WO2025088065A1

WO2025088065A1 - Diagnosis and therapy of aml

Info

Publication number: WO2025088065A1
Application number: PCT/EP2024/080130
Authority: WO
Inventors: Mark Hartmann; Daniel LIPKA; Mariam HAKOBYAN; Maximilian SCHÖNUNG; Maximilian MÖNNIG; Simon RAFFEL
Original assignee: Deutsches Krebsforschungszentrum DKFZ; Universitaet Heidelberg; Universitaetsklinikum Heidelberg
Current assignee: Deutsches Krebsforschungszentrum DKFZ; Universitaet Heidelberg; Universitaetsklinikum Heidelberg
Priority date: 2023-10-25
Filing date: 2024-10-24
Publication date: 2025-05-01
Anticipated expiration: 2026-04-25

Abstract

The present invention relates to the field of diagnostics and therapeutics against leukemia. More specifically, it relates to a method for assessing acute myeloid leukemia (AML) in a subject suspected to suffer therefrom comprising the steps of determining in a sample of said subject the amount of at least one biomarker selected from the group consisting of: CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RAB11A, SELENOK, HLA- DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMPS, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and J AML,, comparing the said amount of the at least one biomarker to a reference, and assessing AML based on the comparison. Yet, the present invention relates to a method for generating a bispecific binding agent and to the use of such bispecific binding agents for treating leukemia, preferably, AML.

Description

Diagnosis and therapy of AML

The present invention relates to the field of diagnostics and therapeutics against leukemia. More specifically, it relates to a method for assessing acute myeloid leukemia (AML) in a subject suspected to suffer therefrom comprising the steps of determining in a sample of said subject the amount of at least one biomarker selected from the group consisting of CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RAB11A, SELENOK, HLA- DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and JAML, , comparing the said amount of the at least one biomarker to a reference, and assessing AML based on the comparison. Yet, the present invention relates to a method for generating a bispecific binding agent and to the use of such bispecific binding agents for treating leukemia, preferably, AML.

Human leukemias are usually characterized by the accumulation of clonal cells infiltrating the bone marrow, blood, and other tissues, such as lymph nodes, liver or spleen. Leukemias can be subdivided into acute and chronic myeloid or lymphatic leukemia, depending on the transformed cell of origin (Dohner et al., 2015; Goldman et al., 2003; Pui et al., 2004). Acute myeloid leukemia (AML) is the most common acute leukemia in adults (Siegel et al., 2017). In the United States and Europe, the incidence is approximately 3 to 5 cases per 100,000 population and the median age at diagnosis is approximately 65 years (Dores et al., 2012; Sant et al., 2010). The incidence increases with age and reaches up to 20 cases per 100,000 population for those over 65 years. Worldwide, AML accounts for about 20,000 new cases per year, leading to more than 10,000 AML-related deaths (American Cancer Society). AMLs are classified according to WHO (Khoury et al., 2022) or ICC (Arber et al., 2022) and these classifications are based on cytologic and genetic features. While chemotherapy can induce a remission in the majority of AML cases, the probability of relapse is high, leading to poor longterm clinical outcomes with 5-year overall survival rates of 35 to 40% for patients <60 years and 5 to 15% for patients >60 years of age, respectively. Currently, clinical outcome can only be poorly predicted and personalized treatment options are often lacking, despite a highly granular classification and broad understanding of the underlying genetic and non-genetic mechanisms. This insufficiency emphasizes the necessity for suitable molecular approaches, which take the clinical heterogeneity into account, to identify novel biomarkers and therapeutic targets.

The strategy to identify pan-cancer factors, which might enable targeting of central tumorigenic mechanisms shared across entities, failed in a plethora of cases due to resistance mechanisms as well as cellular and molecular heterogeneity across patients. A widely accepted approach to tackle this problem is to define disease subtypes and to identify corresponding biomarkers. Ideally, such factors function as predictive biomarkers for personalized therapies to avoid ineffective treatments. However, personalized oncology strategies require elaborated high- precision analyses and streamlined molecular tumor boards, which are mostly not feasible outside of major cancer centers. In conclusion, there is a high clinical need for straightforward diagnostics and broadly applicable therapeutic strategies to treat leukemia.

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

Thus, the present invention relates to a method for assessing acute myeloid leukemia (AML) in a subject suspected to suffer therefrom comprising the steps of a) determining in a sample of said subject the amount of at least one biomarker selected from the group consisting of CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RABI 1 A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and JAML; b) comparing the said amount of the at least one biomarker to a reference; and c) assessing AML based on the comparison.

