The present invention concerns the diagnosis and assessment of juvenile myelomonocytic leukemia (JMML). In particular, it relates to a method of 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, said at least one biomarker being selected from each of i) group I consisting of CD52, RAMP1, LTB, LST1, J AML, IFITM3, CD7, CD69, CD 164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RABI 1 A, 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 of step a) to a reference, and c) diagnosing JMML based on the comparison of step b). Furthermore, the present invention relates to a method of classifying a subject suffering from JMML into a JMML low- or high-risk group. In addition, the present invention concerns use of at least one biomarker present on or in HSPCs in a biological sample for diagnosing JMML into a JMML low- or high-risk group in a subject having or having a risk of developing JMML. Furthermore, the present invention relates to a kit for diagnosing JMML in a subject or classifying a subject suffering from JMML into a JMML low- or high-risk group. Also, the present invention concerns an inhibitory agent that specifically inhibits at least one biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RABI 1 A, 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) for use in treating and/or preventing JMML. The present invention further relates to a pharmaceutical composition for use in treating and/or preventing JMML comprising at least two inhibitory agents according to the invention. Finally, the present invention envisages a method of treating and/or preventing JMML.

Juvenile myelomoncytic leukemia (JMML) is a rare and aggressive type of blood cancer that occurs in early childhood, with a median age at diagnosis below 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) and shares some features with chronic myelomonocytic leukemia in adults. The disorder originates from multipotent hematopoietic stem/progenitor cells and is characterized by overproduction of mature and immature myeloid cells. It is assumed that hyperactive RAS signalling is the main driving event in JMML. The majority of the patients (about 90%) harbour somatic mutations in the KRAS-, NRAS-, PTPN11-, NF1- and/or CBL-gene, which define genetically and clinically distinct subtypes (Schbnung et al., Clin Cancer Res. 2021 Jan 1; 27(1): 158-168).

JMML is also characterized primarily by its heterogeneity. For instance, the clinical course of the disease is very heterogeneous and can only partly be predicted using clinical features. This clinical heterogeneity is reflected in therapeutic considerations ranging from watchful waiting to early allogeneic hematopoietic stem cell transplantation (HSCT) (Niemeyer & Flotho 2019, Blood). Whereas the disease eventually resolves spontaneously in ~15% of the cases, more than 50% of patients relapse after HSCT (Locatelli et al. 2005 Blood).

This clinical heterogeneity underscores the desperate need for diagnostic approaches that would allow the upfront identification of high-risk patients. Furthermore, novel risk-adapted, molecularly targeted treatment options need to be developed and tested. On the one hand, clinical research on JMML discovered genetic subgroups defined by driver mutations leading to RAS pathway hyperactivation (Neubauer et al. 1991 Blood; Flotho et al. 1999 Leukemia). On the other hand, gene expression-related signatures revealed varying non-genetic characteristics in JMML (Bresolin et al. 2010 Journal of Clinical Oncology; Helsmoortel et al. 2016 Blood). Whereas both genetic and non-genetic features have been demonstrated to bear a certain degree of prognostic power, these biomarkers failed to recapitulate the full spectrum of clinical heterogeneity observed in JMML. As a consequence, clinical research on JMML has been lacking meaningful and robust prognostic biomarkers for a long time.

The missing link between genetic and non-genetic signatures to dissect clinical heterogeneity in JMML was considered to be gene regulatory or epigenetic programs, respectively. Indeed, the stratification of JMML patients by DNA methylation patterns revealed a significant correlation between epigenetics and prognosis in JMML (Olk-Batz et al. 2011, Blood). By use of genome-wide array-based DNA methylation analyses, epigenetic subgroups in JMML were established (Lipka et al. 2017, Nature Communications; Murakami et al. 2018, Blood; Stieglitz et al. 2017, Nature Communications), which revealed DNA methylation as the only significant factor to predict overall survival in JMML. Moreover, DNA methylation appears to be predictive of therapeutic response. Treatment with hypomethylating agents appeared to be particularly effective in low- and intermediate-risk DNA methylation subgroups (Niemeyer et al. 2021 Blood Advances). However, in view of this poor response to hypomethylating agents and the high relapse rate after HSCT, there is a lack of therapeutic options that would allow to target high-risk JMML (Loh 2011, British Journal of Haematology).

Preclinical studies that used patient-derived xenograft mouse models revealed that transplanted JMML cells are capable of reconstituting the entire hematopoietic system (Lapidot et al. 1996 Blood; Iversen et al. 1997, Blood; Krombholz et al. 2016, Haematologica; Krombholz et al. 2019, Leukemia). Moreover, the DNA methylation patterns characteristic for epigenetic subgroups in JMML were re-established in xenograft mice. This indicated that hematopoietic stem cells (HSCs) can be considered as the leukemia initiating cells in JMML, which slowly increased the interest in this cell population in the field (Louka et al. 2021, Journal of Experimental Medicine). Presently, clinical outcome can only insufficiently be explained by clinical or genetic features, whereas dysregulation of the DNA methylation landscape has been demonstrated as a non-random, subgroup-specific prognostic feature. At the moment, a systematic and subgroup-stratified characterization of HSPCs in JMML is lacking.

In principle, JMML is a rare aggressive clonal neoplasm (DeVos 2022, Mayerhofer 2021). The incidence of JMML is estimated at 1.3 cases per 1 million person-years in children from 0 to 14 years (according to the WHO Classification of Tumours). Therefore, contrary to other neoplasms only very limited numbers of patient samples are available. In addition, samples biobanked in the European JMML biobank are typically DNA samples rather than viably frozen cells. Nevertheless, there is an ongoing need for therapies for pediatric cancers and, in particular, for aggressive types of cancer such as JMML occurring in early childhood with limited treatment options so far (Laetsch 2021).

Thus, there is a need for prognostic and diagnostic means for risk stratification of JMML and for the development of novel subgroup-specific therapies.

The technical problem underlying the present invention may be seen as the provision of means and methods for complying with the aforementioned need. The technical problem is solved by the embodiments characterized in the claims and herein below.

The present invention relates to a method of diagnosing juvenile myelomonocytic leukemia (JMML) in a subject, the method comprising: a) determining the amount of at least one biomarker on hematopoietic stem and progenitor cells (HSPCs) in a biological sample, said at least one biomarker being selected from each of: i) group I consisting of: CD52, RAMP1, LTB, LST1, J AML, 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 of step a) to a reference; and c) diagnosing JMML based on the comparison of step b).

It is to be understood that in the specification and in the claims, “a” or “an” can mean one or more of the items referred to in the following depending upon the context in which it is used. Thus, for example, reference to “an” item can mean that at least one item can be utilized.

As used in the following, the terms “have”, “comprise”, or “include” are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements.

The terms "particularly", "more particularly", “typically”, and “more typically” or similar terms are used in conjunction with additional and/or alternative features, without restricting alternative possibilities. Thus, features introduced by these terms are additional and/or alternative features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using further alternative features. Similarly, features introduced by "in an embodiment of the invention" or similar expressions are intended to be additional and/or alternative features, without any restriction regarding alternative embodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such way with other additional and/or alternative or non-additional and/or alternative features of the invention. Further, as used in the following, the terms “preferably”, “more preferably”, “most preferably”, "particularly", "more particularly", “typically”, and “more typically” are used in conjunction with features in order to indicate that these features are preferred features, i.e. the terms shall indicate that alternative features may also be envisaged in accordance with the invention.

Further, it will be understood that the term “at least one” as used herein means that one or more of the items referred to following the term may be used in accordance with the invention. For example, if the term indicates that at least one item shall be used this may be understood as one item or more than one item, i.e. two, three, four, five, or any other number. Depending on the item the term refers to, the skilled person understands as to what upper limit the term may refer, if any.

The term “about” as used herein means that with respect to any number recited after said term an interval accuracy exists within in which a technical effect can be achieved. Accordingly, the term “about” in the context of the present invention means ± 20%, ± 10%, ± 5%, ± 2 %, or ± 1% from the indicated parameters or values. This also takes into account usual deviations caused by measurement techniques and the like.

The method of the present invention may consist of the aforementioned steps or may comprise additional steps, such as steps for further evaluation of the assessment obtained in step (c), steps recommending therapeutic measures such as treatments, or the like. Moreover, it may comprise steps prior to step (a) such as steps relating to sample pre-treatments. However, preferably, it is envisaged that the above-mentioned method is an ex vivo method which does not require any steps being practiced on the human or animal body. Moreover, the method be assisted by automation. Typically, the determination of the biomarkers may be supported by robotic equipment while the comparison and assessment may be supported by data processing equipment such as computers.

The term “diagnosing” as used herein refers to the assessment of the health condition of a subject. Accordingly, “diagnosing” as used herein refers to determining whether a subject suffers from juvenile myelomonocytic leukemia (JMML), or not, predicting the risk for developing JMML and/or predicting any deterioration of the health condition of the subject, in particular, with respect to signs and symptoms accompanying JMML. The most common signs and symptoms of JMML include hepatosplenomegaly, lymphadenopathy and anemia, and thrombocytopenia leading to pallor, fatigue, weakness and bleeding, and/or bruising, respectively. Patients present also with bone and joint pain, abdominal pain, recurrent fevers, infections, and/or dry coughs and difficulty in breathing. As will be understood by those skilled in the art, such a diagnosing, although preferred to be, may usually not be correct for 100% of the investigated subjects. The term, however, requires that a statistically significant portion of subjects can be correctly diagnosed. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test, etc. Details may be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Typically envisaged 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 to be understood that the method for diagnosing of the present invention shall aid a medical practitioner in rendering a final diagnosis. The medical may also consider further factors to increase the correctness of its diagnosis, such as presence of symptoms of JMML as described herein elsewhere.

The term “subject” as referred to herein, can be an animal and, preferably, a mammal. More preferably, the subject is a human. The subject to be investigated by the method of the present invention is, preferably, a juvenile subject, i.e. a child or a young adult. More specifically, the subject is at an age of at most 16 years, at most 15 years, at most 14 years, at most 13 years, at most 12 years, at most 11 years, at most 10 years, at most 9 years, at most 8 years, at most 7 years or at most 6 years, at most 5.5 years, at most 5 years, at most 4.5 years, at most 4 years, at most 3.5 years, at most 3 years, at most 2.5 years, at most 2 years, at most 1.5 years, at most 1 year, at most 6 months, or less than 6 months.

The subject to be investigated by the method of the present invention shall be a subject suffering from or having a risk of developing JMML. Suffering from JMML as used herein, means that the subject shall exhibit clinical parameters, signs and/or symptoms of JMML. Thus, the subject according to the invention is, typically, a subject that suffers from a JMML or is suspected to suffer from JMML. Having a risk of developing JMML as used herein, refers to an apparently healthy subject which does not yet show clinical signs or symptoms of JMML but which has an increased risk of developing JMML or to a subject which shows weak signs or symptoms of the disease but which is at risk of developing a worsening of the disease or signs or symptoms associated therewith.

The term “biomarker” relates in accordance with the present invention to a biological molecule the presence, absence or abundance of which is indicative for a health condition. In accordance with the present invention, the said health condition may be JMML or the absence of JMML, a low risk JMML and/or a high risk JMML. A biomarker in accordance with the present invention may be a protein or fragment thereof selected from those proteins referred to elsewhere herein in more detail. Yet, the biomarker may be a transcribed nucleic acid molecule the presence, absence or abundance of which can be used as a surrogate for the protein. Preferably, such transcribed nucleic acid molecules are the messenger RNA molecules (mRNA) or any precursor or variant thereof, including pre-mRNA or mRNA for splice variants. Those RNA nucleic acid molecules may be determined as biomarkers in accordance with the present invention as well. Thus, it will be understood that if, e.g., CD52 shall be determined as biomarker in accordance with the present invention, either CD52 protein may be determined or a transcribed nucleic acid molecule encoding the CD52 protein such as CD52 mRNA. The same applies for all other biomarkers referred to herein except specified otherwise. In the specification, it is referred to the proteins, however, the skilled person is well aware of the transcribed nucleic acid molecules belonging to said proteins and the genes encoding them.

The biomarkers to be used in accordance with the present invention encompass biomarkers associated with high-risk JMML or low-risk JMML.

High-risk JMML biomarkers are those of group I as referred to in accordance with the present invention and are selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD 164, 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 of group I as referred to in accordance with the present invention and are selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RABI 1 A, SELL, VAMP5, FCMR, CLEC7A, NDFIP1, CLEC9A, HCST, LPAR6, HLA-DQA1, HLA-DRB5, and CD34. Preferably, high-risk JMML biomarkers are those of group I as referred to in accordance with the present invention and are selected from the group consisting of CD52, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, CD82, IGHM, RALA, HLA-DRA, SELL, CLEC7A, CLEC9A, and HCST. More preferably, high-risk biomarkers of group I are selected from the group consisting of: CD52, CD69, CD164, IGHM, RALA, and HLA-DRA.

Low risk JMML biomarkers are those of group II as referred to in accordance with the present invention and are selected from the group consisting of IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G. More preferably, low-risk biomarkers of group II are selected from the group consisting of: IGLL1 and HLA-G.

Thus, in order to diagnose JMML in a subject, it shall be necessary to determine at least one biomarker from the high-risk JMML biomarkers (group I) referred to before and at least one biomarker from the low-risk JMML biomarkers (group II) referred to before. More preferably, all of the aforementioned biomarkers of any one of the aforementioned group I and group II may be determined. Thereby, the diagnosis with respect to JMML including all kind of disease stages from low-risk to high-risk can be improved.

The term “CD52” as used herein, refers to the cluster of differentiation 52 glycoprotein encoded in humans by the CD52 gene and is also known as CAMPATH- 1 antigen. CD52 is typically localized on the surface of mature lymphocytes, monocytes, and dendritic cells. It is involved in positive regulation of cytosolic calcium ion concentration. CD52 is a peptide consisting of 61 amino acids, anchored to glycosylphosphatidylinositol (GPI). It is assumed that it functions as an anti-adhesive protein that allows cells to freely move around since it is highly negatively charged and present on sperm cells and lymphocytes. Several orthologues of CD52 have been reported in various animal species.

The CD52 protein referred to in accordance with the present invention is, preferably, human CD52 having an amino acid sequence as deposited under UniProt accession number P31358. It will be understood that the term “CD52” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD52 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the human CD52 protein, preferably over the entire length of the said CD52 proteins, respectively.

The term “RAMP1” as used herein, refers to the receptor activity modifying protein 1 encoded in humans by the RAMP1 gene. It belongs to the RAMP family of single-transmembrane- domain proteins called receptor (calcitonin) activity modifying proteins comprising the three members RAMP1, RAMP2, and RAMP3. RAMPs are considered type I transmembrane proteins with an extracellular N terminus and a cytoplasmic C terminus. One important function of RAMP1 is to control the glycosylation of calcitonin receptors (CRL) and thus, its transportation to the cell membrane. RAMP1 is widely expressed in the brain, spinal cord, gastrointestinal tract, adrenal gland, perivascular nerve, and smooth muscles of the arteries. Several orthologues of RAMP 1 have been reported in various animal species.

The RAMP1 protein referred to in accordance with the present invention is, preferably, human RAMP1 having an amino acid sequence as deposited under UniProt accession number 060894. It will be understood that the term “RAMP1” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned RAMP1 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the human RAMP1 protein, preferably over the entire length of the said RAMP1 proteins, respectively.

