the population) and LD 50 (the dose that is lethal to 50% of the population). The dose ratio between the therapeutic and toxic effects is the therapeutic index, which can be expressed as the ratio LD 50 / ED 50 . The dosage regimen will be determined by the attending physician and other clinical factors; preferably according to any of the above methods.

[0234] All interpretations and definitions of the above terms apply, mutatis mutandis in detail, to the following embodiments.

[0235] The following embodiments are particularly preferred embodiments according to the present invention.

[0236] Embodiment 1: A method for diagnosing juvenile myelomonocytic leukemia (JMML) in a subject, the method comprising:

[0237] a) determining the amount of at least one biomarker present on or in hematopoietic stem and progenitor cells (HSPCs) in a biological sample, the at least one biomarker being selected from each of the following:

[0238] 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

[0239] ii) Group II consisting of: IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G;

[0240] b) comparing the determined amount of step a) with a reference; and

[0241] c) diagnosing JMML based on the comparison of step b).

[0242] Embodiment 2: The method according to Embodiment 1, wherein the biological sample is a tissue sample or a body fluid sample.

[0243] 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.

[0244] Embodiment 4: The method according to any one of Embodiments 1 to 3, wherein the body fluid sample is a peripheral blood sample or a cord blood sample.

[0245] Embodiment 5: The method according to any one of Embodiments 1 to 4, wherein the subject is a human.

[0246] Embodiment 6: The method according to Embodiment 5, wherein the age of 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 4.5 years old, at most 4 years old, at most 3.5 years old, at most 3 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 or less than 6 months.

[0247] 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 have JMML.

[0248] Embodiment 8: The method according to Embodiment 7, wherein the amount determined in step a) is the same as or greater than the reference indicating that the subject has JMML, or wherein the amount determined in step a) is lower than the reference value indicating that the subject does not have JMML.

[0249] 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.

[0250] Embodiment 10: The method according to Embodiment 9, wherein the amount determined in step a) is the same as or lower than the reference indicating that the subject does not have JMML, or wherein the amount determined in step a) is higher than the reference indicating that the subject has JMML.

[0251] Embodiment 11: The method according to any one of Embodiments 1 to 10, wherein the at least one biomarker is determined by flow cytometry, quantitative PCR (qPCR) or transcriptome sequencing, preferably by bulk RNA-seq or scRNA-seq.

[0252] Embodiment 12: A method for classifying a subject with JMML into a low-risk or high-risk group of JMML, the method comprising:

[0253] a) determining the amount of at least one biomarker present on or in hematopoietic stem and progenitor cells (HSPCs) in a biological sample, the at least one biomarker being selected from each of the following:

[0254] 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

[0255] ii) Group II consisting of: IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1 and HLA-G;

[0256] b) Comparing the determined amount in step a) with a reference; and

[0257] c) Classifying the subject into a JMML low-risk group or a JMML high-risk group based on the comparison in step b).

[0258] Embodiment 13: The method according to embodiment 12, wherein, if at least one biomarker selected from Group I is determined in step a),

[0259] - If the reference is from at least one subject known to have high-risk JMML and the amount of the determined at least one biomarker is the same as or increased compared to the reference, then classify the subject into the JMML high-risk group; or

[0260] - If the reference is from at least one subject known not to have high-risk JMML and the amount of the determined at least one biomarker is the same as or decreased compared to the reference, then do not classify the subject into the JMML high-risk group.

[0261] Embodiment 14: The method according to any one of embodiments 12 to 13, wherein, if at least one biomarker selected from Group II is determined in step a),

[0262] - If the reference is from at least one subject known to have low-risk JMML and the amount of the determined at least one biomarker is the same as or increased compared to the reference, then classify the subject into the JMML low-risk group; or

[0263] - If the reference is from at least one subject known not to have low-risk JMML and the amount of the at least one biomarker determined is the same as or lower than the reference, the subject is not classified into the JMML low-risk group.

[0264] Embodiment 15: A method for identifying whether a subject belongs to the JMML high-risk group, the method comprising:

[0265] a) Determining the amount of at least one biomarker present on or in hematopoietic stem and progenitor cells (HSPCs) in a biological sample, the at least one biomarker being 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;

[0266] b) Comparing the determined amount in step a) with a reference; and

[0267] c) Based on the comparison in step b), identifying whether the subject belongs to the JMML high-risk group.