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” are used in conjunction with additional features, without restricting alternative possibilities. Thus, features introduced by these terms are additional 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 “assessing” as used herein assessing comprises (i) diagnosing AML, (ii) determining whether the subject is at risk of worsening of AML or symptoms associated therewith or (iii) determining whether the subject is at risk of death. Diagnosing as used herein also comprises monitoring and/or staging AML. Diagnosing as used herein refers to determining whether a subject suffers from AML, or not. Determining whether the subject is at risk of worsening of AML or symptoms associated therewith comprises predicting whether AML or symptoms associated therewith will worsen within a predictive window in the future starting from the time point onwards when the sample investigated by the method of the invention has been taken. Similar, determining whether the subject is at risk of death comprises predicting the risk of death within a predictive time window in the future starting from the time point onwards when the sample investigated by the method of the invention has been taken. Preferably, the predictive time window in accordance with the present invention is at least about 3 months, at least about 6 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years. The most common signs and symptoms of AML include anemia, paleness, fatigue, shortness of breath, fever, loss of weight, loss of appetite, bleeding, and/or bruising, respectively.

As will be understood by those skilled in the art, such assessing, 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 assessed. 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 assessing 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 assessment, such as presence of symptoms of AML 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, an adult human, preferably, having an age of at least 17 years, at least 18 years, at least 19 years or at least 20 years, or a juvenile human having an age of less than 17 years, preferably, 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 AML. Suffering from AML as used herein, means that the subject shall exhibit clinical parameters, signs and/or symptoms of AML. Thus, the subject according to the invention is, typically, a subject that suffers from AML or is suspected to suffer from AML. Having a risk of developing AML as used herein, refers to an apparently healthy subject which does not yet show clinical signs or symptoms of AML but which has an increased risk of developing AML 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. Typical risk factors for AML include existing other blood disorders, exposure to toxic chemicals, such as chloramphenicol, benzene or phenylbutazene, exposure to ionizing radiation, and/or presence of genetic risk factors, such as Down syndrome, Fanconi anemia, Bloom syndrome, ataxia-telangiectasia or Kostmann syndrome. A secondary AML may also arise as a consequence of previous treatments such as chemotherapy or radiotherapy. Thus, patients subjected to such therapies may also be at risk of developing AML.

The term “acute myeloid leukemia (AML)” as used herein refers to a cancer of the myeloid cell lineage. It is characterized by rapid and abnormal growth of myeloid stem cells or myeloid blasts in the bone marrow. AML is a heterogeneous cancer since it may develop from different myeloid cells at different stages within their differentiation process towards mature white blood cells such as eosinophils, basophiles, neutrophils or monocytes or towards erythrocytes or platelets.

Typically, AML can be diagnosed by abnormal white blood cell count, i.e. leukocytosis or leukopenia as well as anemia and/or thrombocytopenia. Auer rods present in a sample further strengthen the AML diagnosis. In addition to blood cell analysis, AML is typically diagnosed by an analysis of bone marrow samples using microscopy and/or flow cytometry. Bone marrow or blood samples may also be analyzed for chromosomal abnormalities by cytogenetics or fluorescent in situ hybridization, e.g., to look for specific mutations in genes such as FLT3, nucleophosmin, and KIT, which may influence the outcome of the disease. Also, next generation sequencing panels may be used for AML diagnosis and risk stratification. Cytochemical stains on blood and bone marrow smears are helpful in the distinction of AML from acute lymphoblastic leukemia and in sub-classification of AML. A combination of a myeloperoxidase or Sudan black stain and a nonspecific esterase stain typically provides the desired diagnosis.

The standard classification scheme for AML is the World Health Organization (WHO) system or the ICC system. According to the WHO criteria, the diagnosis of AML is established by demonstrating involvement of >20% of the blood and/or bone marrow by leukemic myeloblasts, except in three forms of acute myeloid leukemia with recurrent genetic abnormalities: t(8;21), inv(16) or t(16;16), and acute promyelocytic leukemia with PML- RARA, in which the presence of the genetic abnormality is diagnostic irrespective of blast percentage. Myeloid sarcoma is also considered a subtype of AML independently of the blast count. According to the 2022 classification of the WHO, six types of AML are recognized: Acute myeloid leukemia with recurrent genetic abnormalities, AML with myelodysplasia- related changes, Therapy-related myeloid neoplasms, Myeloid sarcoma, Myeloid proliferations related to Down syndrome, and AML not otherwise categorized. Details may be found in, e.g., Khoury et al. 2022.