The term “LTB” (LTP) as used herein, refers to the lymphotoxin-beta protein, also known as tumor necrosis factor C (TNF-C), encoded in humans by the LTB gene. It is a type II membrane protein of the TNF superfamily and is the primary ligand for the lymphotoxin-beta receptor. LTB interacts with two ligands: membrane heterotrimeric lymphotoxin alpha (LTa) and homotrimeric LIGHT. Typically, it is expressed by epithelial cells, stromal cells, dendritic cells (DCs), and macrophages, but is absent on lymphocytes. LTB is known as key regulator of lymphoid organogenesis and inflammation. Moreover, it has been established that LTB has pro- tumorigenic function. For instance, mice with overexpression of LTa or LTP showed increased tumor growth and metastasis in several models of cancer. For LTB, there are two isoforms known in humans (UniProt accession numbers Q06643-1 and Q06643-2). Several orthologues of RAMP 1 have been reported in various animal species.

The LTB protein referred to in accordance with the present invention is, preferably, human LTB having an amino acid sequence as deposited under UniProt accession number Q06643. It will be understood that the term “LTB” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned LTB protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the human LTB protein, preferably over the entire length of the said LTB proteins, respectively.

The term “LST1” as used herein, refers to the leukocyte-specific transcript 1 protein encoded in humans by the LST1 gene. It is a membrane protein that has a possible role in modulating immune responses. So far, 13 isoforms have been described, that are produced by alternative splicing. For example, isoform 1 and isoform 2 have an inhibitory effect on lymphocyte proliferation. Several orthologues of LST1 have been reported in various animal species.

The LST1 protein referred to in accordance with the present invention is, preferably, human LST1 having an amino acid sequence as deposited under UniProt accession number 000453. It will be understood that the term “LST1” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned LST1 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the human LST1 protein, preferably over the entire length of the said LST1 proteins, respectively.

The term “JAML” as used herein, refers to the junction adhesion molecule like protein or AMICA1 encoded in humans by the JAML gene. JAML is a transmembrane protein of the plasma membrane of leukocytes that control their migration and activation through interaction with CXADR, a plasma membrane receptor found on adjacent epithelial and endothelial cells. The interaction between both receptors mediates the activation of gamma-delta T-cells, a subpopulation of T-cells residing in epithelia and involved in tissue homeostasis and repair. Upon epithelial CXADR-binding, JAML induces downstream cell signalling events in gammadelta T-cells through PI3-kinase and MAP kinases. It results in proliferation and production of cytokines and growth factors by T-cells that in turn stimulate epithelial tissues repair. It also controls the transmigration of leukocytes within epithelial and endothelial tissues through adhesive interactions with epithelial and endothelial CXADR. Typically, JAML is located in bicellular tight junctions, nucleoplasm, and plasma membrane. Four isoforms of JAML are described and several orthologues of JAML have been reported in various animal species.

The JAML protein referred to in accordance with the present invention is, preferably, human JAML having an amino acid sequence as deposited under UniProt accession number Q86YT9. It will be understood that the term “JAML” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned JAML protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the human JAML protein, preferably over the entire length of the said JAML proteins, respectively.

The term “IFITM3” as used herein, refers to the interferon induced transmembrane protein 3 encoded in humans by the IFITM3 gene. IFITM3 proteins are a family of interferon induced antiviral proteins. The family contains five members, including IFITM1, IFITM2 and IFITM3 and belong to the CD225 superfamily. The protein restricts cellular entry by diverse viral pathogens, such as influenza A virus, Ebola virus and Sars-CoV-2. Several orthologues of IFTIM3 have been reported in various animal species.

The IFITM3 protein referred to in accordance with the present invention is, preferably, human IFITM3 having an amino acid sequence as deposited under UniProt accession number Q01628. It will be understood that the term “IFITM3” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned IFITM3 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the human IFITM3 protein, preferably over the entire length of the said IFITM3 proteins, respectively.

The term “CD7” as used herein, refers to the cluster of differentiation 7 protein encoded in humans by the CD7 gene. CD7 is a transmembrane protein found on thymocytes and mature T cells. It belongs to the immunoglobulin superfamily and plays an essential role in T-cell interactions and T-cell/B-cell interaction during early lymphoid development. There are 5 potential isoforms known so far and several orthologues of CD7 have been reported in various animal species.

The CD7 protein referred to in accordance with the present invention is, preferably, human CD7 having an amino acid sequence as deposited under UniProt accession number P09564. It will be understood that the term “CD7” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD7 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the CD7 protein, preferably over the entire length of the said CD7 proteins, respectively.

The term “CD69” as used herein, refers to cluster of differentiation 69 protein encoded in humans by the CD69 gene. It is a disulphide-linked homodimer protein with two different subunits. Each subunit consists of an extracellular C-type lectin domain (CTLD) connected with a single- spanning transmembrane region followed by a short cytoplasmic tail. It is an early activation marker that is expressed in hematopoietic stem cells, T cells, and many other cell types in the immune system. The activation of T lymphocytes and natural killer (NK) cells, both in vivo and in vitro, induces expression of CD69. It is involved in lymphocyte proliferation and functions as a signal-transmitting receptor in lymphocytes. It is also implicated in T cell differentiation as well as lymphocyte retention in lymphoid organs. One potential isoform is known so far and several orthologues of CD69 have been reported in various animal species.

The CD69 protein referred to in accordance with the present invention is, preferably, human CD69 having an amino acid sequence as deposited under UniProt accession number Q07108. It will be understood that the term “CD69” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD69 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the CD69 protein, preferably over the entire length of the said CD69 proteins, respectively.

The term “CD 164” as used herein, refers to the cluster of differentiation 164 protein or sialomucin core protein 24 (also known as endolyn) encoded in humans by the CD164 gene. 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 the C-X-C chemokine receptor type 4 (CXCR4) and may regulate muscle development. Elevated expression of this gene has been observed in human patients with Sezary syndrome, a type of blood cancer, and a mutation in this gene may be associated with impaired hearing. Five isoforms are known so far and several orthologues of CD 164 have been reported in various animal species.

The CD 164 protein referred to in accordance with the present invention is, preferably, human CD 164 having an amino acid sequence as deposited under UniProt accession number Q04900. It will be understood that the term “CD 164” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD 164 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the CD 164 protein, preferably over the entire length of the said CD 164 proteins, respectively.

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 antigens-associated invariant chain) encoded in humans by the CD74 gene. The protein encoded by this gene associates with class II major histocompatibility complex (MHC) and is an important chaperone that regulates antigen presentation for immune response. It also serves as cell surface receptor for the cytokine macrophage migration inhibitory factor (MIF) which, when bound to the encoded protein, initiates survival pathways and cell proliferation. Multiple alternatively spliced transcript variants encoding five different isoforms have been identified and several orthologues of CD74 have been reported in various animal species.

The CD74 protein referred to in accordance with the present invention is, preferably, human CD74 having an amino acid sequence as deposited under UniProt accession number P04233. It will be understood that the term “CD74” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD74 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the CD74 protein, preferably over the entire length of the said CD74 proteins, respectively.

The term “TNF as used herein, refers to the tumor necrosis factor protein encoded in humans by the TNF gene. TNF belongs to the TNF superfamily, which consists of various transmembrane proteins with a homologous TNF domain. It is an adipokine and a cytokine, and is produced by a broad variety of cell types including lymphoid cells, mast cells, endothelial cells, cardiac myocytes, 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 cell signalling in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. This cytokine has been implicated in a variety of diseases, including autoimmune diseases, insulin resistance, psoriasis, rheumatoid arthritis ankylosing spondylitis, tuberculosis, autosomal dominant polycystic kidney disease, and cancer. Mutations in this gene affect susceptibility to cerebral malaria, septic shock, and Alzheimer disease. Knockout studies in mice also suggested the neuroprotective function of this cytokine.

The TNF protein referred to in accordance with the present invention is, preferably, human TNF having an amino acid sequence as deposited under UniProt accession number P01375. It will be understood that the term “TNF” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned TNF protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the TNF protein, preferably over the entire length of the said TNF proteins, respectively. Several orthologues of TNF have been reported in various animal species.

The term “TFPI” as used herein, refers to the tissue factor pathway inhibitor protein encoded in humans by the TFPI gene. This gene encodes a Kunitz-type serine protease inhibitor that regulates the tissue factor (TF)-dependent pathway of blood coagulation. Specifically, TFPI is a single-chain polypeptide, which can reversibly inhibit Factor Xa of the coagulation cascade. Two different isoforms have been identified and several orthologues of TFPI have been reported in various animal species. The TFPI protein referred to in accordance with the present invention is, preferably, human TFPI having an amino acid sequence as deposited under UniProt accession number Pl 0646. It will be understood that the term “TFPI” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned TFPI protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the TFPI protein, preferably over the entire length of the said TFPI proteins, respectively.

The term “DLK1” as used herein, refers to the Protein delta homolog 1 encoded in humans by the DLK1 gene. This gene encodes a transmembrane protein that contains multiple epidermal growth factor repeats and functions as a regulator of cell growth. A soluble form of DLK1 cleaved off by ADAMI 7 is involved in inhibiting adipogenesis, the differentiation preadipocytes into adipocytes. DKL1 is a member of the EGF-like family of homeotic proteins. Two different isoforms have been identified and several orthologues of DKL1 have been reported in various animal species.

The DLK1 protein referred to in accordance with the present invention is, preferably, human DLK1 having an amino acid sequence as deposited under UniProt accession number P80370. It will be understood that the term “DLK1” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned DLK1 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the DLK1 protein, preferably over the entire length of the said DLK1 proteins, respectively.

The term “CD82” as used herein, refers to the cluster of differentiation 82 protein encoded in humans by the CD82 gene. CD82 belongs to the tetraspanin/transmembrane 4 superfamily. The protein acts as metastasis suppressor. The expression of this gene has been shown to be downregulated in tumor progression of human cancers and can be activated by p53 through a consensus binding sequence in the promoter. Its expression and that of p53 are strongly correlated, and the loss of expression of these two proteins is associated with poor survival for prostate cancer patients. Two alternatively spliced transcript variants encoding distinct isoforms have been identified and several orthologues of CD82 have been reported in various animal species.

The CD82 protein referred to in accordance with the present invention is, preferably, human CD82 having an amino acid sequence as deposited under UniProt accession number P22701. It will be understood that the term “CD82” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD82 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the CD82 protein, preferably over the entire length of the said CD82 proteins, respectively.

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

The IGHM protein referred to in accordance with the present invention is, preferably, human IGHM having an amino acid sequence as deposited under UniProt accession number P01871. It will be understood that the term “IGHM” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned IGHM protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the IGHM protein, preferably over the entire length of the said IGHM proteins, respectively.

The term “CALCRL” as used herein, refers to the calcitonin gene-related peptide type 1 receptor protein encoded in humans by the CALCRL gene. It is a G protein-coupled receptor related to the calcitonin receptor. Typically, CALCRL is located in the endoplasmic reticulum, endosome and lysosome and presumably active in the plasma membrane. It is suggested that the protein may modulate a variety of physical functions in all major systems (e.g. respiratory, endocrine, gastrointestinal, immune, and cardiovascular). More particularly, it is assumed to be involved in several processes including G protein-coupled receptor signalling pathway, cellular response to sucrose stimulus, and receptor internalization. Several orthologues of CALCRL have been reported in various animal species.

The CALCRL protein referred to in accordance with the present invention is, preferably, human CALCRL having an amino acid sequence as deposited under UniProt accession number QI 6602. It will be understood that the term “CALCRL” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CALCRL protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the CALCRL protein, preferably over the entire length of the said CALCRL proteins, respectively.

The term “RALA” as used herein, refers to the Ras-related protein Ral-A encoded in humans by the RALA gene on chromosome 7. RALA is one of two paralogs of the Ral protein, the other being RalB. The product of this gene belongs to the small GTPase superfamily, Ras family of proteins. 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 RalGEFs and, in turn, activate 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 orthologues of RALA have been reported in various animal species.

The RALA protein referred to in accordance with the present invention is, preferably, human RALA having an amino acid sequence as deposited under UniProt accession number Pl 1233. It will be understood that the term “RALA” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned RALA protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the RALA protein, preferably over the entire length of the said RALA proteins, respectively.

The term “SLC2A5” (also known as GLUT5) as used herein, refers to the solute carrier family 2, facilitated glucose transporter member 5 protein encoded in humans by the SLC2A5 gene. Typically, SCL2A5 is expressed on the apical border of enterocytes in the small intestine, in skeletal muscle, testis, kidney, fat tissue, and brain. The protein encoded by this gene is a fructose transporter responsible for fructose uptake by the small intestine. SLC2A5 is also necessary for the increase in blood pressure due to high dietary fructose consumption. Two alternatively spliced transcript variants encoding two distinct isoforms have been identified and several orthologues of SLC2A5 have been reported in various animal species.

The SLC2A5 protein referred to in accordance with the present invention is, preferably, human SLC2A5 having an amino acid sequence as deposited under UniProt accession number P22732. It will be understood that the term “SLC2A5” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned SLC2A5 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the SLC2A5 protein, preferably over the entire length of the said SLC2A5 proteins, respectively.

The term “HSPA5” as used herein, refers to the heat shock 70 kDa protein 5 encoded in humans by the HSPA5 gene. It is also known as binding immunoglobulin protein (BiP) or 78 kDa glucose-regulated protein (GRP-78). Typically, it is located in the lumen of the endoplasmic reticulum (ER) where it operates as a HSP70 chaperone involved in the folding and assembly of proteins and is a master regulator of ER homeostasis. Elevated expression and atypical translocation of this protein to the cell surface has been reported in viral infections and some types of cancer cells. Several orthologues of HSPA5 have been reported in various animal species.

The HSPA5 protein referred to in accordance with the present invention is, preferably, human HSPA5 having an amino acid sequence as deposited under UniProt accession number Pl 1021. It will be understood that the term “HSPA5” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned HSPA5 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the HSPA5 protein, preferably over the entire length of the said HSPA5 proteins, respectively.

The term “HLA-DRA” as used herein, refers to the HLA class II histocompatibility antigen, DR alpha chain protein encoded in humans by the HLA-DRA gene. This protein is a heterodimer consisting of an alpha and a beta chain, both anchored in 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 derived from extracellular proteins, in particular, pathogen-derived peptides to T cells. Several orthologues of HLA-DRA have been reported in various animal species.

The HLA-DRA protein referred to in accordance with the present invention is, preferably, human HLA-DRA having an amino acid sequence as deposited under UniProt accession number P01903. It will be understood that the term “HLA-DRA” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned HLA-DRA protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the HLA-DRA protein, preferably over the entire length of the said HLA-DRA proteins, respectively. The term “RAB11A” as used herein, refers to the Ras-related protein Rab-l lA encoded in humans by the RAB11A gene. The protein encoded by this gene belongs to the Rab family of the small GTPase superfamily. It is associated with both constitutive and regulated secretory pathways, and may be involved in protein transport. Rab-l la controls intracellular trafficking of the innate immune receptor TLR4, and thereby also receptor signalling. Two isoforms are known and several orthologues of RABI 1 A have been reported in various animal species.

The RABI 1 A protein referred to in accordance with the present invention is, preferably, human RABI 1 A having an amino acid sequence as deposited under UniProt accession number P62491. It will be understood that the term “RABI 1A” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned RABI 1 A protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the RABI 1 A protein, preferably over the entire length of the said RABI 1 A proteins, respectively.