[0268] Embodiment 16: The method according to Embodiment 15, wherein

[0269] - If the reference is from at least one subject known to have high-risk JMML and the amount of the at least one biomarker determined is the same as or increased compared to the reference, the subject is classified into the JMML high-risk group; or

[0270] - If the reference is from at least one subject known not to have high-risk JMML and the amount of the at least one biomarker determined is the same as or lower than the reference, the subject is not classified into the JMML high-risk group.

[0271] Embodiment 17: A method for identifying whether a subject belongs to the JMML low-risk group, the method comprising:

[0272] a) Determine the amount of at least one biomarker present on or in hematopoietic stem and progenitor cells (HSPCs) in a biological sample, said at least one biomarker being selected from Group II consisting of: IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G;

[0273] b) Compare the determined amount in step a) with a reference, wherein the reference is derived from at least one subject known not to have JMML; and

[0274] c) Based on the comparison in step b), if the amount determined in step a) is the same as or lower than the reference, indicating that the subject does not have JMML, then determine whether the subject belongs to the low-risk group of JMML.

[0275] Embodiment 18: The method according to Embodiment 17, wherein

[0276] - If the reference is derived from at least one subject known to have low-risk JMML and the determined amount of the at least one biomarker is the same as or increased compared to the reference, then classify the subject into the low-risk group of JMML; or

[0277] - If the reference is derived from at least one subject known not to have low-risk JMML and the determined amount of the at least one biomarker is the same as or decreased compared to the reference, then do not classify the subject into the low-risk group of JMML.

[0278] Embodiment 19: The method according to any one of Embodiments 12 to 18, further comprising selecting a therapy for a subject with JMML based on the identified JMML risk group in step c).

[0279] Embodiment 20: At least one biomarker present on or in hematopoietic stem and progenitor cells (HSPCs) in a biological sample, for use in diagnosing juvenile myelomonocytic leukemia (JMML) in a subject having or at risk of developing JMML or classifying a subject with JMML into a low-risk or high-risk group of JMML, said biomarker being selected from one of the following groups:

[0280] 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

[0281] b) Group II consisting of: IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G.

[0282] Embodiment 21: A kit for diagnosing juvenile myelomonocytic leukemia (JMML) in a subject or classifying a subject with JMML into a low-risk or high-risk group of JMML, comprising at least one detection agent and instructions for performing the method according to any one of Embodiments 1 to 18, wherein the at least one detection agent is capable of specifically detecting 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, SELK, PRRG3, NINJ1, MGST1, and HLA-G, present on or in hematopoietic stem and progenitor cells (HSPCs).

[0283] Embodiment 22: Use of an inhibitor for the treatment and / or prevention of juvenile myelomonocytic leukemia (JMML), wherein the inhibitor specifically inhibits at least one biomarker selected from the group consisting of those present on or in hematopoietic stem and progenitor cells (HSPCs): 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.

[0284] Embodiment 23: The inhibitor for use according to Embodiment 22, wherein the inhibitor specifically binds to and inhibits the at least one biomarker.

[0285] Embodiment 24: The inhibitor for use according to Embodiment 22 or 23, wherein the inhibitor is a peptide, protein, small molecule, lipid, or aptamer.

[0286] Embodiment 25: The inhibitor for use according to Embodiment 22 or 23, wherein the inhibitor is an antibody or an antigen-binding fragment thereof.

[0287] Embodiment 26: The inhibitor for use according to Embodiment 25, wherein the antibody or the antigen-binding fragment thereof is alemtuzumab.

[0288] Embodiment 27: The inhibitor according to Embodiment 22, wherein the inhibitor specifically binds to and inhibits the at least one biomarker, and the at least one biomarker is an expressed nucleic acid, preferably mRNA.

[0289] 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 oligonucleotide.

[0290] Embodiment 29: Use of a pharmaceutical composition for the treatment and / or prevention of juvenile myelomonocytic leukemia (JMML), the pharmaceutical composition comprising at least two inhibitors as defined in any one of Embodiments 22 to 28, wherein each of the inhibitors specifically inhibits different biomarkers selected from the group consisting of those present on or in hematopoietic stem and progenitor cells (HSPCs): 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.