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 AML, the absence of AML, being at risk of developing AML or not being at risk of developing AML. 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 as an example for a biomarker according to the invention 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 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 “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 Rai 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 1 A” 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 “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 “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 hematopoiesis 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 “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 SLC5 A3 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 “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 NINJ 1 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 P10620. 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 “HCST” refers to the Hematopietic Cell Signal Transducer, a transmembrane signaling adaptor that contains a YxxM motif in its cytoplasmic domain. HCST protein may form part of the immune recognition receptor complex with the C-type lectin-like receptor NKG2D. As part of this receptor complex, it may activate phosphatidylinositol 3-kinase dependent signaling through its intracytoplasmic YxxM motif. The immune recognition complex containing HCST may have a role in cell survival and proliferation by activation of NK and T cell responses. Alternative splicing results in two transcript variants encoding different isoforms.

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 “CD47” refers to Cluster of Differentiation 47 which is also known as integrin associated protein (IAP). It is a transmembrane protein and belongs to the immunoglobulin superfamily. It binds to membrane integrins and its ligands thrombospondin- 1 (TSP-1) and signal-regulatory protein alpha (SIRPa). CD47 prevents phagocytosis by macrophages. CD47 is involved in a range of cellular processes, including apoptosis, proliferation, adhesion, and migration. Furthermore, it plays a key role in immune and angiogenic responses. CD47 is ubiquitously expressed in human cells and has been found to be overexpressed in many different tumor cells.

The CD47 protein referred to in accordance with the present invention is, preferably, human CD47 having an amino acid sequence as deposited under UniProt accession number Q08722. It will be understood that the term “CD47” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD47 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 CD47 protein, preferably over the entire length of the said CD47 proteins, respectively.

The term “SELENOK” refers to selenoprotein K and belongs to the selenoprotein K protein family. 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 Ca²⁺ flux in immune cells and mounting effective immune response. This protein is a selenoprotein, containing the rare amino acid selenocysteine. The SELENOK protein referred to in accordance with the present invention is, preferably, human SELENOK having an amino acid sequence as deposited under UniProt accession number Q9Y6D0. It will be understood that the term “SELENOK” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned SELENOK 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 SELENOK protein, preferably over the entire length of the said SELENOK proteins, respectively.

The term “CD96” refers to Cluster of Differentiation 96 also called Tactile (T cell activation, increased late expression). It is a receptor protein which is expressed on T cells and NK cells. The protein belongs to the immunoglobulin superfamily. It is a type I membrane protein. The protein may play a role in the adhesion of activated T and NK cells to their target cells during the late phase of the immune response. Alternative splicing occurs at this locus and two transcript variants encoding distinct isoforms have been identified. CD96 is a transmembrane glycoprotein that has three extracellular immunoglobulin-like domains and is expressed by all resting human and mouse NK cells. CD96 main ligand is CD155. CD 96 has approximately 20% homology with CD226 and competed for binding to CD155 with CD226.

The CD96 protein referred to in accordance with the present invention is, preferably, human CD96 having an amino acid sequence as deposited under UniProt accession number Q8WUE2 or P40200. It will be understood that the term “CD96” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD96 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 CD96 protein, preferably over the entire length of the said CD96 proteins, respectively.

The term “NDFIP1” refers to NEDD4 family-interacting protein 1. It is known to activate ELECT domain-containing E3 ubiquitin-protein ligases, including NEDD4 and ITCH, and thereby to modulate stability of their targets. The protein is, thus, involved in the control of many cellular processes.

The NDFIP1 protein referred to in accordance with the present invention is, preferably, human NDFIPlhaving an amino acid sequence as deposited under UniProt accession number Q9BT67. 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 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. Moreover, it will be understood that when referring to the determination of at least one biomarker from the aforementioned group of biomarkers, the present invention envisages determining one or more of the said biomarkers and even the determination of all of said biomarkers.

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 analyzing devices such as FACS analyzers or devices for PCR analysis, such as devices for single cell PCR, qPCR or bulk PCR, or sequencing devices. Preferably, the at least one biomarker in accordance with the method of the present invention is determined by flow cytometry, quantitative PCR (qPCR) or transcriptome sequencing, preferably, bulk RNA- seq or scRNA-seq.