The term “SELL” as used herein, refers to the L-selectin protein, also known as CD62L, encoded in humans by the SELL gene. This gene encodes a cell surface adhesion molecule that belongs to a family of adhesion/homing receptors. 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 binding and subsequent rolling of leucocytes on endothelial cells, facilitating their migration into secondary lymphoid organs and inflammation sites. Single-nucleotide polymorphisms in this gene have been associated with various diseases including immunoglobulin A nephropathy. Two isoforms are known and several orthologues of SELL have been reported in various animal species.

The SELL protein referred to in accordance with the present invention is, preferably, human SELL having an amino acid sequence as deposited under UniProt accession number P14151. It will be understood that the term “SELL” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned SELL protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the SELL protein, preferably over the entire length of the said SELL proteins, respectively.

The term “VAMP5” as used herein, refers to the vesicle-associated membrane protein 5 encoded in humans by the VAMP5 gene. Synaptobrevins/VAMPs, syntaxins, and the 25-kD synaptosomal-associated protein are the main components of a protein complex involved in the docking and/or fusion of vesicles and cell membranes. The VAMP5 gene is a member of the vesicle-associated membrane protein (VAMP)/synaptobrevin family and the SNARE superfamily. This VAMP family member may participate in vesicle trafficking events that are associated with myogenesis. Several orthologues of VAMP5 have been reported in various animal species.

The VAMP5 protein referred to in accordance with the present invention is, preferably, human VAMP5 having an amino acid sequence as deposited under UniProt accession number 095183. It will be understood that the term “VAMP5” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned VAMP5 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the VAMP5 protein, preferably over the entire length of the said VAMP5 proteins, respectively.

The term “FCMR” as used herein, refers to the FC fragment of IgM receptor protein encoded in humans by the FCMR gene. This protein may play a role in the immune system processes. It protects from FAS-, TNF alpha- and FADD-induced apoptosis without increasing expression of the inhibitors of apoptosis BCL2 and BCLXL and seems to activate an inhibitory pathway that prevents CASP8 activation following FAS stimulation, rather than blocking apoptotic signals downstream. FCMR is also implicated in inhibiting FAS-induced apoptosis by preventing CASP8 processing through CFLAR up-regulation. Three isoforms are known and several orthologues of FCMR have been reported in various animal species. The FCMR protein referred to in accordance with the present invention is, preferably, human FCMR having an amino acid sequence as deposited under UniProt accession number 060667. It will be understood that the term “FCMR” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned FCMR protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the FCMR protein, preferably over the entire length of the said FCMR proteins, respectively.

The term “CLEC7A” as used herein, refers to the C-type lectin domain family 7 member A protein encoded in humans by the CLEC7A gene. 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 functions as a pattern-recognition receptor that recognizes a variety of beta- 1,3 -linked and beta-l,6-linked glucans from fungi and plants, and in this way plays a role in innate immune response. Ten isoforms are known so far and several orthologues of CLEC7A have been reported in various animal species.

The CLEC7A protein referred to in accordance with the present invention is, preferably, human CLEC7A having an amino acid sequence as deposited under UniProt accession number Q9BXN2. It will be understood that the term “CLEC7A” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CLEC7A protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the CLEC7A protein, preferably over the entire length of the said CLEC7A proteins, respectively.

The term “NDFIP1” as used herein, refers to the Nedd4 family interacting protein 1 encoded in humans by the NDFIP1 gene. 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 family of proteins. NDFIP1 is suggested to be part of a family of integral Golgi membrane proteins. Eight isoforms are known so far and several orthologues of NDFIP1 have been reported in various animal species.

The NDFIP1 protein referred to in accordance with the present invention is, preferably, human NDFIP1 having an amino acid sequence as deposited under UniProt accession number Q96PU5. It will be understood that the term “NDFIP1” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned NDFIP1 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the NDFIP1 protein, preferably over the entire length of the said NDFIP1 proteins, respectively.

The term “CLEC9A” as used herein, refers to the C-type lectin domain family 9 member A protein encoded in humans by the CLEC9A gene. Typically, the protein is expressed by myeloid lineage cells. CLEC9A is a group V C-type lectin-like receptor (CTLR) that functions as an endocytic receptor on a small subset of myeloid cells specialized for the uptake and processing of material from dead cells. It recognizes filamentous form of actin in association with particular actin-binding domains of cytoskeletal proteins, including spectrin, exposed when cell membranes are damaged, and mediate the cross-presentation of dead-cell associated antigens in a Syk-dependent manner.

The CLEC9A protein referred to in accordance with the present invention is, preferably, human CLEC9A having an amino acid sequence as deposited under UniProt accession number Q6UXN8. It will be understood that the term “CLEC9A” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CLEC9A protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the CLEC9A protein, preferably over the entire length of the said CLEC9A proteins, respectively. Several orthologues of CLEC9A have been reported in various animal species.

The term “HCST” as used herein, refers to the hematopoietic cell signal transducer protein encoded in humans by the HCST gene. This gene encodes a transmembrane signaling adaptor that contains a YxxM motif in its cytoplasmic domain. The encoded protein may form part of the immune recognition receptor complex with the C-type lectin-like receptor NKG2D. As part of this receptor complex, this protein may activate phosphatidylinositol 3-kinase dependent signaling pathways through its intracytoplasmic YxxM motif. This receptor complex may have a role in cell survival and proliferation by activation of NK and T cell responses. Two isoforms are known and several orthologues of HCST have been reported in various animal species.

The HCST protein referred to in accordance with the present invention is, preferably, human HCST having an amino acid sequence as deposited under UniProt accession number Q9UBK5. It will be understood that the term “HCST” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned HCST protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the HCST protein, preferably over the entire length of the said HCST proteins, respectively.

The term “LPAR6” as used herein, refers to the lysophosphatidic acid receptor 6 encoded in humans by the LPAR6 gene. LPAR6 is also known as LPAe, P2RY5, and GPR87. The protein encoded by this gene belongs to the family of G-protein coupled receptors that are preferentially activated by adenosine and uridine nucleotides. Mutations in this gene are implicated to cause a rare, inherited form of hair loss (hypotrichosis simplex). Several orthologues of LPAR6 have been reported in various animal species.

The LPAR6 protein referred to in accordance with the present invention is, preferably, human LPAR6 having an amino acid sequence as deposited under UniProt accession number P43657. It will be understood that the term “LPAR6” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned LPAR6 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the LPAR6 protein, preferably over the entire length of the said LPAR6 proteins, respectively.

The term “HLA-DQA1” as used herein, refers to the major histocompatibility complex, class II, DQ alpha 1 protein encoded in humans by the HLA-DQA1 gene on chromosome 6. HLA- DQA1 is a heterodimer consisting of an alpha (DQA) and a beta chain (DQB), both anchored in 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 derived from extracellular proteins. Several orthologues of HLA-DQA1 have been reported in various animal species.

The HLA-DQA1 protein referred to in accordance with the present invention is, preferably, human HLA-DQA1 having an amino acid sequence as deposited under UniProt accession number P01909. It will be understood that the term “HLA-DQA1” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned HLA-DQA1 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the HLA-DQA1 protein, preferably over the entire length of the said HLA-DQA1 proteins, respectively.

The term “HLA-DRB5” as used herein, refers to the HLA class II histocompatibility antigen, DRB5 beta chain protein encoded in humans by the HLA-DRB5 gene. This class II molecule is a heterodimer consisting of an alpha (DRA) and a beta (DRB) chain, both anchored in the membrane. It plays a central role in the immune system by presenting peptides derived from extracellular proteins. Diseases associated with HLA-DRB5 include Pityriasis Rosea and Multiple Epiphyseal Dysplasia Due To Collagen 9 Anomaly. Several orthologues of HLA- DRB5 have been reported in various animal species.

The HLA-DRB5 protein referred to in accordance with the present invention is, preferably, human HLA-DRB5 an amino acid sequence as deposited under UniProt accession number Q30154. It will be understood that the term “HLA-DRB5” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned HLA-DRB5 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the HLA-DRB5 protein, preferably over the entire length of the said HLA-DRB5 proteins, respectively.

The term “CD34” as used herein, refers to the cluster of differentiation 34 protein encoded in humans by the CD34 gene. This transmembrane phosphoglycoprotein may play a role as adhesion molecule in early haematopoiesis by mediating the attachment of stem cells to the bone marrow extracellular matrix or directly to stromal cells. Alternatively spliced transcript variants encoding two different isoforms have been identified and several orthologues of CD34 have been reported in various animal species.

The CD34 protein referred to in accordance with the present invention is, preferably, human CD34 having an amino acid sequence as deposited under UniProt accession number P28906. It will be understood that the term “CD34” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD34 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the CD34 protein, preferably over the entire length of the said CD34 proteins, respectively.

The term “IGLL1” as used herein, refers to the immunoglobulin lambda-like polypeptide 1 protein encoded in humans by the IGLL1 gene. This protein is involved in transduction of signals for cellular proliferation, differentiation from the proB cell to the preB cell stage, allelic exclusion at the Ig heavy chain gene locus and promotion of Ig light chain gene rearrangements. Mutations in this gene can result in B cell deficiency and agammaglobulinemia, an autosomal recessive disease in which few or no gamma globulins or antibodies are made. Two different isoforms have been identified and several orthologues of IGLL1 have been reported in various animal species.

The IGLL1 protein referred to in accordance with the present invention is, preferably, human IGLL1 having an amino acid sequence as deposited under UniProt accession number P15814. It will be understood that the term “IGLL1” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned IGLL1 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the IGLL1 protein, preferably over the entire length of the said IGLL1 proteins, respectively.

The term “BEST1” as used herein, refers to the bestrophin 1 protein encoded in humans by the BEST1 gene. It belongs to the bestrophin family, which comprises four related genes that code for integral membrane proteins. BEST1 is characterized by a highly conserved N-terminus with four to six transmembrane domains. Bestrophins may form chloride ion channels or may regulate voltage-gated L-type calcium-ion channels. Bestrophins are generally believed to form calcium-activated chloride-ion channels in epithelial cells but they have also been shown to be highly permeable to bicarbonate ion transport in retinal tissue. Mutations in this gene are responsible for juvenile-onset vitelliform macular dystrophy (VMD2), also known as Best macular dystrophy, in addition to adult-onset vitelliform macular dystrophy (AVMD) and other retinopathies. Mutations in the BEST1 gene have also been identified as the primary cause for at least five different degenerative retinal diseases. Two different isoforms have been identified and several orthologues of BEST 1 have been reported in various animal species.

The BEST1 protein referred to in accordance with the present invention is, preferably, human BEST1 having an amino acid sequence as deposited under UniProt accession number 076090. It will be understood that the term “BEST1” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned BEST1 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the BEST1 protein, preferably over the entire length of the said BEST1 proteins, respectively.

The term “EREG” as used herein, refers to the epiregulin protein encoded in humans by the EREG gene. This gene encodes a secreted peptide hormone and member of the epidermal growth factor (EGF) family of proteins. The encoded protein is a ligand of the epidermal growth factor receptor (EGFR) and the structurally related erb-b2 receptor tyrosine kinase 4 (ERBB4). The encoded protein may be involved in a wide range of biological processes including inflammation, wound healing, oocyte maturation, and cell proliferation. Additionally, the encoded protein may promote the progression of cancers of various human tissues. Several orthologues of EREG have been reported in various animal species.

The EREG protein referred to in accordance with the present invention is, preferably, human EREG having an amino acid sequence as deposited under UniProt accession number 014944. It will be understood that the term “EREG” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned EREG protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the EREG protein, preferably over the entire length of the said EREG proteins, respectively.

The term “SLC5A3” as used herein, refers to the sodium/myo-inositol cotransporter protein encoded in humans by the SLC5A3 gene. Typically, it is located in the plasma membrane. SLC5A3 is a sodium/myo-inositol co-transporter It is also said to act upstream of or within several processes, including peripheral nervous system development, positive regulation of reactive oxygen species biosynthetic process, and regulation of respiratory gaseous exchange. Several orthologues of SLC5A3 have been reported in various animal species.

The SLC5A3 protein referred to in accordance with the present invention is, preferably, human SLC5A3 having an amino acid sequence as deposited under UniProt accession number P53794. It will be understood that the term “SLC5A3” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned SLC5A3 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the SLC5A3 protein, preferably over the entire length of the said SLC5A3 proteins, respectively.

The term “SELK” as used herein, refers to the selenoprotein K encoded in humans by the SELK gene. It is a transmembrane protein that is localized in the endoplasmic reticulum (ER), and is involved in ER-associated degradation (ERAD) of misfolded, glycosylated proteins. It also has a role in the protection of cells from ER stress-induced apoptosis. Knockout studies in mice show the importance of this gene in promoting Ca ²⁺ flux in immune cells and mounting effective immune response. SELK also plays a role in T-cell proliferation and in T-cell and neutrophil migration. Several orthologues of SELK have been reported in various animal species.

The SELK protein referred to in accordance with the present invention is, preferably, human SELK having an amino acid sequence as deposited under UniProt accession number Q9Y6D0. It will be understood that the term “SELK” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned SELK protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the SELK protein, preferably over the entire length of the said SELK proteins, respectively.

The term “PRRG3” as used herein, refers to the transmembrane gamma-carboxyglutamic acid protein 3 encoded in humans by the PRRG3 gene. This gene encodes a protein which contains a vitamin K-dependent carboxylation/gamma-carboxyglutamic domain. The encoded protein is a member of a family of vitamin K-dependent transmembrane proteins which contain a glutamate-rich extracellular domain. Diseases associated with PRRG3 include Hereditary Combined Deficiency of Vitamin K-Dependent Clotting Factors and Vitamin K Deficiency Bleeding. Several orthologues of PRRG3 have been reported in various animal species. The PRRG3 protein referred to in accordance with the present invention is, preferably, human PRRG3 having an amino acid sequence as deposited under UniProt accession number Q9BZD7. It will be understood that the term “PRRG3” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned PRRG3 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the PRRG3 protein, preferably over the entire length of the said PRRG3 proteins, respectively.

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

The NINJ1 protein referred to in accordance with the present invention is, preferably, human NINJ1 having an amino acid sequence as deposited under UniProt accession number Q92982. It will be understood that the term “NINJ1” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned NINJ1 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino sequence of the NINJ1 protein, preferably over the entire length of the said NINJ1 proteins, respectively.

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

The MGST1 protein referred to in accordance with the present invention is, preferably, human MGST1 having an amino acid sequence as deposited under UniProt accession number Pl 0620. It will be understood that the term “MGST1” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned MGST1 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the MGST1 protein, preferably over the entire length of the said MGST1 proteins, respectively.

The term “HLA-G” as used herein, refers to the HLA-G histocompatibility antigen, class I, G, also known as human leukocyte antigen G, a protein encoded in humans by the HLA-G gene. HLA-G belongs to the HLA class I heavy chain paralogues. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). The heavy chain is anchored in the membrane. HLA-G is expressed on fetal derived placental cells. It functions as a major immune checkpoint, e.g. it downregulates the immune system’s response. HLA-G also plays a role in immune tolerance in pregnancy, modulation of the immune response to parasitic diseases and has been shown to be associated with tumor escape in cancers as well as allergenic responses. Seven isoforms are known and several orthologues of HLA-G have been reported in various animal species.