[0291] Embodiment 30: The pharmaceutical composition for the use according to Embodiment 29, further comprising a pharmaceutically acceptable carrier.

[0292] Embodiment 31: A method for the treatment and / or prevention of juvenile myelomonocytic leukemia (JMML), the method comprising administering to a subject in need thereof a therapeutically effective amount of at least one inhibitor as defined in any one of Embodiments 22 to 28.

[0293] All references cited in this specification are incorporated herein by reference in their entirety for the specific disclosures mentioned therein. BRIEF DESCRIPTION OF THE DRAWINGS

[0294] Figure 1 : Patients were selected for in-depth multimodal analysis to detect novel biomarkers and therapeutic targets for JMML stem cells. DNA methylation array analysis (EPIC array) was performed on total hematopoietic cells from 8 different patients diagnosed with JMML. The heatmap shows the average DNA methylation values (β-values) of 124 classifier CpGs from a previously published JMML DNA methylation classifier ( et al. 2021 Clinical Cancer Research). The epigenotype refers to the consensus definition of DNA methylation subgroups in JMML. The high-risk group refers to HM patients, while the low-risk group refers to LM and IM patients.

[0295] Figure 2: Biomarker identification and validation. (A) Dot plot summarizing scRNA-seq expression data of identified high-risk and low-risk HSC biomarkers. These biomarkers represent differentially expressed genes in HSCs between high-risk and low-risk JMML patients, which encode putative cell surface markers. The high-risk and low-risk groups are defined as explained above Figure 1 and described. The normal group represents HSCs from healthy donor cord blood. The color of the dots represents the normalized mean expression value of single-cell data for each group. The dot size represents the percentage of cells in each group that detected the expression of the marker gene. (B) As Figure 1 determined, FACS analysis histograms of high-risk JMML HSC surface markers CD52, CD69, and CD164 on JMML Lin - CD34 + HSPCs from low-risk to high-risk patients confirmed the expected increase in cell surface protein expression in high-risk patients.

[0296] Figure 3 : Functional validation of anti-CD52 treatment on xenograft (PDX) mice derived from JMML patients. (A) Representative FACS plots of control and anti-CD52-treated (alemtuzumab) JMML PDX mice. Anti-CD52-FITC was used to detect extracellular CD52 levels on human hematopoietic cells. FSC-A is the forward scatter area. (B) Quantification of live human hematopoietic CD45 + cells in the bone marrow of control and anti-CD52-treated (alemtuzumab) PDX mice. (n = 10 mice / group)

[0297] Figure 4 : Depletion of total human hematopoietic function in JMML PDX mice after anti-CD52 treatment. Panels (A) to (G) represent the log-scale numbers of live human cells across different hematopoietic lineages in total bone marrow quantification of anti-CD52-treated (alemtuzumab) or control JMML PDX mice by FACS analysis. Depletion of total hematopoietic function, including CD52-negative cells such as red blood cells, confirmed that JMML stem cells are disease-propagating cells, and the JMML stem cell markers identified here are therapeutic targets for JMML.

[0298] Figure 5 : Disease propagation in JMML was disrupted 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 (alemtuzumab). (A) Engraftment of human CD45 +Cells, revealing the disruption of anti-CD52 therapy on disease spread. (n = 7 - 8 mice / group) (B) Kaplan-Meier curves summarizing the leukemia-free survival of 2° recipients, confirming the efficacy of targeting JMML HSC surface markers. (n = 3 mice / group)

[0299] Figure 6 : Conservation of the JMML epigenotype in stem cells and throughout hematopoiesis. (A) For Lin - CD34 + CD38 - Ultra-low input whole-genome bisulfite sequencing (WGBS) of JMML HSPCs revealed the conservation of the JMML epigenotype in the immature JMML stem cell compartment. (B) Integration of ultra-low WGBS and DNA methylation array analysis revealed that the DNA methylation patterns of JMML stem cells were conserved in the DNA methylomes of a total 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 from 0 to 1 refer to DNA methylation β values. The conservation in 147 patients indicates that the molecular programs in JMML stem cells are generally of prognostic and therapeutic value in JMML. (C) Integration of DNA methylation data (WGBS) from JMML stem cells with DNA methylation array data (Illumina 450k and EPIC) from JMML bulk material (total samples) including mature immune cells. Risk-associated DNA methylation changes in JMML stem cells were used to examine the conservation in bulk patient material from a total of 331 patients. Principal component 1 (PC 1) accurately summarized the epigenomes of all patients determined by using DNA methylation array analysis, confirming the transmission of the epigenome from immature JMML cells to mature JMML cells.