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 at least one biomarker to be determined in accordance with the present invention may be determined as protein. To this end, typically a binding agent is applied that specifically binds to the said biomarker protein and that can be detected either by a detectable label present in the binding agent itself or by a secondary binding molecule that specifically binds to the binding agent and comprises a detectable label.

A “binding agent” refers in this context to any molecule that is capable of specifically binding to the biomarker to be detected. The binding agent is selected based on the type of analysis to be conducted. Binding agents 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 agent 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.

An “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 10th fibronectin type III (10Fn3) 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 a biomarker as specified elsewhere herein.

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 acidocaldarius; lipocalin and anticalins derived from lipocalins; DARPins (designed ankyrin repeat domains); SH3 domain of Fyn; Kunits domain of protease inhibitors; monobodies, e.g. the 10th 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.

A “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.

A “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.

A “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 proteinlike 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, sidechain 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.

Typically, a binding agent to be used in accordance with the present invention for determining at least one biomarker shall comprise a detectable label. 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 enzymesubstrate 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 ultraviolet. 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 agent, 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 bulk RNA sequencing, preferably, transcriptome sequencing, and, more preferably single-cell sequencing (scRNA- seq) including technologies such as next single-multiome assays. 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, transcriptomic, or other multi-omics information. Thus, “transcriptome sequencing” as used herein refers to a method to analyze 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. Platebased 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.

The term “sample” refers to any sample obtainable from the subject to be investigated that contains myeloid cell linage cells. 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. Preferably, the body fluid sample is a blood sample or a liquor 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 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 samples, such as bone marrow samples, may be obtained by, e.g., biopsy. Separated cells may be obtained from the body fluids or the 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.

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). 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 true-positive test results to the product of number of truepositive 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.

In a preferred embodiment of the method of the present invention, the reference is derived from at least one subject known to suffer from AML. More preferably, the amount for the at least one biomarker determined in step a) which is identical or increased compared to the reference is indicative for a subject suffering from AML whereas an amount for the at least one biomarker determined in step a) which is reduced compared to the reference is indicative for a subject not suffering from AML.

In yet a preferred embodiment of the method of the present invention, the reference is derived from at least one subject known not to suffer from AML. More preferably, an amount for the at least one biomarker determined in step a) which is identical or reduced compared to the reference is indicative for a subject not suffering from AML whereas an amount for the at least one biomarker determined in step a) which is increased compared to the reference is indicative for a subject suffering from AML.

In another preferred embodiment, the at least one biomarker to be determined in accordance with the method of the present invention is a combination of biomarkers consisting of: SLC2A5, IFITM3, LST1, CALCRL, CD52, and MGST1.

In a further preferred embodiment, the at least one biomarker to be determined in accordance with the method of the present invention is a combination of biomarkers consisting of: CLECL7A, CLEC9A, HCST, LST1, LTB, and IFITM3.

Moreover, a TNF may be preferably be determined together with the aforementioned group of biomarkers as a further biomarker.

TNF as referred to herein relates to Tumor Necrosis Factor. TNF is a cytokine. It is mainly secreted by macrophages and plays a role in various local as well as systemic inflammatory processes. TNF can stimulate cell proliferation and induce cell differentiation but also apoptosis.

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 Q5STB3. 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. The aforementioned combination of biomarkers, preferably, together with TNF, can be used in accordance with the present invention to establish a score as described in the accompanying Examples, below, and in Figure 1. The score has been shown to correlate with the risk for mortality in the future. Thus, the said score can be, preferably used for determining whether the subject is at risk of death when assessing AML according to the method of the present invention.

Advantageously, it has been found in the studies underlying the present invention that the biomarkers CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RABI 1 A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and J AML can be used for assessing AML as specified elsewhere herein. This includes diagnosis of AML as well as risk stratification of AML subjects. Moreover, the said biomarkers may also be used as therapeutic targets for therapeutic agents either binding to two or more of said targets individually, i.e. as single target drugs, or together, i.e. as a multi- and, in particular, dual-target binding drug, such as bispecific antibodies specified elsewhere herein.

In the studies underlying the present invention, two approaches were combined to identify key risk factors across entities in a stepwise manner:

First, a multi-modal cellular and molecular analysis was used to identify prognostic biomarkers across subtypes in juvenile myelomonocytic leukemia (JMML), which is a rare myeloproliferative neoplasm (MPN) of early childhood. A transcriptome- and DNA methylome-wide sequencing with flow cytometry were combined to determine JMML stem cell-specific cell surface markers.