The HLA-G protein referred to in accordance with the present invention is, preferably, human HLA-G having an amino acid sequence as deposited under UniProt accession number Pl 7693. It will be understood that the term “HLA-G” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned HLA-G protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs 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 with the specific amino acid sequence of the HLA-G protein, preferably over the entire length of the said HLA-G proteins, respectively.

The degree of identity between two amino acid sequences in accordance with the present invention can be determined by algorithms well known in the art. Preferably, the degree of identity is to be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm disclosed by Smith, by the homology alignment algorithm of Needleman, by the search for similarity method of Pearson, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI) or by visual inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment and, thus, the degree of identity. Preferably, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. Variants referred to above may be allelic variants or any other species-specific homologs, paralogs, or orthologues. Variants referred to above may be allelic variants or any other species-specific homologs, paralogs, or orthologues.

The term “determining the amount of at least one biomarker” as used in accordance with the present invention refers to qualitative and quantitative determination of biomarkers, i.e. the term encompasses the determination of the presence or absence or the determination of the absolute or relative amount of said biomarkers. The term further encompasses measuring the amount or concentration, preferably, semi-quantitatively or quantitatively.

The term “amount” as used herein refers to the absolute amount of the biomarker, the relative amount or concentration of the biomarker as well as any value or parameter, which correlates thereto or can be derived therefrom. Such values or parameters comprise intensity signal values from all specific physical or chemical properties obtained from the said biomarker or a detection molecule and/or detectable label. The values or parameters can be obtained by direct or indirect measurement. Direct measuring relates to measuring the amount or concentration of the biomarker based on a signal which is obtained from the biomarker molecule itself and the intensity of which directly correlates with the number of molecules of the biomarker present in the sample. Such a signal - sometimes referred to herein as intensity signal - may be obtained, e.g., by measuring an intensity value of a specific physical or chemical property of the biomarker molecule. Indirect measuring includes measuring of a signal obtained from a secondary component, i.e. a component not being the biomarker molecule itself. It is to be understood that values correlating to the aforementioned amounts or parameters can also be obtained and/or modified by all standard mathematical operations.

Determining the amount in the method of the present invention may be carried out by any technique, which allows for detecting the presence or absence or the amount of said biomarker. Suitable techniques depend on the molecular nature and the properties of the biomarkers. For example, a protein biomarker may be determined by measuring properties other than in the case of a transcribed nucleic acid molecule biomarker. The skilled artisan is well aware of those differences in the measurable properties. Moreover, it will be understood that a protein biomarker may be detected by using detection agents and/or techniques, which differ from those used for transcribed nucleic acid molecule biomarkers. The skilled artisan is, however, also well aware of said different detection agents and/or techniques.

In accordance with the present invention, determining the amount of a biomarker can be achieved by all known means for determining such amounts in a sample. Said means comprise immunoassay devices and methods, which may utilize labelled molecules in various sandwich, competition, or other assay formats. Said assays will develop a signal, which is indicative for the presence or absence of the protein. Moreover, the signal strength can, preferably, be correlated directly or indirectly (e.g. reverse- proportional) to the amount of the biomarker present in a sample. Further suitable methods comprise measuring a physical or chemical property specific for the biomarkers. Said methods comprise, preferably, biosensors, optical devices coupled to immunoassays, biochips, or other analytical devices such as chromatography devices or single cell analysing devices such as FACS analysers or devices for PCR analysis, such as devices for single cell PCR, qPCR or bulk PCR, or sequencing devices.

In a preferred embodiment of the method of the present invention, the amount of a 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 within a cell population by counting positive versus negative sorting events. Thus, biomarker data for a clinical sample is not a single value representing an overall staining intensity, but instead a value reflecting the proportion in which individual cells surpass an intensity threshold for the particular biomarker. The gating criterion for a positive sorting event can be set as a combination of desired signal intensities for the protein biomarkers being used. Preferably, the biomarker(s) to be determined in accordance with the present invention may be determined as protein. To this end, typically a binding molecule is applied that specifically binds to the said biomarker protein and that can be detected either by a detectable label present in the binding molecule or by a secondary binding molecule that specifically binds to the first binding molecule and comprises a detectable label. The binding molecule can be exposed to the biomarker in solution or while the binding molecule is immobilized on a solid support.

A binding molecule refers in this context to any molecule that is capable of specifically binding to the biomarker to be detected. The binding molecule is selected based on the type of analysis to be conducted. 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 peptidomimetics. Preferably, such a binding molecule may be an antibody or an antigen-binding fragment thereof.

An “antibody” in accordance with the present invention may encompass all types of antibodies, which specifically bind to the biomarker protein. Preferably, the antibody of the present invention is a monoclonal antibody, a polyclonal antibody, a single chain antibody, a chimeric antibody or any fragment or derivative of such antibodies being still capable of binding to the biomarker protein specifically.

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 within the term “antigen binding fragment” include a fragment antigen binding (Fab) fragment, a Fab’ fragment, a F(ab’)2 fragment, a heavy chain antibody, a single-domain antibody (sdAb), a single-chain fragment variable (scFv), a fragment variable (Fv), a VH domain, a VL domain, a single domain antibody, a nanobody, an IgNAR (immunoglobulin new antigen receptor), a di-scFv, a bispecific T-cell engager (BITEs), a dual affinity re-targeting (DART) molecule, a triple body, a diabody, a single-chain diabody, an alternative scaffold protein, and a fusion protein thereof.

Specific binding as used in the context of the antibody of the present invention means that the antibody does not cross react with other molecules present in the sample to be investigated. Specific binding can be tested by various well-known techniques. Antibodies or fragments thereof, in general, can be obtained by using methods, which are described in standard text books, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies can be prepared by the techniques, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals and, preferably, immunized mice. Preferably, an immunogenic peptide is applied to a mammal. The said 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 increase the immunological response. Such adjuvants encompass, preferably, 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. Monoclonal antibodies, which specifically bind to an analyte can be subsequently prepared using the well-known hybridoma technique, the human B cell hybridoma technique, and the EBV hybridoma technique. Detection systems using antibodies are based on the highly specific binding affinity of antibodies for a specific antigen, i.e. the biomarker protein. Binding events result in a physicochemical change that can be detected as described elsewhere herein.

An “adnectin” as used herein, refers to a synthetic binding protein, also known as monobody, based on the 10 ^th fibronectin type III ( ¹⁰ Fn3) domain. It is a member of the immunoglobulin superfamily and contains a “beta sandwich” protein fold that bears striking resemblance to an antibody domain. As such, adnectins represent a simple and robust alternative to antibodies for creating target-binding proteins. A major advantage of adnectins over conventional antibodies is that adnectins can readily be used as genetically encoded intracellular inhibitors, that is one can express an adnectin inhibitor in a cell of choice by simply transfecting the cell with an adnectin expression vector. Preferably, the adnectin as used herein, shall bind specifically to CD52, RAMP1, LTB, LST1, J AML, 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.

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

“Antibody mimetics” as used herein, refer to compounds, which can specifically bind antigens, similar to an antibody, but are not structurally related to antibodies. Usually, antibody mimetics are artificial peptides or proteins with a molar mass of about 3 to 20 kDa, which comprise one, two or more exposed domains specifically binding to an antigen. Examples include inter alia the LACI-Dl (lipoprotein-associated coagulation inhibitor); affilins, e.g. human-y B crystalline or human ubiquitin; cystatin; Sac7D from Sulfolobus acidocaldctriiis lipocalin and anticalins derived from lipocalins; DARPins (designed ankyrin repeat domains); SH3 domain of Fyn; Kunits domain of protease inhibitors; monobodies, e.g. the 10 ^th type III domain of fibronectin; adnectins: knottins (cysteine knot miniproteins); atrimers; evibodies, e.g. CTLA4-based binders, affibodies, e.g. three-helix bundle from Z-domain of protein A from Staphylococcus aureus,' Trans-bodies, e.g. human transferrin; tetranectins, e.g. monomeric or trimeric human C-type lectin domain; microbodies, e.g. trypsin-inhibitor-II; affilins; armadillo repeat proteins. Nucleic acids and small molecules are sometimes considered antibody mimetics as well (aptamers), but not artificial antibodies, antibody fragments and fusion proteins composed from these. Common advantages over antibodies are better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs. Preferably, the antibody mimetics as used herein, shall bind specifically to at least one biomarker described herein elsewhere.

A ’’scaffold protein” as used herein, refers to a specific protein whose main function is to mediate protein complexes. Scaffold proteins usually have multiple protein domains that mediate binding to other proteins. Examples of scaffold proteins include but are not limited to the protein inaD from rhabdomeres of Drosophila melanogaster or titin, a protein found in muscles.

The term “lectin” as used herein, refers to carbohydrate-binding proteins that are highly specific for sugar groups. They occur ubiquitously in nature and may bind to soluble carbohydrates or carbohydrate moieties that are part of a glycoprotein or glycolipid. Lectins typically agglutinate certain cells and/or precipitate glycoconjugates. As such, they find use in medicine, particularly for blood typing. Lectins are also used in neuroscience for anterograde labelling to trace the path of efferent axons. Preferably, the lectin as used herein, shall bind specifically to at least one biomarker as described herein elsewhere.

An “affibody” as used herein are small, highly robust proteins with high affinity to target proteins. In contrast to antibodies, affibodies are composed of alpha helices and lack disulphide bridges. In particular, they are based on a three-helix bundle domain with 58 amino acids and have a molar mass of about 6 kDa. They can be expressed in soluble and proteolytically stable forms in various host cells on its own or via fusion with other protein partners. Affibodies can be used for protein purification, enzyme inhibition, research reagents for protein capture and detection, diagnostic imaging, and targeted therapy. For example, the second generation affibody, ABY-025 binds selectively to HER2 receptors with picomolar affinity. Preferably, the affibodies as used herein, shall bind specifically to at least one biomarker described herein elsewhere.

The term “nanobody” as used herein, refers to tiny, recombinantly produced antigen binding fragments, typically consisting of a single monomeric variable antibody domain. Although nanobodies lack the light chains and heavy chain constant domain, the antigen-binding capacity remains similar to that of conventional antibodies. Typically, the complementarity-determining region 3 (CDR3) of nanobodies is similar or even longer than that of human variable domain of the heavy immunoglobulin chain (VH). They can form finger-like structures to recognize cavities or hidden epitopes that are not available to monoclonal antibodies, a feature that enhances the binding affinity and specificity of nanobodies. Preferably, the nanobodies as used herein, shall bind specifically to at least one biomarker described herein elsewhere.

An “avimer” (short of avidity multimer) as used herein, refers to artificial proteins with multiple binding sites for specific binding to certain antigens. They are not structurally related to antibodies and thus, are classified as antibody mimetics. Typically, they consist of two or more peptide sequences of 30 to 35 amino acids, connected by linker peptides. The individual sequences are derived from A domains of various membrane receptors and have a rigid structure, stabilised by disulfide bonds and calcium. Each A domain can bind to a certain epitope of the target protein. The combination of domains binding to different epitopes of the same protein increases affinity to this protein, an effect known as avidity. Avimers are widely used in early detection in tissue imaging, treatment, and study on carcinogenesis. Preferably, the avimers as used herein, shall bind specifically to at least one biomarker described herein elsewhere.

The term “peptidomimetic” as used herein, refers to compound that mimics one or more structural aspects or biological activities of a naturally-occurring polypeptide, but which comprises one or more non-peptide or non-naturally occurring chemical structures or bonds. Peptidomimetics are frequently used to mimic the biological action of a peptide, thus they may be small protein-like chain designed to mimic one or more peptides. Peptidomimetics are often synthesized based on existing peptides of interest with one or more modifications to alter the molecule's structure or properties. Modifications can change the peptide molecule's stability, half-life, biological activity, absorption, or side-effects (e.g., toxicity, solubility, hydrophobicity, side-chain charge, or flexibility) of a peptide. Peptidomimetics can be useful as medicaments or drug-like compounds developed rationally, or based on modification of an existing peptide with known or putative biological activity. Preferably, the peptidomimetics as used herein, shall bind specifically to at least one biomarker described herein elsewhere. A “detectable label” as referred to herein, which may be used in accordance with the invention include gold particles, latex beads, acridan ester, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels, e.g., magnetic beads, including paramagnetic and superparamagnetic labels, and fluorescent labels. Enzymatically active labels include e.g. horseradish peroxidase, alkaline phosphatase, beta-Galactosidase, Luciferase, and derivatives thereof. Suitable substrates for detection include di-amino-benzidine (DAB), 3,3'-5,5'- tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3- indolyl-phosphate. A suitable enzyme- substrate combination may result in a coloured reaction product, fluorescence or chemiluminescence, which can be measured according to methods known in the art (e.g. using a light-sensitive film or a suitable camera system). As for measuring the enzymatic reaction, the criteria given above apply analogously. Typical fluorescent labels include e.g. fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red, Fluorescein, the Alexa dyes, brilliant violet or brilliant ulraviolet. Also, the use of quantum dots as fluorescent labels is contemplated. Typical radioactive labels include 35S, 1251, 32P, 33P, and the like. A radioactive label can be detected by any method known and appropriate, e.g. a light-sensitive film or a phosphor imager. Suitable labels may also be or comprise tags, such as biotin, digoxygenin, His-, GST-, FLAG-, GFP-, MYC-tag, influenza A virus hemagglutinin (HA), maltose binding protein, and the like.

The amount of a biomarker can be detected using a biomarker/binding molecule complex. The amount may also be detected indirectly from the biomarker/binding molecule complex, for example, as a result of a reaction that is subsequent to the biomarker/binding molecule interaction, but is dependent on the formation of the biomarker/binding molecule complex. In some examples, the amount of a biomarker may be detected directly from the biomarker in a biological sample. The amounts of biomarkers can also be detected using a multiplexed format that allows for the simultaneous detection of two or more biomarkers in a biological sample. In the multiplexed format, binding molecules are immobilized, directly or indirectly, covalently or non-covalently, in discrete locations on a solid support.

Also preferably, the biomarker(s) to be determined in accordance with the present invention may be determined as transcribed nucleic acid molecules. To this end, a binding molecule, such as a nucleic acid molecule that hybridizes specifically to the transcribed nucleic acid molecule or any molecule derived therefrom by, e.g., reverse transcription reactions, may be applied for detection. The hybridizing binding molecule itself may comprise a detectable label or it may specifically bind to a secondary molecule comprising such a detectable label.

Also preferably, the amount of a biomarker may be also detected by PCR based techniques such as quantitative polymerase chain reaction (qPCR). “Quantitative PCR” or “real-time PCR” is a well-known technique used for the detection and quantification of nucleic acids (DNA or RNA) in a sample. Typically, a fluorescent reporter dye is used as an indirect measure of the amount of nucleic acid present during each amplification cycle. The increase in the fluorescent signal is directly proportional to the amount of exponentially increased PCR product molecules (amplicons) produced during the repetitive phases of the reaction.

Yet more preferably, the amount of a biomarker may be detected by transcriptome sequencing, also known as single-cell sequencing (scRNA-seq). The transcriptome sequencing technology is a well-known method and refers to the sequencing of a single-cell genome or transcriptome in order to obtain genomic, transcriptome, or other multi-omics information. Thus, “transcriptome sequencing” as used herein refers to a method to analyse the RNA expression from large populations of cells, preferably populations of hematopoietic stem and progenitor cells. Typically, the method comprises 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 the isolation of single-cells, e.g. by FACS. Droplet-based methods, however, use lipid droplet formation to separate single-cells by phase separation in bulk samples of single-cells in suspension. The latter technique allows analysis of thousands of cells, whereas the plate-based is typically suitable for hundreds.