[0300] Figure 7 : Association between DNA methylation and surface marker gene expression. (A) Ultra-low input WGBS data revealed differential methylation of JMML stem cell surface markers. (B) scRNA-seq revealed methylation-related differential gene expression of JMML stem cell surface markers. The heatmap aligned the differentially expressed gene expression with the associated differentially methylated regions (DMRs) in a 200 Mb region around each transcription start site (TSS). Generalized linear models with stepwise feature elimination were used to identify DMR-associated DEGs using AIC, thereby identifying methylation-related gene expression changes across JMML phenotypes. Examples

[0301] The examples will only illustrate the present invention. In no way should they be construed as limiting the scope.

[0302] Example 1: Enrichment of hematopoietic stem cells and progenitors

[0303] To classify the epigenotypes and risk groups of JMML patients, primary hematopoietic cells were analyzed by DNA methylation array (Infinium Human Methylation EPIC Bead Chip (EPIC) array) as previously described ( et al. 2021, Clinical Cancer Research). Briefly, 100 - 250 ng of genomic DNA (gDNA) was placed in the Genomics and Proteomics Core Facility of the German Cancer Research Center (Heidelberg, Germany), and the data was analyzed using the "RnBeads" bioconductor package as previously described (Lipka et al. 2018, Nature Communications, et al. 2021 Clinical Cancer Research). Patients were classified into one of three epigenetically defined subgroups (hypomethylated (LM), intermediate methylated (IM), or hypermethylated (HM)) according to a previously published JMML 124 CpG DNA methylation classifier ( et al. 2021, Clinical Cancer Research).

[0304] To obtain materials for single - cell RNA sequencing (scRNA - seq) of JMML hematopoietic stem cells and progenitors (HSPC), primary cells from JMML patients were isolated across epigenetic risk groups from biopsies using fluorescence - activated cell sorting (FACS). Table 1 summarizes the antibodies used to enrich Lin - CD34 + CD38 - HSPC:

[0305] Table 1: Antibodies used to enrich JMML HSPC.

[0306] antigen dye dilute company CD34 APC-Cy7 1:20 Invitrogen CD38 PE 1:30 Invitrogen CD4 APC 1:25 BD Biosciences CD8 APC 1:20 BD Biosciences CD11b APC 1:20 BD Biosciences CD14 APC 1:50 Invitrogen CD19 APC 1:50 Invitrogen CD20 APC 1:25 BD Biosciences CD56 APC 1:20 BD Biosciences CD235a APC 1:50 Invitrogen CD52 BV421 1:20 BD Biosciences CD69 BB700 1:40 BD Biosciences CD164 BV786 1:40 BD Biosciences

[0307] Example 2: Single - cell sequencing

[0308] Approximately 10,000 JMML HSPCs were applied to single-cell RNA sequencing (scRNA-seq) using the 10X Genomics chromium platform according to the manufacturer's instructions. Single-cell sequencing libraries were generated using the Chromium Single Cell 3’ Library & Gel Bead Kit v2 and the SingleCell A Chip Kit. All libraries were sequenced in paired-end mode with 26+74bp on an Illumina HiSeq 4000 at the Genomics and Proteomics Core Facility of the German Cancer Research Center (Heidelberg, Germany).

[0309] Example 3: Data evaluation of single-cell sequencing

[0310] Approximately 10,000 JMML HSPCs were applied to single-cell RNA sequencing (scRNA-seq) using the 10X Genomics chromium platform according to the manufacturer's instructions. Single-cell sequencing libraries were generated using the Chromium Single Cell 3’ Library & Gel Bead Kit v2 and the SingleCell A Chip Kit. All libraries were sequenced in paired-end mode with 26+74bp on an Illumina HiSeq 4000 at the Genomics and Proteomics Core Facility of the German Cancer Research Center (Heidelberg, Germany).