Second, to assess the clinical relevance of JMML stem cell biomarkers across entities, those genes bearing prognostic value in AML were tested. Furthermore, a prognostic JMML stem cell score was developed to predict risk in AML, which represents the first JMML-based AML classification and is therefore exemplary for the existence of shared prognostic factors across leukemias.

Following the assessment of JMML stem cell surface markers as prognostic biomarkers in AML, gene pairs were selected based on their suitability as bispecific therapeutic targets. For this purpose, the following steps were applied:

(1) Relevant expression in immature AML cells as the target population for leukemia therapy.

(2) Co-expression of gene pairs in immature AML cells on a single-cell level.

(3) The absence of co-expression of the very same gene pairs in healthy cells of AML patients and/or of healthy donors to minimize the risk of potential therapeutic side effects. 39 of these genes were expressed in at least one of the tested AML datasets. 10 of these genes were significant prognostic biomarkers in AML. And 7 of these genes were integrated into a JMML stem cell score to predict outcome in AML. Overall, 44 cell surface markers were subtype-specifically expressed in JMML and hence identified as prognostic biomarkers. Of those, 39 genes were expressed at relevant frequencies in immature AML pairs, and 633 gene pairs were significantly co-expressed in immature AML cells in at least one AML dataset tested.

The high success rate in identifying high confident candidates for therapeutic purposes is based on the multi-modal molecular characterization of cancer stem cells, which integrates different layers of malignant gene regulation programs. On the one hand, by taking the large degree of heterogeneity into account, this strategy reduces the probability of identifying false positive candidates. On the other hand, the comparative candidate selection across entities increases the chance of identifying true positive candidates for central tumorigenic mechanisms. The proof- of-concept was provided by pre-clinical evaluation of anti-CD52 treatment as a high-risk stem cell biomarker in JMML.

The identification of cross-entity prognostic biomarkers enables quick and economic, but at the same time accurate risk stratification in AML without the requirement for expensive omics- based analysis pipelines. Additionally, the biomarkers identified in the studies underlying the present serve as potential novel drug targets for bispecific therapies. Thanks to the present invention, AML assessment can greatly benefit by a reliable, biomarker-based diagnosis and risk stratification. Moreover, more specific therapeutic approaches towards AML treatment have become feasible.

The definitions and explanations of the terms made above apply mutatis mutandis to the following embodiments except if specified otherwise.

The present invention relates to a device adopted for carrying out the method of the present invention said device comprising:

(a) an analysing unit comprising a binding agent for at least one biomarker selected from the group consisting of CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA- DRA, CD 164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RAB11A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA- DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and JAML, wherein binding of the binding agent to the at least one biomarker when present in a sample of a subject can be detected; and (b) an evaluation unit comprising a processor and a database comprising a stored reference for the at least one biomarker, wherein said evaluation unit is adapted for providing the assessment of AML.

The term “device” as used herein refers to a system comprising the aforementioned units operatively linked to each other as to allow the determination of the presence, absence or abundance of biomarkers and evaluation thereof according to the method of the invention such that an assessment can be provided.

The analyzing unit, typically, comprises at least one detection element being capable of detecting the biomarkers present in the sample. This is typically achieved by specific binding of the binding agent to the at least one biomarker and subsequent detection by the detector element of the complex formed. Prior to introducing the sample into the detection element, the sample may be pre-treated by detection molecules in order to generate detectable signals, e.g., by allowing the formation of biomarker-binding agent complexes whereby the binding agent may comprises a detectable label that can be detected by the detection element. The detection element may also comprise a reaction zone that allows carrying out a chemical detection reaction such as a PCR. The detection element shall be adapted to determine the amount of the biomarkers. The determined amount can be subsequently transmitted to the evaluation unit.