When a biomarker indicates an abnormal process or a disease in a subject, that biomarker is generally described as being either over-expressed as compared to an expression level or amount of the biomarker that indicates a normal process or an absence of a disease or other condition in a subject. “Over-expression” refers to a level or amount of a biomarker in a biological sample that is greater than a level or amount of the biomarker that is typically detected in similar biological samples from healthy or normal subjects.

The biomarkers of the invention shall be present on or in hematopoietic stem and progenitor cells (HSPCs). A biomarker being displayed on the surface of HSPCs, for example in the form of a surface protein, a receptor, a lipid and the like is present on a HSPC in accordance with the present invention. A biomarker being produced inside HSPCs and are intended to remain intracellular, such as a transcribed nucleic acid molecule, and thus, is present in a HSPC. Hematopoietic stem and progenitor cells (HSPCs) are a heterogenous population of cells that are responsible for the production of virtually all kind of mature blood and immune cells. HSPCs encompass a continuum of lowly primed undifferentiated cells, including hematopoietic stem cells (HSCs) (Velten et al. 2017 Nature Cell Biology), which are believed to represent the origin of postnatal hematopoietic differentiation. HSCs are capable to self-renew and to gradually acquire lineage biases along multiple directions within the HSPC compartment, which gives rise to committed or unilineage-restricted progenitor cells, respectively. For instance, myeloid progenitors ultimately give rise to differentiated cells such as monocytes, macrophages, neutrophils, or dendritic cells, whereas lymphoid progenitors ultimately give rise to e.g. T-cells, B-cells, or natural killer cells. The term „HSPCs“ as used herein, refers to CD34 ⁺ -cells, preferably, CD34 CD38 cells, and, more preferably, Lin'CD34 ⁺ CD38' -cells. Said cells can be, preferably, isolated from a sample as referred to herein, preferably, a tissue sample or a body fluid sample by, e.g., fluorescence-activated cell sorting (FACS) or other cell sorting techniques known from literature. Lineage-negative (Lin') cells are a mix of all cells which express almost no or even no mature cell lineage markers. These lineage markers can be, preferably, selected from the group consisting of CD4, CD8, CDl lb, CD14, CD19, CD20, CD56, and CD235a. More preferably, these lineage markers consist of CD4, CD8, CDl lb, CD14, CD19, CD20, CD56, and CD235a. In the latter case, a Lin'CD34 ⁺ CD38' -cell corresponds to a CD4-CD8-CDl lb'CD14-CD19-CD20'CD56'CD235a'CD34 ⁺ CD38' -cell.

The terms “sample” refers to any sample obtainable from the subject to be investigated that contains HSPCs. Preferably, said sample may be a tissue or a 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 umbilical cord blood sample. The blood sample also includes fractions thereof. For example, a blood sample can be fractionated into serum, plasma or into fractions containing particular types of blood cells, such as red blood cells or white blood cells (leukocytes). The term “sample” also includes materials containing homogenized solid material, such as from a tissue sample, or a tissue biopsy. Samples of body fluids can be obtained by well-known techniques and include, preferably, samples of blood. Tissue or organ samples, such as bone marrow samples, may be obtained by, e.g., biopsy. Separated cells may be obtained from the body fluids or the tissues or organs by separating techniques such as centrifugation or cell sorting. Accordingly, the term refers to the biological sample per se, which is used directly during the determination of the amounts of biomarkers. However, the term may also refer to samples that have to undergo various steps prior to determining of the amounts of biomarkers, for example, isolating cells from biological material.

The determined amounts of the biomarkers are compared to a reference in accordance with the method of the present invention. The term “reference” as used herein relates to an amount or value, which allows for allocation of a subject into either a group of subjects suffering from a disease or condition or being at risk for developing it, or a group of subjects, which do not suffer from said disease or condition or are not at risk for developing it. Such a reference can be a threshold amount, which separates these groups from each other. Accordingly, the reference shall be an amount, which allows for allocation of a subject into a group of subjects suffering from a disease or condition or being at risk for developing it, or not. A suitable threshold amount separating the two groups can be calculated without further ado by the statistical tests referred to herein elsewhere based on amounts of biomarkers from either a subject or group of subjects known to suffer from a disease or condition or being at risk for developing it or a subject or group of subjects known not to suffer from a disease or condition or being at risk for developing it. The reference amount applicable for an individual subject may vary depending on various physiological parameters such as age, gender, or subpopulation. Said references may be, preferably, references for each biomarker derived from at least one subject known to suffer from JMML. Yet preferably, references for each biomarker derived from at least one subject not known to suffer from JMML. More preferably, the references for each biomarker are derived from at least one subject that was diagnosed to suffer from JMML or to belong into either the high- or low-risk JMML group by DNA methylome analysis, preferably as carried out in the accompanying Examples, below.

Reference amounts can, in principle, be calculated for a cohort of subjects based on the average or mean values for a given parameter such as biomarker amount by applying standard statistically methods. In particular, accuracy of a test such as a method aiming to diagnose an event, or not, is best described by its receiver-operating characteristics (ROC) (see especially Zweig 1993, Clin. Chem. 39:561-577). The ROC graph is a plot of all of the sensitivity/ specificity pairs resulting from continuously varying the decision threshold over the entire range of data observed. The clinical performance of a diagnostic method depends on its accuracy, i.e. its ability to correctly allocate subjects to a certain prognosis or diagnosis. The ROC plot indicates the overlap between the two distributions by plotting the sensitivity versus 1 -specificity for the complete range of thresholds suitable for making a distinction. On the y- axis is sensitivity, or the true-positive fraction, which is defined as the ratio of number of truepositive test results to the product of number of true-positive and number of false-negative test results. This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup. On the x-axis is the false-positive fraction, or 1 -specificity, which is defined as the ratio of number of false-positive results to the product of number of true-negative and number of false-positive results. It is an index of specificity and is calculated entirely from the unaffected subgroup. Because the true- and false-positive fractions are calculated entirely separately, by using the test results from two different subgroups, the ROC plot is independent of the prevalence of the event 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 distributions of results) has an ROC plot that passes 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 test with no discrimination (identical distributions of results for the two groups) is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes. If the ROC plot falls completely below the 45° diagonal, this is easily remedied by reversing the criterion for "positivity" from "greater than" to "less than" or vice versa. Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test. Dependent on a desired confidence interval, a threshold can be derived from the ROC curve allowing for the diagnosis or prediction for a given event with a proper balance of sensitivity and specificity, respectively. Accordingly, the reference to be used for the aforementioned method of the present invention, i.e. a threshold, which allows to discriminate between subjects suffering from coagulation defects and those who do not suffer therefrom can be generated, usually, by establishing a ROC for said cohort as described above and deriving a threshold amount therefrom. Dependent on a desired sensitivity and specificity for a diagnostic method, the ROC plot allows deriving suitable thresholds. It will be understood that an optimal sensitivity is desired for excluding a subject for being at increased risk (i.e. a rule out) whereas an optimal specificity is envisaged for a subject to be assessed as being at an increased risk (i.e. a rule in).

The term “comparing” as used herein encompasses comparing the determined amount for a biomarker as referred to herein to a reference. It is to be understood that comparing as used herein refers to any kind of comparison made between the values for the amount with the reference. However, it is to be understood that, preferably, identical types of values are compared with each other, e.g., if an absolute amount is determined and to be compared in the method of the invention, the reference shall also be an absolute amount, if a relative amount is determined and to be compared in the method of the invention, the reference shall also be a relative amount, etc. The comparison may be carried out manually or computer assisted. The value of the amount and the reference can be, e.g., compared to each other and the said comparison can be automatically carried out by a computer program executing an algorithm for the comparison. The computer program carrying out the said evaluation will provide the desired assessment in a suitable output format.

The term “JMML” as referred to in accordance with the present invention encompasses JMML, JMML-like neoplasms, and JMML associated with neurofibromatosis or Noonan syndrome- associated myeloproliferative disorder (CBL-syndrome). Typical symptoms and signs at diagnosis of JMML are splenomegaly, hepatomegaly, lymphadenopathy, pallor, and fever. Less frequent are infection, bleeding, cough, and skin rash. Infrequently, cafe au lait spots, abdominal pain, xanthoma, bone pain, diarrhea, and CNS infiltration can occur (Arber et al. 2022 Blood). Typically, a subject is known to suffer from JMML if certain diagnostic criteria for JMML can be verified by specific assays, such as blood tests, bone marrow aspiration, cytogenetic, and molecular tests. Diagnostic criteria for JMML in accordance with the present invention may be, preferably, those of the International Consensus Classification (ICC) of myeloid neoplasms and acute leukemias. The ICC categorizes JMML and related disorders as MDS/MPN and groups them with pediatric and/or germline mutation-associated disorders (Arber loc. cit.). JMML is a clonal disorder of childhood characterized by constitutive activation of the RAS signal transduction pathway. More than 95% of the patients harbor mutations in the RAS pathway. Canonical mutations affect PTPN11, NRAS, KRAS, NF1, CBL, and rarely RRAS genes. Clonal diseases that phenotypically mimic JMML but do not harbor one of these mutations are classified as JMML-like neoplasms. The polyclonal Noonan syndrome-associated myeloproliferative disorder can clinically resemble JMML. All of these cases, i.e. JMML, JMML-like neoplasms, Noonan syndrome-associated myeloproliferative disorder, share typical characteristics.

The diagnostic criteria for JMML listed by the ICC comprise genetic, as well as clinical and hematologic features. Required are blast percentages in peripheral blood and bone marrow <20% and the absence of a.BCR::ABLl fusion (or Philadelphia chromosome). Most of the cases have splenomegaly (absent in ~3% of cases) and monocyte counts > 1 x 10 ⁹ /L (not reached in ~7% of cases). Genetically, usually one finding of the following is observed: somatic mutations in PTPN11, KRAS, NRAS, or RRAS,' or a germline mutation in NF1 and loss of heterozygosity or clinical diagnosis of neurofibromatosis type 1; or a CBL mutation and loss of heterozygosity of CBL.

Furthermore, monosomy 7 or any other chromosomal abnormality, higher levels of hemoglobin F than is normal for the age of the patient, myeloid precursors in the blood, granulocytemacrophage colony- stimulating factor (GM-CSF) hypersensitivity in colony assay, hyperphosphorylation of STAT5 can be indicative of JMML, whereas KMT2A rearrangements are excluded (Niemeyer & Flotho 2019 Blood). While GM-CSF hypersensitivity and hematological parameters were most important earlier, the current WHO classification highlights the RAS pathway mutation as most important criterion. JMML-like neoplasms lack a RAS pathway mutation but phenotypically resemble JMML. This group includes rearrangements like ALK, ROS1, FIP1LL:RARA, or CCDC88C::FLT3 fusions (Arber loc. cit.).

Diagnostic criteria for JMML in accordance with the present invention may be, also preferably, those of the WHO, Chapter 5, Myelodysplastic/ myeloproliferative neoplasms (Orazi et al. 2016). Accordingly, from the following clinical and hematological criteria all 4 are required: peripheral blood monocyte count > 1 x 10 ⁹ cells/L; - blast percentage in peripheral blood and bone marrow of < 20%; splenomegaly; and no Philadelphia (Ph) chromosome or BCR-ABL1 fusion.

From the following genetic criteria, at least one criterion is sufficient: somatic mutation in PTPN 11 , KRAS, or NRAS, germline mutations (indicating Noonan syndrome) must be excluded; clinical diagnosis of neurofibromatosis type 1 or NF1 mutation; and germline CBL mutation and LOH of CBL, occasional cases have heterozygous splicesite mutations.

For cases that do not meet any of the genetic criteria above, the following criteria must, in addition to the clinical and hematological criteria above, be fulfilled:

Monosomy 7 or any other chromosomal abnormality, or at least two of the following criteria: increased haemoglobin F for age; myeloid or erythroid precursors on peripheral blood smear; granulocyte-macrophage colony-stimulating factor (also called CSF2) hypersensitivity in colony assay; and hyperphosphorylation of STAT5.

More preferably, a subject suffering from JMML is identified by DNA methylome analysis as described, e.g., in the accompanying Examples, below.

In one embodiment, the reference is derived from at least one subject known to suffer from JMML.

Preferably, a subject 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: (i) group I consisting of: CD52, RAMP1, LTB, LST1, J AML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RABI 1 A, 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 to a reference.

In an embodiment of the method of the present invention, wherein the reference is derived from at least one subject known to suffer from JMML, the determined amount of at least one of aforementioned markers, which is identical or larger than the reference, is indicative for a subject suffering from JMML. A determined amount of at least one of aforementioned markers, which is lower than the reference, may be indicative for a subject not suffering from JMML.

In another embodiment, the reference is derived from at least one subject known not to suffer from JMML.

Typically, a subject is known not to suffer from JMML if the aforementioned diagnostic criteria for JMML referred to above are not met. Preferably, a subject not suffering from JMML can be identified by DNA methylome analysis as well as described, e.g., in the accompanying Examples, below.

Preferably, a subject known not to suffer from JMML may be also characterized by the physiological abundance of a biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, J AML, IFITM3, CD7, CD69, CD 164, 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 on or in HSPCs in a biological sample of said subject.

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: (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, RABI 1 A, 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 to a reference.

In an embodiment wherein the reference is derived from at least one subject known not to suffer from JMML, the determined amount of at least one of aforementioned markers, which is identical or below the reference, is indicative for a subject not suffering from JMML. A determined amount of at least one of aforementioned markers, which is larger than the reference, may be indicative for a subject suffering from JMML.

It will be understood that in accordance with 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 or the presence or abundance of one or more biomarkers of group II, i.e. low-risk JMML biomarkers on or in HSPCs. The method of the present invention shall also provide an aid for diagnosing the absence of JMML, i.e. if no biomarker of group I and no biomarker of group II are determined on or in HSPCs. By investigating the biomarkers of group I and II, thus, a subject can be diagnosed for JMML. Thanks to the finding in the studies underlying the present invention it is possible to efficiently screen for JMML in a population of juvenile subjects or to more reliable render a diagnosis for an individual subject. Therefore, treatment efforts may be improved. As discussed already before, JMML is a rare aggressive cancer of childhood. The availability of patient samples for investigation is limited given the low incidence and prevalence of the disease which makes diagnostic and therapeutic developments difficult and cumbersome. In the studies underlying the present invention, it was possible to isolate viable cells from a limited number of patient samples, representing all known JMML subtypes. Among the viable cells, the number of HSPCs (Lin- CD34+ CD38- HSPCs) is typically low. Nevertheless, it was possible to obtain rich data sets by applying single cell technologies, including scRNA-seq or FACS techniques from the material available.

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

Therefore and in light with the foregoing, the present invention also contemplates a method of classifying a subject suffering from JMML into a JMML low- or 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, said at least one biomarker being selected from each of: i) group I consisting of: CD52, RAMP1, LTB, LST1, J AML, 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 of step a) to a reference; and c) classifying the subject into a JMML low or high-risk group based on the comparison of step b). The term “classifying” as used herein refers to allocating a subject into a group of subjects exhibiting a similar or identical disease status. The said disease status into which the subject can be classified by applying the method of the present invention is either the JMML high-risk group or the JMML low-risk group.