[0311] The scRNA-seq data was aligned and quantified using the Cell Ranger single-cell software suite (10X Genomics) with GRCh38 as the human reference genome. As 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 to remove outliers or bimodality, respectively.

[0312] Downstream analysis was performed using Seurat V3 or V4 (Stuart and Butler et al. 2019 Cell; Hao and Hao et al. 2021 Cell), with default parameters of NormalizeData (LogNormalize), ScaleData, and FindVariableGenes. RunPCA was applied to perform data dimensionality reduction from variable genes, and the number of PCs was selected based on elbow plot analysis.

[0313] As healthy normal controls, scRNA-seq data from human umbilical cord blood was used, which was publicly available from the Human Cell Atlas (HCA) data portal (https: / / data.humancellatlas.org / explore / projects / cc95ff89-2e68-4a08-a234-480eca21ce79 )

[0314] To generate consistent cell type definitions across datasets after data integration, Seurat's label transfer function was applied using published human reference scRNA-seq data of healthy adult hematopoiesis in the HCA project (Hay et al. 2018, Experimental Hematology). Thus, hematopoietic stem cells (HSCs) could be identified in both JMML and healthy reference data.

[0315] Example 4: Identification of Biomarkers Specific to Risk Groups

[0316] To determine risk group-specific biomarkers, patients in the HM subgroup were considered high risk, while patients in the IM and LM subgroups were considered low risk. To screen for differentially expressed genes (DEGs) between high-risk and low-risk HSCs in JMML, the FindMarkers function of Seurat was applied with the following default settings: genes were expressed in at least 10% of the cells, the expression difference on the natural logarithm scale was at least 0.25, a one-tailed Wilcoxon rank sum test, and a P value adjusted for multiple testing using Bonferroni correction. To identify DEGs encoding cell surface markers, the entire DEG list was used as input to SurfaceGenie to calculate the surface protein consensus (SPC) score (Waas et al. 2020, Bioinformatics). This yielded a list of a total of 61 genes with an SPC score greater than 0. By manual screening, this gene list was further refined to exclude those genes that were either most highly expressed in the JMML IM subgroup or had higher expression in normal control HSCs than in the JMML risk group with the highest expression of the corresponding gene. In this way, statistically significant DEGs specific to the risk groups were identified, presumably encoding cell surface factors. These genes represent (1) prognostic intracellular and extracellular biomarkers for high-risk and low-risk groups in JMML, and (2) potential drug targets on the cell surface of JMML HSCs.

[0317] Example 5: Evaluation of the Functional Role of Biomarkers

[0318] To evaluate the functional role of these 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. Flow cytometry using FACS was used to quantify human CD45 + cell tissue infiltration and the amount of human CD52 + cells. To evaluate the clinical efficacy of anti-CD52 treatment, the total bone marrow of 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 the PDX mice.

[0319] Table 2: Antibodies for quantifying human hematopoietic cells and CD52 + cells in PDX mice.

[0320] antigen dye reactivity company CD45 Horizon BV605 mouse Biolegend CD11b AxF488 mouse, human Biolegend CD45 PE human BD Biosciences CD38 PerCP / Cy5.5 human BD Biosciences CD33 APC human BD Biosciences CD235a Horizon BV421 human BD Biosciences CD34 PE / Cy7 human Biolegend CD13 APC / Cy7 human Biolegend CD14 Pacific Blue human Biolegend CD19 FITC human Biolegend CD66b PerCP / Cy5.5 human Biolegend CD3 PE / Cy7 human Biolegend CD71 APC / Cy7 human Biolegend CD52 FITC human Biolegend CD45RA PerCP / Cy5.5 human Biolegend CD90 APC / Cy7 human Biolegend CD38 APC human Biolegend

[0321] Figure 1 The heatmap of DNA methylation array analysis (EPIC array) in shows the average methylation values of 124 classifier CpGs of total hematopoietic cells from 8 different JMML patients. Based on these values, the patients were classified into 2LM patients (P1 - 2), 2IM patients (P3 - 4), and 4HM patients (P5 - 8). The subset of HM patients is considered high - risk JMML, while LM and IM patients are grouped as low - risk JMML.

[0322] Hematopoietic stem and progenitor cells (HSPCs) were defined as Lin - CD34 + CD38 - cells and were enriched from each of the eight patients by flow cytometry. These HSPCs were analyzed using droplet - based single - cell RNA sequencing (scRNA - seq). A total of 13,594 hematopoietic stem cells (HSCs) were identified by using reference - based cell - type annotation. These JMML HSCs were used to screen (call) differentially expressed genes (DEGs) across the defined risk groups (high - risk vs. low - risk). From this transcriptome - wide list of DEGs, genes encoding putative cell - surface markers were selected. To identify genes abnormally upregulated in JMML, the average gene expression values of JMML HSCs were compared with 1,069 HSCs isolated from human umbilical cord blood. Figure 2 A of Figure 2 summarizes the average expression values of 41 identified surface - marker genes, which show (1) risk - group - specific differential expression patterns and (2) disease - specific overexpression. These genes represent prognostic biomarkers and therapeutic targets for JMML HSCs. Figure 2 B of Figure 2 illustrates the corresponding protein expression of the high - risk JMML biomarkers CD52, CD69, and CD164.

[0323] To functionally evaluate the role of these surface - marker genes and assess their therapeutic potential, the established JMML pre - clinical patient - derived xenograft (PDX) mouse model was used to apply alemtuzumab, which is a monoclonal therapeutic anti - CD52 antibody. Figure 3 A of Figure 3 shows that anti - CD52 treatment was able to specifically deplete human CD52 + cells relative to control mice not administered alemtuzumab. This treatment led to human CD45 in treated mice +Overall depletion of cells, while the number of leukemia cells implanted in control mice was significantly higher ( Figure 3 ) of B).

[0324] To further evaluate the therapeutic potential of anti-CD52 targeted therapy, flow cytometry was used to analyze alemtuzumab-treated and untreated JMML PDX mice. FACS not only confirmed the efficient depletion of human CD52 + , but also confirmed the efficient depletion of CD52 - cells (including human CD34 + CD38 - cells) and mature hematopoietic cells in all hematopoietic lineages ( Figure 4 ). Thus, alemtuzumab treatment targets human HSPCs, resulting in depletion of almost the entire hematopoietic system, indicating that JMML HSPCs contain disease-propagating cells that are therapeutically vulnerable.

[0325] Secondary transplantation of whole bone marrow from alemtuzumab-treated animals and control animals confirmed that anti-CD52 treatment prevented leukemia engraftment in 2° recipients. FACS analysis revealed a significant reduction in the number of human (leukemia) cells engrafted in blood, bone marrow, spleen, liver, and lung ( Figure 5 ) of A). In addition, in the secondary transplantation experiment, the leukemia-free survival rate was significantly improved in mice receiving bone marrow from initially treated mice compared to mice receiving bone marrow from untreated PDX mice ( Figure 5 ) of B). This result not only confirmed the functional relevance of these surface markers. In addition, it also demonstrated the therapeutic potential of the biomarkers identified here. In summary, targeting CD52 leads to efficient depletion of leukemia-propagating stem cells in vivo, providing a preclinical rationale for further evaluating anti-CD52 treatment in JMML patients. In addition, this example demonstrates the power of this molecularly precise approach.

[0326] In summary, the present inventors used FACS or expression analysis to identify novel intracellular and extracellular biomarkers for rapid, reliable, and cost-effective risk stratification. In addition, the functional relevance and therapeutic potential of such surface markers were demonstrated by anti-CD52 treatment of patient-derived xenografts. Thus, CD52 is a new therapeutic target and a prognostic and predictive biomarker for high-risk JMML. In summary, the present inventors identified the first drug target specific for high-risk JMML.

[0327] Example 6: In a large patient cohort, the methylation patterns of patients showing expression of prognostic biomarkers are conserved

[0328] To evaluate the conservation of molecular programs in JMML stem cells, the inventors herein applied ultra-low input whole-genome bisulfite sequencing (WGBS) for the first time on JMML HSPCs enriched by flow cytometry to profile the entire methylome of JMML stem cells. The inventors modified the single-cell bisulfite protocol (Clark et al. 2017 Nature Protocols) to apply the above sorting strategy to analyze up to 100 highly purified JMML HSPCs.