The term “JMML high-risk group” as used herein refers to a group of subjects known to suffer from JMML that have a high-risk of worsening of JMML or any sign or symptom associated therewith and/or lethality from JMML without treatment within a period of at most 8 years, at most 7 years, at most 6.5 years, at most 6 years, at most 5.5 years, at most 5 years, at most 4.5 years, at most 4 years, at most 3.5 years, at most 3 years, at most 2.5 years, at most 2 years, at most 1.5 years, at most 1 year, at most 6 months, or less than 6 months after diagnosis. The term also refers to a group of subjects known to suffer from JMML that have a high risk of relapse. A subject known to suffer from JMML may also be classified into a JMML high-risk group if specific biomarkers on HSPCs are detected.

Thus, in one embodiment of the aforementioned method of the invention, wherein if at least one biomarker selected from group I is determined in step a), the subject is classified into a JMML high-risk group. In such a case, it will be understood that, preferably, the reference for the at least one group I biomarker can be obtained from at least one subject known to be from the high-risk JMML group. A determined amount for the at least one biomarker from group I which is identical or increased compared to the reference shall be indicative for a high-risk subject. Yet, it will be understood that, preferably, the reference for the at least one group I biomarker can be obtained from at least one subject known to be not from the JMML high-risk group. A determined amount for the at least one biomarker from group I which is identical or decreased compared to the reference shall be indicative for a subject not being a high-risk subject.

The term “JMML low-risk group” as used herein refers to a group of subjects known to suffer from JMML that have a low risk of worsening of JMML or any sign or symptom associated therewith and/or lethality from JMML without treatment within a period of at most 8 years, at most 7 years, at most 6.5 years, at most 6 years, at most 5.5 years, at most 5 years, at most 4.5 years, at most 4 years, at most 3.5 years, at most 3 years, at most 2.5 years, at most 2 years, at most 1.5 years, at most 1 year, at most 6 months, or less than 6 months after diagnosis. The term also refers to a group of subjects known to suffer from JMML, which often show spontaneous remission. A subject known to suffer from JMML may also be classified into a JMML low-risk group if specific biomarkers on HSPCs are detected. Thus, in one embodiment of the aforementioned method of the invention, wherein if at least one biomarker selected from group II is determined in step a), the subject is classified into a JMML low-risk group. In such a case, it will be understood that, preferably, the reference for the at least one group II biomarker can be obtained from at least one subject known to be from the JMML low-risk group. A determined amount for the at least one biomarker from group I which is identical or increased compared to the reference shall be indicative for a low-risk subject. Yet, it will be understood that, preferably, the reference for the at least one group II biomarker can be obtained from at least one subject known to be not from the low-risk JMML group. A determined amount for the at least one biomarker from group II which is identical or decreased compared to the reference shall be indicative for a subject not being a low-risk subject.

The present invention further relates to a method of identifying whether a subject belongs into a JMML high-risk group, the method comprising: 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 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 of step a) to a reference; and c) identifying whether the subject belongs into a JMML high-risk group based on the comparison of step b).

The reference may be, preferably, obtained from at least one subject known to belong into the JMML high-risk group. If the determined at least one biomarker is identical or increased compared to the reference, it will be understood that the subject investigated belongs into the JMML high-risk group as well. Yet, the reference may be, preferably, obtained from at least one subject known to not belong into the JMML high-risk group. If the determined at least one biomarker is identical or decreased compared to the reference, it will be understood that the subject investigated belongs not into the JMML high-risk group as well.

The present invention further relates to a method of identifying whether a subject belongs into a JMML low-risk group, the method comprising: 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 ofIGLLI, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G; b) comparing the determined amount of step a) to a reference; and c) identifying whether the subject belongs into a JMML low-risk group based on the comparison of step b).

The reference may be, preferably, obtained from at least one subject known to belong into the JMML low-risk group. If the determined at least one biomarker is identical or increased compared to the reference, it will be understood that the subject investigated belongs into the JMML low-risk group as well. Yet, the reference may be, preferably, obtained from at least one subject known to not belong into the JMML low-risk group. If the determined at least one biomarker is identical or decreased compared to the reference, it will be understood that the subject investigated belongs not into the JMML low-risk group as well.

The methods of the present invention, preferably, further comprise selecting a therapy for the subject suffering from JMML based on the identified JMML risk group of step c).

As used herein, the term “therapy” refers to any measure aiming at ameliorating and/or curing JMML or any sign or symptom associated therewith. Such measures can be selected from administering a drug, administering a disease-modulating radiation therapy, surgery or transplantation, scheduling a further appointment with a medical practitioner or any combination thereof.

Preferably, a suitable therapy is selected depending on whether the subject suffering from JMML is identified to belong into a JMML low- or high-risk group. Typically, one of the following JMML treatment regimen are considered: i) allogeneic stem cell transplantation or ii) drug therapy. Drug therapy typically includes administering the approved azacitidin (Vidaza®), a chemical analogue of the nucleoside cytidine. Preferably, drug therapy includes administering an inhibitory agent that specifically inhibits at least one group I or II biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RABI 1 A, 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) as specified elsewhere herein. The present invention further contemplates use of at least one biomarker present on or in hematopoietic stem and progenitor cells (HSPCs) in a biological sample 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; for diagnosing juvenile myelomonocytic leukemia in a subject or classifying a subject suffering from JMML into a JMML low- or high-risk group in a subject having or having a risk of developing JMML.

The present invention further relates to a kit for diagnosing JMML in a subject or classifying a subject suffering from JMML into a JMML low- or high-risk group comprising at least one detection agent and instructions to carry out the method of the present invention, wherein the at least one detection agent is capable of specifically detecting a group I or II biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA- DRA, RABI 1 A, 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.

The term “kit” as used herein refers to collection of the aforementioned components, typically, provided in separately or within a single container. The container also typically comprises 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 a computer program code, which is capable of carrying out or supports the determination of the biomarkers referred to in the methods of the present invention when implemented on a computer or a data processing device. The computer program code may be provided on a data storage medium or device such as an optical storage medium (e.g., a Compact Disc) or directly on a computer or data processing device or may be provided in a download format such as a link to an accessible server or cloud. Moreover, the kit may usually comprise standards for reference amounts of biomarkers for calibration purposes as described elsewhere herein in detail. The kit according to the present invention may also comprise further components, which are necessary for carrying out the method of the invention such as solvents, buffers, washing solutions and/or reagents required for detection of the released second molecule. Further, it may comprise the device of the invention either in parts or in its entirety.

The term “detection agent” as used herein refers to a detection agent referred to in accordance with the methods of the present invention described elsewhere in this specification. In particular, a 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 being capable of specifically hybridizing to said transcribed nucleic acid, such as an antisense nucleic acid probe or oligonucleotide primer may be used as detection agent whereas for protein biomarker aptamers, antibodies, adnectins, ankyrins, antibody mimetics and other protein scaffolds, small molecules, nucleic acids, lectins, affybodies, nanobodies, avimers and peptidomimetics may be used.

The present invention further contemplates an inhibitory agent that specifically inhibits at least one group I or II biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, J AML, IFITM3, CD7, CD69, CD 164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RABI 1 A, 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), for use in treating and/or preventing juvenile myelomonocytic leukemia (JMML).

The term “inhibitory agent” refers to a substance that influences one or more biological or chemical reactions elicited by the at least one biomarker referred to above, i.e. its biological activity, in such a way that they are slowed down, impeded or prevented. Depending on the substance type, reversible or irreversible inhibition may occur. It will be understood that the said one or more biological or chemical reactions are associated with JMML, i.e. they may contribute directly or indirectly to the occurrence or progression of the disease.

The term "treating” as used herein relates to ameliorating and/or curing JMML as referred to herein, preventing progression of the disease or at least an amelioration of at least one symptom associated with the said disease to a significant extent. Said treating as used herein also encompasses an entire restoration of health with respect to JMML. It will be understood that a treatment as referred to herein will, in all likelihood, not be successful in all subjects which received the treatment. However, it is envisaged that the treatment is effective in at least a statistically significant portion of the subjects that are treated. Whether a statistically significant portion, e.g., of a cohort of subjects, can be successfully treated may, preferably, be determined, e.g., by statistical tests using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc. Preferably, the treatment shall be effective for 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 of a given cohort or population.

The term “preventing” refers to retaining health with respect to JMML for a certain period of time in a subject. It will be understood that the said period of time may be dependent on the therapy used or on the amount of the drug compound which has been administered. It is to be understood that prevention may not be effective in all subjects that have been administered a binding agent according to the present invention. However, the term requires that, preferably, a statistically significant portion of subjects of a cohort or population are effectively prevented from suffering from a disease or disorder referred to herein or its accompanying symptoms. Preferably, a cohort or population of subjects is envisaged in this context, which normally, i.e. without preventive measures according to the present invention, would develop JMML. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well-known statistic evaluation tools discussed elsewhere in this specification.

In particular, the present invention envisages the group I or II biomarker being 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, RABI 1 A, 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 agent is, thus, a peptide, a protein, a small molecule, a lipid or an aptamer.

A “peptide” or a “protein” as referred to herein relates to a molecule consisting of amino-acid residues joined by peptide bonds. Molecules consisting of short amino acid chains, e.g. up to about 100 amino acids, are referred to as peptides, whereas molecules having larger amino acid chains are referred to as proteins. Preferably, the peptide or protein shall inhibit the biological activity of at least one biomarker directly or indirectly. More preferably, the said inhibitory agent shall specifically bind to and thereby inhibit the biological activity of at least one biomarker.

The term “small molecule” as used herein relates to a molecule with a low molecular weight. Typically, a small molecule is an organic compound with a molecular weight of less than 900 Daltons. Small molecules include, for example, small secondary metabolites such as alkaloids, lipids, glycosides, terpenes, tetrapyrroles, phenazines, oliogonucleotides, or small peptide-like molecules. Preferably, the small molecule shall inhibit the biological activity of at least one biomarker directly or indirectly. More preferably, the said inhibitory agent shall specifically bind to and thereby inhibit the biological activity of at least one biomarker.

A “lipid” as used herein relates to hydrophobic or amphiphilic small molecules. Lipids include fatty acids and their derivates such as triglycerides, diglycerides, monoglycerides, phospholipids, lysophospholipids such as lysophosphatidylcholin (LPC), glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, prenol lipids, and sterol lipids. Preferably, the lipid shall inhibit the biological activity of at least one biomarker directly or indirectly. More preferably, the said inhibitory agent shall specifically bind to and thereby inhibit the biological activity of at least one biomarker.

As used herein, the term “aptamer” refers to a polynucleotide or polypeptide binding specifically to a target molecule by virtue of its three-dimensional structure. Peptide aptamers, preferably, are peptides comprising 8-80 amino acids, more preferably 10-50 amino acids, and most preferably 15-30 amino acids. They can e.g. be isolated from randomized peptide expression libraries in a suitable host system like baker’s yeast (see, for example, Klevenz et al., Cell Mol Life Sci. 2002, 59: 1993-1998). A peptide aptamer, preferably, is a free peptide; it is, however, also contemplated that a peptide aptamer is fused to a polypeptide serving as “scaffold”, meaning that the covalent linking to said polypeptide serves to fix the three- dimensional structure of said peptide aptamer to a specific conformation. Preferably, the aptamer specifically binds to and inhibits the biological activity of at least one biomarker.

In another embodiment, the inhibitory agent is an antibody or antigen-binding fragment thereof as defined elsewhere herein in detail. In a more preferred embodiment, the antibody or antigenbinding fragment thereof is alemtuzumab. Alemtuzumab refers to a humanized monoclonal antibody that is commercially obtainable under the brand names Campath and Lemtrada. Said 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).

The present invention also envisages biomarkers being a transcribed nucleic acid, preferably, an mRNA. Preferably, the inhibitory agent is, thus, a ribozyme, an inhibitory RNA molecule, an antisense oligonucleotide or a morpholino.

The term "ribozyme" as used herein refers to catalytic RNA molecules possessing a well- defined tertiary structure that allows for catalyzing either the hydrolysis of one of their own phosphodiester bonds (self-cleaving ribozymes), or the hydrolysis of bonds in other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome. The ribozymes envisaged in accordance with the present invention are, preferably those, which specifically hydrolyse the target transcripts. In particular, hammerhead ribozymes are preferred in accordance with the present invention. How to generate and use such ribozymes is well- known in the art (see, e.g., 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).

The term “inhibitory RNA molecule” as used herein, refers to an RNA molecule that inhibits gene expression in a sequence-specific manner. Inhibitory RNA molecules include, for example, small interfering RNA (siRNA), small hairpin RNA (shRNA) and microRNA (miRNA). The inhibitory RNA molecule typically induces a process known as RNA interference (RNAi), leading to cleavage and/or translational inhibition of a target mRNA with a complementary sequence. It is known to those skilled in the art that the inhibitory RNA molecule can show perfect or imperfect base-pairing to a complementary target sequence. siRNA and shRNAs typically base-pair perfectly and induce mRNA cleavage only in a single, specific target. On the contrary, miRNAs usually have incomplete base pairing to a target and often inhibit the translation of many different mRNAs with similar sequences. An inhibitory RNA molecule may be chemically synthesized or expressed within the cell, for example by introduction of a respective recombinant DNA construct. It will be understood that such a DNA construct may contain additional regulatory elements such as an enhancer, a constitutive or inducible promoter or a terminator. The inhibitory RNA molecule shall be a direct or indirect inhibitor of at least one group I or II biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RABI 1 A, 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 for at least one biomarker shall impair translation of the respective mRNA into a functional protein and thus, inhibiting activity of the respective proteins or it may degrade or facilitate degradation of the said mRNA.

An “antisense oligonucleotide” as used herein, refers to a single strand DNA and/or RNA molecule that is capable of interfering with DNA and/or RNA processing. Antisense oligonucleotides comprise a nucleic acid sequence, which is complementary to a specific RNA or DNA sequence. Typically, an antisense oligonucleotide will bind, in a sequence-specific manner, to their respective complementary oligonucleotides, DNA, or RNA, thereby interfering with DNA and/or RNA processing. It is known to those skilled in the art that antisense oligonucleotides may interfere with mRNA processing through RNase H-mediated degradation, translational arrest, modulation of splicing or they may act through steric hindrance of proteins. Means and methods for the design and synthesis of antisense oligonucleotides are well-known in the art and include, for example, rational design, chemical modifications and design of antisense oligonucleotides containing locked nucleic acids (LNA) as well as solid-phase chemical synthesis. Antisense oligonucleotides can be chemically synthesized or expressed within the cell, for example by introduction of respective recombinant DNA construct. It will be understood by those skilled in the art that such a DNA construct may contain additional regulatory elements such as an enhancer, a constitutive or inducible promoter or a terminator. Preferably, the antisense oligonucleotide has a length of at least 8, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50 or more nucleotides. The antisense oligonucleotide may comprise deoxyribonucleotides, ribonucleotides, or a combination of both. Preferably, the antisense oligonucleotide is a DNA and/or RNA molecule that interferes with expression and/or translation of one of the aforementioned biomarkers, so that a functional protein cannot be made.

The term “morpholino” as used herein relates to a molecule that blocks access of other molecules to small, specific sequences of the base-pairing surfaces of an RNA. Typically, said small specific sequences have a length of about 25 nucleotides. In general, a morpholino comprises a backbone of methylenemorpholine rings and phosphorodiamidate linkages. Morpholinos are commonly also known as morpholino oligomers (MO nucleic acid analogs) and phosphorodiamidate morpholino oligomers (PMO). Morpholinos typically do not lead to degradation of their target RNA molecules, but rather act by sterical blocking, i.e. binding to a target sequence within an RNA and thereby getting in the way of molecules that may otherwise interact with said RNA. Preferably, the morpholino is directly binding to the pre-mRNA and/or mRNA of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA-DRA, RABI 1 A, 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, interferes with their translation and/or splicing leading to production of a less or non-fimctional protein. Thus, activity of any of the respective proteins is inhibited.

The present invention further envisages a pharmaceutical composition for use in treating and/or preventing JMML comprising at least two inhibitory agents as elsewhere defined, wherein each of the inhibitory agents 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, RABI 1 A, 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 in hematopoietic stem and progenitor cells (HSPCs).

Thus, the aforementioned pharmaceutical composition of the present invention comprises two or more different inhibitory agents that inhibit two or more different biomarkers as referred to before.

The term “pharmaceutical composition” as used herein, relates to compositions comprising the compounds of the present invention and, preferably, one or more pharmaceutically acceptable carrier. The compounds of the present invention can be formulated as pharmaceutically acceptable salts. Preferred acceptable salts are acetate, HC1, sulphate, chloride and the like. The pharmaceutical compositions are, preferably, administered systemically. Suitable routes of administration conventionally used for drug administration are oral, intravenous, subcutaneous, or parenteral administration as well as inhalation. However, depending on the nature and mode of action of a compound, the pharmaceutical compositions may be administered by other routes as well. Moreover, the compounds can be administered in combination with other drugs either in a common pharmaceutical composition or as separated pharmaceutical composition, wherein said separated pharmaceutical compositions may be provided in form of a kit.

The compounds are, preferably, administered in conventional dosage forms prepared by combining the drugs with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate for the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well- known variables.

The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof. The pharmaceutical carrier employed may be, for example, a solid, a gel or a liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid, degradable polymers like PLGA (DeYoung at al. (2011), DIABETES TECHNOLOGY & THERAPEUTICS 13: 1145; Ramazani et al., (2016), Int J Pharm. 499(1-2): 358-367, and the like. Exemplary liquid carriers are phosphate buffered saline solution, syrup, oil such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions, and the like. Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax. Said suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

The diluent(s) is/are selected so as not to affect the biological activity of the compound or compounds. Examples of such diluents are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, non- immunogenic stabilizers, reactive oxygen scavengers, and the like.

The pharmaceutical composition is, preferably, administered in conventional dosage forms prepared by combining the active compound with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing or dissolving the ingredients as appropriate to obtain the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well- known variables. Similarly, the carrier or diluent may include time delay material well known in the art, such as glyceryl mono-stearate, or glyceryl distearate alone or with a wax. A therapeutically effective dose refers to an amount of the active compound to be used in a pharmaceutical composition of the present invention which provides the effect referred to in this specification. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, which may include the patient's 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 assessment. A typical dose can be, for example, in the range of 1 pg to 1000 mg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 pg to 100 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 pg to 1 mg units per kilogram of body weight per minute, respectively. Preferably, the pharmaceutical composition is administered once to the subject, i.e., preferably, is used as a one-time treatment. Depending on the subject and the mode of administration, the quantity of substance administration may vary over a wide range to provide from about 0.01 mg per kg body mass to about 100 mg per kg body mass. The pharmaceutical compositions and formulations referred to herein are administered at least once in order to treat or ameliorate or prevent a disease or condition recited in this specification. However, the said pharmaceutical compositions may be administered more than one time, for example from two to 50 times, more preferably from five to 50 times. Preferably, administration is adjusted to maintain an effective concentration in the body of a subject for the time period intended. Progress can be monitored by periodic assessment.

The present invention further relates to a method of treating and/or preventing JMML comprising administering to a subject in need thereof a therapeutically effective amount at least one inhibitory agent as defined elsewhere herein.

A “therapeutically effective amount” refers to an amount of the at least one inhibitory agent of the invention which prevents, ameliorates or cures JMML or the symptoms accompanying said disease referred to in this specification. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. The dosage regimen will be determined by the attending physician and other clinical factors; preferably in accordance with any one of the above-described methods.

All explanations and definitions of the terms made above apply mutatis mutandis for the following embodiments.

The following embodiments are particular preferred embodiments according to the invention.

Embodiment 1 : A method of 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, said at least one biomarker being selected from each of i) group I consisting of CD52, RAMP1, LTB, LST1, J AML, IFITM3, CD7, CD69, CD 164, 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 of step a) to a reference; and c) diagnosing JMML based on the comparison of step b).

Embodiment 2: The method of embodiment 1, wherein the biological sample is a tissue sample or a body fluid sample.

Embodiment 3: The method of any one of embodiments 1 and 2, wherein the tissue sample is a connective tissue sample, preferably, bone marrow.

Embodiment 4: The method of any one of embodiments 1 to 3, wherein the body fluid sample is a peripheral blood sample or umbilical cord blood sample.

Embodiment 5: The method of any one of embodiments 1 to 4, wherein the subject is a human.

Embodiment 6: The method of embodiment 5, wherein the subject is at an age of at most 16 years, at most 15 years, at most 14 years, at most 13 years, at most 12 years, at most 11 years, at most 10 years, at most 9 years, at most 8 years, at most 7 years, at most 6 years, at most 5.5 years, at most 5 years, at most 4.5 years, at most 4 years, at most 3.5 years, at most 3 years, at most 2.5 years, at most 2 years, at most 1.5 years, at most 1 year, at most 6 months, or less than 6 months.

Embodiment 7: The method of any one of embodiments 1 to 6, wherein the reference is derived from at least one subject known to suffer from JMML.

Embodiment 8: The method of embodiment 7, wherein the amount determined in step a) which is identical to or larger than the reference is indicative for a subject suffering from JMML or wherein an amount determined in step a) which is lower than the reference is indicative for a subject not suffering from JMML.

Embodiment 9: The method of any one of embodiments 1 to 8, wherein the reference is derived from at least one subject known not to suffer from JMML.

Embodiment 10: The method of embodiment 9, wherein an amount determined in step a) which is identical to or below the reference is indicative for a subject not suffering from JMML or wherein an amount determined in step a) which is larger than the reference is indicative for a subject suffering from JMML. Embodiment 11 : The method of 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, bulk RNA-seq or scRNA-seq.

Embodiment 12: A method of classifying a subject suffering from JMML into a JMML low- or 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, said at least one biomarker being selected from each of: i) group I consisting of: CD52, RAMP1, LTB, LST1, J AML, IFITM3, CD7, CD69, CD 164, 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 of step a) to a reference; and c) classifying the subject into a JMML low-risk group or a JMML high-risk group based on the comparison of step b).

Embodiment 13: The method of embodiment 12, wherein if at least one biomarker selected from group I is determined in step a),

- the subject is classified into a JMML high-risk group if the reference is derived from at least one subject known to suffer from high-risk JMML and the determined amount for the at least one biomarker is identical or increased compared to the reference; or

- the subject is classified not into JMML high-risk group if the reference is derived from at least one subject known not to suffer from high-risk JMML and the determined amount for the at least one biomarker is identical or decreased compared to the reference.

Embodiment 14: The method of any one of embodiment 12 to 13, wherein if at least one biomarker selected from group II is determined in step a),

- the subject is classified into a JMML low-risk group if the reference is derived from at least one subject known to suffer from low-risk JMML and the determined amount for the at least one biomarker is identical or increased compared to the reference; or

- the subject is classified not into JMML low-risk group if the reference is derived from at least one subject known not to suffer from low-risk JMML and the determined amount for the at least one biomarker is identical or decreased compared to the reference. Embodiment 15: A method of identifying whether a subject belongs into a 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, said at least one biomarker being selected from group I consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD 164, 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 of step a) to a reference; and c) identifying whether the subject belongs into a JMML high-risk group based on the comparison of step b).

Embodiment 16: The method of embodiment 15, wherein,

- the subject is classified into a JMML high-risk group if the reference is derived from at least one subject known to suffer from high-risk JMML and the determined amount for the at least one biomarker is identical or increased compared to the reference; or

- the subject is classified not into JMML high-risk group if the reference is derived from at least one subject known not to suffer from high-risk JMML and the determined amount for the at least one biomarker is identical or decreased compared to the reference.

Embodiment 17: A method of identifying whether a subject belongs into a 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, said at least one biomarker being selected from group II consisting of IGLL1, BEST1, EREG, SLC5A3, SELK, PRRG3, NINJ1, MGST1, and HLA-G; b) comparing the determined amount of step a) to a reference, wherein the reference is derived from at least one subject known not to suffer from JMML; and c) identifying whether the subject belongs into a JMML low-risk group if the amount determined in step a), which is identical to or below the reference is indicative for a subject not suffering from JMML, based on the comparison of step b).

Embodiment 18: The method of embodiment 17, wherein

- the subject is classified into a JMML low-risk group if the reference is derived from at least one subject known to suffer from low-risk JMML and the determined amount for the at least one biomarker is identical or increased compared to the reference; or the subject is classified not into JMML low-risk group if the reference is derived from at least one subject known not to suffer from low-risk JMML and the determined amount for the at least one biomarker is identical or decreased compared to the reference.

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

Embodiment 20: Use of at least one biomarker present on or in hematopoietic stem and progenitor cells (HSPCs) in a biological sample 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, for diagnosing juvenile myelomonocytic leukemia (JMML) in a subject or classifying a subject suffering from JMML into a JMML low- or high-risk group in a subject having or having a risk of developing JMML.

Embodiment 21 : Kit for diagnosing juvenile myelomonocytic leukemia (JMML) in a subject or classifying a subject suffering from JMML into a JMML low- or high-risk group comprising at least one detection agent and instructions to carry out the method of 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, RABI 1 A, 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).

Embodiment 22: An inhibitory agent that specifically inhibits at least one biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD164, CD74, TNF, TFPI, DLK1, CD82, IGHM, CALCRL, RALA, SLC2A5, HSPA5, HLA- DRA, RABI 1 A, 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) for use in treating and/or preventing juvenile myelomonocytic leukemia (JMML). Embodiment 23 : The inhibitory agent for use of embodiment 22, wherein said inhibitory agent specifically binds to and inhibits the at least one biomarker.

Embodiment 24: The inhibitory agent for use of embodiment 22 or 23, wherein said inhibitory agent is a peptide, a protein, a small molecule, a lipid or an aptamer.

Embodiment 25: The inhibitory agent for use of embodiment 22 or 23, wherein said inhibitory agent is an antibody or antigen-binding fragment thereof.

Embodiment 26: The inhibitory agent for use of embodiment 25, wherein said antibody or antigen-binding fragment thereof is alemtuzumab.

Embodiment 27: The inhibitory agent of embodiment 22, wherein said inhibitory agent specifically binds to and inhibits translation of the at least one biomarker, said at least one biomarker being an expressed nucleic acid, preferably, mRNA.

Embodiment 28: The inhibitory agent for use of embodiment 27, wherein said inhibitory agent is a ribozyme, an inhibitory RNA molecule, an antisense oligonucleotide or a morpholino.

Embodiment 29: A pharmaceutical composition for use in treating and/or preventing juvenile myelomonocytic leukemia (JMML) comprising at least two inhibitory agents as defined in any one of embodiments 22 to 28, wherein each of said inhibitory agents specifically inhibits a different biomarker selected from the group consisting of CD52, RAMP1, LTB, LST1, JAML, IFITM3, CD7, CD69, CD 164, 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).

Embodiment 30: The pharmaceutical composition for use of embodiment 29, further comprising a pharmaceutically acceptable carrier.

Embodiment 31 : A method of treating and/or preventing juvenile myelomonocytic leukemia (JMML) comprising administering to a subject in need thereof a therapeutically effective amount at least one inhibitory agent as defined in any one of embodiments 22 to 28. All references cited throughout the specification are herewith incorporated by reference with respect to the specifically mentioned disclosure content as well as in their entireties.

FIGURES

Figure 1: Patient selection for an in-depth multi-modal analysis to detect novel biomarkers and therapeutic targets of JMML stem cells. DNA methylation array analysis (EPIC-array) of total hematopoietic cells from 8 different patients diagnosed for JMML. The heatmap shows average DNA methylation values (P-values) of 124 classifier CpGs from the previously published DNA methylation classifier for JMML (Schbnung 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 and the low-risk group refers to LM and IM patients.

Figure 2: Biomarker identification and validation. (A) Dotplot summarizing the scRNA-seq expression data of identified high- and low-risk HSC biomarkers. The biomarkers represent differentially expressed genes in HSCs between high- and low-risk JMML patients, which encode for putative cell surface markers. The high- and low-risk groups are defined as explained above in Figure 1. The normal group represents HSCs from umbilical cord blood from healthy donors. The colour of the dots represents the normalized average expression values of the single-cell data per group. The dot size represents the percentage of cells detected to express the marker genes per group. (B) Histograms of FACS analyses of high-risk JMML HSC surface markers CD52, CD69, and CD 164 on JMML Lin CD34 ⁺ HSPCs from low- to high-risk patients as determined in figure 1, confirming the expected increased cell surface protein expression in high-risk patients.

Figure 3: Functional validation applying an anti-CD52 treatment on JMML patient-derived xenograft (PDX) mice. (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 forward scatter area. (B) Quantification of viable human hematopoietic CD45 ⁺ cells in the bone marrow of control and anti-CD52 treated (Alemtuzumab) PDX mice, (n = 10 mice per group)

Figure 4: Depletion of total human hematopoiesis in JMML PDX mice upon anti-CD52 treatment. Panels (A) to (G) represent log scaled number of living human cells across different hematopoietic lineages as quantified via FACS analysis of total bone marrow from anti-CD52 treated (Alemtuzumab) or control JMML PDX mice. The depletion of total hematopoiesis including CD52-negative cells such as Erythrocytes confirms JMML stem cells as the disease- propagating cells and the here identified JMML stem cell markers as therapeutic targets in JMML.

Figure 5: Disease propagation of JMML is disrupted in serial transplantation experiments upon anti-CD52 treatment. 2° recipient mice received either total bone marrow of control mice (PBS) or of mice treated with anti-CD52 (Alemtuzumab). (A) Engraftment of human CD45 ⁺ cells in 2° recipients, revealing the disruption of disease propagation upon anti-CD52 treatment, (n = 7-8 mice per group) (B) Kaplan-Meier curve summarizing the leukemia-free survival of 2° recipients, confirming the efficacy of targeting JMML HSC surface markers, (n = 3 mice per group)

Figure 6: Conservation of JMML epigenotypes in stem cells and total hematopoiesis. (A) Ultralow input whole-genome bisulfite sequencing (WGBS) of Lin'CD34 ⁺ CD38‘ JMML HSPCs reveals the conservation of JMML epigenotypes in the immature JMML stem cell compartment. (B) Integration of ultra-low WGBS and DNA methylation array analysis reveals that DNA methylation patterns of JMML stem cells are conserved in DNA methylomes of overall 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 beta-values. The conservation in 147 patients demonstrates that molecular programs in JMML stem cells are generally of prognostic and therapeutic value in JMML. (C) Integration of DNA methylation data from JMML stem cells (WGBS) with DNA methylation array data (Illumina 450k and EPIC) from JMML bulk material including mature immune cells. Risk-related DNA methylation changes in JMML stem cells were used to examine conservation in bulk patient material of overall 331 patients. Principal component 1 (PC 1) accurately recapitulates the epigenotypes of all patients as determined by use of DNA methylation array analysis, confirming the transmission of epigenotypes from immature to mature JMML cells.