[0329] The DNA methylome of JMML stem cells revealed the conservation of the JMML epigenome, as Figure 1 determined by bulk DNA methylation array analysis in Figure 6 A). Integration of ultra-low input WGBS and DNA methylation array data from a total of 147 JMML patients revealed the conservation of disease-specific DNA methylation signatures in bulk JMML samples, indicating the transmission of the epigenotype from immature to mature JMML cells Figure 6 in B). To evaluate JMML stem cells as the origin of disease-specific epigenetic phenotypes, the inventors demonstrated that JMML stem cell-specific DNA methylation signatures could recapitulate the epigenetic phenotypes of a total of 331 JMML patients Figure 6 in C).

[0330] Furthermore, the inventors could demonstrate that DNA methylation changes were 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 ). Collectively, the conservation of disease-specific aberrations in over 300 patients confirmed the functional value of JMML stem cell signatures as relevant prognostic biomarkers and JMML therapeutic targets.

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Claims

1. A method for diagnosing juvenile myelomonocytic leukemia (JMML) in a subject, the method comprising: a) determining the amount of at least one biomarker present on or in hematopoietic stem and progenitor cells (HSPCs) in a biological sample, the at least one biomarker being 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, 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).

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 and the age of 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 4.5 years old, at most 4 years old, at most 3.5 years old, at most 3 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, or less than 6 months.

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 wherein the amount determined in step a) being the same as or greater than the reference indicates that the subject has JMML, or wherein the amount determined in step a) being 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 subjects known not to have JMML, and wherein the amount determined in step a) being the same as or lower than the reference indicates that the subject does not have JMML, or wherein the amount determined in step a) being higher than the reference indicates that the subject has JMML.

6. A method for classifying a subject with JMML into a low-risk or high-risk group of JMML, the method comprising: a) determining the amount of at least one biomarker present on or in hematopoietic stem and progenitor cells (HSPCs) in a biological sample, the at least one biomarker being 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, SELK, PRRG3, NINJ1, MGST1, and HLA-G; b) comparing the determined amount in step a) with a reference; and c) classifying the subject into a JMML low or high risk group based on the comparison in step b).

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 JMML high risk group, or wherein, if at least one biomarker selected from Group II is determined in step a), the subject is classified into the JMML low risk group.

8. A method for identifying whether a subject belongs to the JMML high risk group, the method comprising: a) determining the amount of at least one biomarker present on or in hematopoietic stem and progenitor cells (HSPCs) in a biological sample, the at least one biomarker 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) comparing the determined amount in step a) with a reference; and c) identifying whether the subject belongs to the JMML high risk group based on the comparison in step b).

9. A method for identifying whether a subject belongs to the JMML low risk group, the method comprising: a) determining the amount of at least one biomarker present on or in hematopoietic stem and progenitor cells (HSPCs) in a biological sample, the at least one biomarker selected from 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) identifying whether the subject belongs to the JMML low risk group based on the comparison in step b).

10. The method according to any one of claims 8 to 9, further comprising selecting a therapy for a subject with JMML based on the JMML risk group identified in step c).

11. At least one biomarker present on or in hematopoietic stem and progenitor cells (HSPCs) in a biological sample, for use in diagnosing juvenile myelomonocytic leukemia (JMML) in a subject having or at risk of developing JMML or classifying a subject with JMML into a JMML low-risk group or a high-risk group, said biomarker being selected from one of the following 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, SELK, PRRG3, NINJ1, MGST1, and HLA-G.

12. A kit for diagnosing juvenile myelomonocytic leukemia (JMML) in a subject or classifying a subject with JMML into a JMML low-risk or high-risk group, 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 is capable of specifically detecting 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, SELK, PRRG3, NINJ1, MGST1, and HLA-G present on or in hematopoietic stem and progenitor cells (HSPCs).

13. Use of an inhibitor for the treatment and / or prevention of juvenile myelomonocytic leukemia (JMML), wherein the inhibitor 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 is present on or in hematopoietic stem and progenitor cells (HSPCs).

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

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

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

17. Use of a pharmaceutical composition for the treatment and / or prevention of juvenile myelomonocytic leukemia (JMML), wherein the pharmaceutical composition comprises at least two inhibitors as defined in any one of claims 20 to 26, wherein each of the inhibitors 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 in hematopoietic stem and progenitor cells (HSPCs).

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