Figure 7: Association of DNA methylation and surface marker gene expression. (A) Ultra-low input WGBS data reveals differential methylation of JMML stem cell surface markers. (B) scRNA-seq reveals methylation-associated differential gene expression of JMML stem cell surface markers. The heatmap aligns differentially expressed gene expression to the associated differentially methylated regions (DMR) in a region of 200 Mb around each of the transcription start sites (TSS). The identification of methylation-associated gene expression changes across JMML epitypes was performed by use of a generalized linear model with stepwise feature elimination using AIC to identify DEGs associated with DMRs.

EXAMPLES The Examples shall merely illustrate the invention. They shall, whatsoever, not be construed as limiting the scope.

Example 1: Enrichment of hematopoietic stem and progenitor cells

To classify the epigenotype and hence the risk-group of JMML patients, DNA methylation array analysis (Infmium Human Methyl ationEPIC Bead Chip (EPIC) array) of primary hematopoietic cells was performed as described before (Schbnung et al. 2021, Clinical Cancer Research). In brief, 100-250 ng of genomic DNA (gDNA) was subjected to the Genomics and Proteomics Core Facility at the German Cancer Research Center (Heidelberg, Germany) and the data were analysed using the “RnBeads” Bioconductor package as described before (Lipka et al. 2018, Nature Communications, Schbnung et al. 2021 Clinical Cancer Research). The patients were classified into one of three epigenetic subgroups (low methylation (LM), intermediate methylyation (IM), or high methylation (HM)) based on the previously published 124 CpG DNA methylation classifier for JMML (Schbnung et al. 2021, Clinical Cancer Research).

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

Table 1 : Antibodies used to enrich JMML HSPCs.

Example 2: Single-cell sequencing

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

Example 3: Data evaluation of single-cell sequencing

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

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

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

As healthy normal controls, scRNA-seq data from human umbilical cord blood was used, which is publicly available from the human cell atlas (HCA) data portal https://data.humancellatlas.org/explore/projects/cc95ff89-2e 68-4a08-a234-480eca21ce79). To generate a consensus cell type definition across datasets after data integration, Seurat’s label transfer function was applied by use of published human reference scRNA-seq data of healthy adult hematopoiesis from the HCA project (Hay et al. 2018, Experimental Hematology). As a result, hematopoietic stem cells (HSCs) could be identified in both JMML and healthy reference data.

Example 4: Identification of biomarkers specific for a risk-group

To identify risk group-specific biomarkers, patients from the HM subgroup were considered as high-risk, whereas patients from the IM and LM subgroup were considered as low-risk. To call differentially expressed genes (DEGs) between JMML high- and low-risk HSCs, Seurat’s FindMarkers function was applied with default settings: genes expressed in at least 10% of cells, expression difference on a natural log scale of at least 0.25, one-tailed Wilcoxon rank sum test, and P values adjusted for multiple testing using the Bonferroni correction. To identify DEGs which encode for cell surface markers, the entire list of DEGs was used as an input for SurfaceGenie to calculate the Surface Protein Consensus (SPC) score (Waas et al. 2020, Bioinformatics). This resulted in a list of overall 61 genes with a SPC score above 0. This list was further reduced by manual curation to exclude genes which are either highest expressed in the JMML IM subgroup or higher expressed in the normal control HSCs than in the JMML risk-group having the highest expression of the corresponding gene, respectively. Like this, the statistically significant, risk-group-specific DEGs were identified, which presumably encode for cell surface factors. Those genes represent (1) prognostic intra- and extracellular biomarkers for high- and low-risk groups in JMML and (2) potential drug targets on the cell surface of JMML HSCs.

Example 5: Evaluation of the functional role of biomarkers

To evaluate the functional role of such surface markers in molecular pathogenesis of JMML, patient-derived xenograft mice were generated as described before (Krombholz et al. 2016 Haematologica; Krombholz et al. 2019 Leukemia). One to four days after birth, primary JMML cells were transplanted into 68mmunodeficient Rag2-/-yc-/- mice. Seven weeks after transplantation, an anti-CD52 treatment was performed by application of Alemtuzumab (Campath, Sanofi) for 4 rounds of 100 pg/kg (i.v.) once per week. Tissue infiltration of human CD45 ⁺ cells and the amount of human CD52 ⁺ cells of treated vs untreated mice were flow cytometrically quantified using FACS. To evaluate the clinical impact of the anti-CD52 treatment, total bone marrow of treated and untreated mice was applied to secondary transplantations and the overall survival of the secondary recipients was monitored. Table 2 contains the antibodies used to analyze the PDX mice.

Table 2: Antibodies used to quantify human hematopoietic cells and CD52 ⁺ cells in PDX mice.

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

Hematopoietic stem and progenitor cells (HSPCs) were defined as Lin CD34 ⁺ CD38‘ cells and flow cytometrically enriched from each of the eight patients. These HSPCs were analysed using droplet-based single-cell RNA-sequencing (scRNA-seq). In total, 13,594 hematopoietic stem cells (HSCs) were identified by use of a reference-based cell type annotation. These JMML HSCs were used to call differentially expressed genes (DEGs) across risk-groups as defined above (high-risk vs low-risk). Of this transcriptome-wide list of DEGs, genes that encode for putative cell surface markers were selected. To determine genes aberrantly upregulated in JMML, average gene expression values of JMML HSCs were compared to 1,069 HSCs isolated from human umbilical cord blood. Figure 2 A summarizes the average expression values of 41 determined surface marker genes, which show (1) risk-group-specific differential expression patterns and (2) disease-specific overexpression. These genes represent both prognostic biomarkers and therapeutic targets of JMML HSCs. Figure 2 B exemplifies corresponding protein expression for the high risk JMML biomarkers CD52, CD69 and CD 164.

To functionally evaluate the role of those surface marker genes and to assess their therapeutic potential, an established preclinical patient-derived xenograft (PDX) mouse model of JMML was used to apply alemtuzumab, which is a monoclonal therapeutic anti-CD52 antibody. Figure 3 A demonstrates that the anti-CD52 treatment was able to specifically deplete human CD52 ⁺ cells relative to control mice without Alemtuzumab administration. This treatment led to the global depletion of human CD45 ⁺ cells in treated mice, whereas the number of engrafted leukemia cells in control mice were distinctly higher (Figure 3 B).

To further assess the therapeutic potential of an anti-CD52 targeted treatment, alemtuzumab- treated and -untreated JMML PDX mice were analysed applying flow cytometry. FACS confirmed not only an efficient depletion of human CD52 ⁺ but also CD52' cells including human CD34 ⁺ CD38‘ cells as well as mature hematopoietic cells across all hematopoietic lineages (Figure 4). Thus, alemtuzumab-treatment targeted human HSPCs, resulting in the depletion of virtually the entire hematopoietic system and, which demonstrated that JMML HSPCs contain therapeutically vulnerable disease-propagating cells.

Secondary transplantation of total bone marrow from alemtuzumab-treated and control animals confirmed that anti-CD52 treatment obstructed leukemic engraftment in 2° recipients. FACS analysis revealed a significantly reduced number of engrafted human (leukemic) cells in blood, bone marrow, spleen, liver, and lung (Figure 5 A). Moreover, leukaemia-free survival of mice receiving bone marrow from primarily treated mice was strongly improved relative to mice receiving bone marrow from untreated PDX mice in a secondary transplantation experiment (Figure 5 B). This result does not only confirm the functional relevance of such surface markers. Additionally, it exemplifies the therapeutic potential of the here identified biomarkers. In conclusion, targeting of CD52 leads to efficient depletion of leukemia-propagating stem cells in vivo, which provides a pre-clinical rationale to further assess anti-CD52 treatment for JMML patients. Furthermore, this example demonstrates the power of such a molecular high-precision approach.

In summary, the inventors identified novel intra- and extracellular biomarkers for quick, reliable, and economic risk stratification using FACS or expression analyses. Moreover, the functional relevance and the therapeutic potential of such surface markers was demonstrated by an anti-CD52 treatment of patient-derived xenografts. Hence, CD52 constitutes a novel therapeutic target, as well as a prognostic and predictive biomarker of high-risk JMML. In conclusion, the inventors identified the first drug target specific for high-risk JMML.

Example 6: Methylation patterns of patients exhibiting prognostic biomarker expression is conserved in a large patient cohort

To assess the conservation of molecular programs in JMML stem cells, the inventors analysed for the first time the entire methylomes of JMML stem cells applying ultra-low input wholegenome bisulfite sequencing (WGBS) on flow cytometrically enriched JMML HSPCs. The inventors modified a single-cell bisulfite protocol (Clark et al. 2017 Nature Protocols) to analyse a maximum of 100 highly purified JMML HSPCs applying the above-mentioned sorting strategy.

DNA methylomes of JMML stem cells revealed the conservation of JMML epigenotypes as determined via bulk DNA methylation array analysis in Figure 1 (Figure 6 A). Integrating ultralow input WGBS and DNA methylation array data of overall 147 JMML patients revealed the conservation of disease-specific DNA methylation signatures in bulk JMML samples, indicating the transmission of epigenotypes from immature to mature JMML cells (Figure 6 B). To evaluate JMML stem cells as the origin of disease-specific epigenotypes, the inventors demonstrated that JMML stem cell-specific DNA methylation signatures are able to recapitulate the epigenotypes of overall 331 JMML patients (Figure 6 C).

Moreover, the inventors could demonstrate by integration of ultra-low WGBS and scRNA-seq data of JMML stem cells that DNA methylation changes are associated with the expression of JMML stem cell surface markers such as CD52 (Figure 7). In conclusion, the conservation of disease-specific aberrations in more than 300 patients confirms the functional value of JMML stem cell signatures as relevant prognostic biomarkers as well as therapeutic targets in JMML. Cited literature

Arber DA, Orazi A, Hasseijian RP et al. Blood 2022 Sep 15; 140(11): 1200-1228

Orazi A et al., Chapter 5: Myelodysplastic/myeloproliferative neoplasms, Swerdlow SH, editor. WHO classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th edn. Lyon, France: International Agency for Research on Cancer; 2017

Schonung M, Meyer J, Nollke P et al. “International Consensus Definition of DNA Methylation Subgroups in Juvenile Myelomonocytic Leukemia”; Clin Cancer Res. 2021 Jan 1; 27(1): 158- 168

Lipka DB, Witte T, Toth R etal. “RAS-pathway mutation patterns define epigenetic subclasses in juvenile myelomonocytic leukemia”. Nat Commun 8, 2126 (2017)

Krombholz CF, Gallego- Villar L, Sahoo SS et al. “Azacitidine is effective for targeting leukemia-initiating cells in juvenile myelomonocytic leukemia.” Leukemia; 2019

Jul;33(7): 1805-1810

Louka E, Povinelli B, Rodriguez-Meira A et al. “Heterogeneous disease-propagating stem cells in juvenile myelomonocytic leukemia”; J Exp Med. 2021 Feb l;218(2):e20180853

DeVos N., Hofmans, M., Lammens, T., DeWilde, B., VanRoy N., DeMoerloose, B. "Targeted therapy in juvenilemyelomonocytic leukemia: Where arewe now?” Pediatr Blood Cancer. 2022; e29930.

Mayerhofer, C., Niemeyer, C.M., Flotho, C. “Current Treatment of Juvenile Myelomonocytic Leukemia” J. Clin. Med. 2021, 10, 3084.

Laetsch TW., DuBois, SG., Glade Bender, J., Macy, M.E., Moreno, L. “Opportunities and Challenges in Drug Development for Pediatric Cancers” Cancer Discov (2021) 11 (3): 545- 559.

Niemeyer CM and Flotho C. “Juvenile myleomonocytic leukemia: who’s the driver at the wheel?”; Blood 2019 133(10): 1060-1070

Niemeyer CM, Arico M, Basso G et al. “Chronic myelomonocytic leukemia in childhood: a retrospective analysis of 110 cases”; Blood 1997 May 15;89(10):3534-43 Locatelli F, Ndllke P, Zecca M, et al. “Hematopoietic stem cell transplantation (HSCT) in children with juvenile myelomonocytic leukemia (JMML): results of the EWOG-MDS/EBMT trial”. Blood 2005 Jan 1; 105(l):410-9

Neubauer A, Shannon K, Liu E. “Mutations of the ras proto-oncogenes in childhood monosomy 7”. Blood. 1991;77(3):594-598.

Flotho C, Valcamonica S, Mach-Pascual S et al. “RAS mutations and clonality analysis in children with juvenile myelomonocytic leukemia (JMML)”. Leukemia 1999 Jan;13(l):32-7

Bresolin S, Zecca M, Flotho C et al. “Gene expression-based classification as an independent predictor of clinical outcome in juvenile myelomonocytic leukemia”. J Clin Oncol. 2010 Apr 10;28(l l):1919-27

Helsmoortel HH, Bresolin S, Lammens T et al. “LIN28B overexpression defines a novel fetal- like subgroup of juvenile myelomonocytic leukemia”. Blood 2016; 127(9): 1663-1172

Olk-Batz C, Poetsch AR, Nollke P et al. „Aberrant DNA methylation characterizes juvenile myelomonocytic leukemia with poor outcome”; Blood 2011 May 5;117(18):4871 -80

Murakami N, Okuno Y, Yoshida K et al. “Integrated molecular profiling of juvenile myelomonocytic leukemia”; Blood 2018 Apr 5; 131 (14): 1576- 1586

Stieglitz E, Mazor T, Olshen AB et al. „Genome-wide DNA methylation is predictive of outcome in juvenile myelomonocytic leukemia”; Nat Commun 8, 2127 (2017)

Loh ML, “Recent advances in the pathogenesis and treatment of juvenile myelomonocytic leukaemia”; British Journal of Haematology, vol. 152, issue 6, p. 677-687

Lapidot T, Grunberger T, Vormoor J, et al. ’’Identification of human juvenile chronic myelogenous leukemia stem cells capable of initiating the disease in primary and secondary SCID mice”; Blood. 1996;88(7):2655-2664.

Iversen PO, Lewis ID, Turczynowicz S et al. “Inhibition of granulocyte-macrophage colonystimulating factor prevents dissemination and induces remission of juvenile myelomonocytic leukemia in engrafted immunodeficient mice”; Blood 1997; 90:4910-7 Krombholz CF, Aumann K, Kollek M et al. “Long-term serial xenotransplantation of juvenile myelomonocytic leukemia recapitulates human disease in Rag2-/-yc-/- mice”; Haematologica 2016 May, vol. 101, no. 5

Velten L, Haas SF, Raffel S, et al. “Human haematopoietic stem cell lineage commitment is a continuous process”; Nature Cell Biology 2017 Apr;19(4):271-281

Hay SB, Ferchen K, Chetal K et al. “The Human Cell Atlas bone marrow single-cell interactive web portal”; Exp Hematol. 2018 Dec;68:51-61

Waas M, Snarrenberg ST, Littrell J et al. “SurfaceGenie: a web-based application for prioritizing cell-type-specific marker candidates”; Bioinformatics. 2020 Jun 1 ;36(11):3447- 3456

Orazi et al. 2016 WHO, Chapter 5, Myelodysplastic/ myeloproliferative neoplasms

Clark SJ, Smallwood SA, Lee HJ, Krueger F, Reik W, Kelsey G, 2017, “Genome-wide baseresolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq)”, Nature Protocols 2017, 12, 534-547