The evaluation unit comprises a data processing element, such as a computer, with an implemented algorithm for determining the amount of biomarkers present in the sample. The processing unit as referred to in accordance with the method of the present invention, typically, comprises a Central Processing Unit (CPU) and/or one or more Graphics Processing Units (GPUs) and/or one or more Application Specific Integrated Circuits (ASICs) and/or one or more Tensor Processing Units (TPUs) and/or one or more field-programmable gate arrays (FPGAs) or the like. A data processing element may be a general purpose computer or a portable computing device, for example. It should also be understood that multiple computing devices may be used together, such as over a network or other methods of transferring data, for performing one or more steps of the methods disclosed herein. Exemplary computing devices include desktop computers, laptop computers, personal data assistants (“PDA”), cellular devices, smart or mobile devices, tablet computers, servers, and the like. In general, a data processing element comprises a processor capable of executing a plurality of instructions (such as a program of software). The evaluation unit, typically, comprises or has access to a memory. A memory is a computer readable medium and may comprise a single storage device or multiple storage devices, located either locally with the computing device or accessible to the computing device across a network, for example. Computer-readable media may be any available media that can be accessed by the computing device and includes both volatile and non-volatile media. Further, computer readable-media may be one or both of removable and non-removable media. By way of example, and not limitation, computer-readable media may comprise computer storage media. Exemplary computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or any other memory technology, CD-ROM, Digital Versatile Disk (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used for storing a plurality of instructions capable of being accessed by the computing device and executed by the processor of the computing device. The evaluation unit may also comprise or has access to an output device. Exemplary output devices include fax machines, displays, printers, and files, for example. According to some embodiments of the present disclosure, a computing device may perform one or more steps of a method disclosed herein, and thereafter provide an output, via an output device, relating to a result, indication, ratio or other factor of the method.

The present invention relates to a kit for assessing AML in a subject comprising at least one binding agent and instructions to carry out the method of the present invention, wherein the at least one binding agent is capable of specifically detecting a biomarker selected from the group consisting of CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RABI 1 A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and J AML.

It will be understood that the binding 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 binding 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. Details on binding agents are to be found elsewhere herein.

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. 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.

The present invention also relates, in general, to at least one biomarker or a binding agent for said at least one biomarker for use in assessing AML in a subject, wherein said at least one biomarker is selected from the group consisting of CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RAB11A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and JAML.

The present invention also relates, in general, to the use of at least one biomarker or a binding agent for the said at least one biomarker for assessing AML in a sample of a subject, wherein said at least one biomarker is selected from the group consisting of CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, HCST, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, LST1, NDFIP1, IFITM3, RALA, RAB11A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, LTB, CD7, SLC2A5, EREG, FCMR, DLK1, CLEC7A, JAML, and CLEC9A.

The present invention also relates to a method for generating a bispecific binding agent comprising the steps of: a) determining the abundance of a plurality of biomarkers present in each single cell of (i) leukemic cells, preferably, AML cells, and control cells and (ii) leukemic stem cells, preferably, AML stem cells, and control cells; b) identifying among the plurality of biomarkers those which are present above a pre-determined threshold on (i) leukemic cells, preferably, AML cells, but not control cells and (ii) leukemic stem cells, preferably, AML stem cells, but not control cells; c) identifying among the biomarkers present above the pre-defined threshold biomarker pairs which are present in one single cell; and d) generating a bispecific binding agent comprising a first target binding domain that specifically binds to a first target and a second target binding domain that specifically binds to a second target, wherein the first and the second target are biomarker pairs identified in step c) and are different from each other. In step a) of the aforementioned method of the invention, the abundance of a plurality of biomarkers present in each single cell of leukemic cells, preferably, AML cells, and control cells, i.e. non leukemic cells is determined by suitable techniques such as FACS analyses or real time PCR based techniques. Moreover, the abundance of a plurality of biomarkers present in each single cell of leukemic stem cells, preferably, AML stem cells, and control cells, i.e. non-leukemic stem cells, is determined as well. Further details are also found in the accompanying Examples, below.

In step b), among the plurality of biomarkers those are identified which are present above a predetermined threshold. Typically, the said threshold represents the upper limit of the physiological abundance for the biomarkers. It will be understood that there might be an individual threshold for each of the biomarkers. The skilled artisan is, however, well aware of how to determine suitable thresholds. Further details are also found in the accompanying Examples, below. It will be understood that a biomarker will be identified if it is present on a leukemic cell or leukemic stem cell at a level above the threshold but is present on the respective control cell below the said threshold. As a result of step b), a list of biomarkers is provided containing those biomarkers out of the plurality of biomarkers which are present on individual leukemic cells or leukemic stem cells above the pre-defined threshold while being present on respective control cells below the said threshold.

In step c), biomarker pairs from said list of biomarkers are identified which are present on one single cell together.

Such bispecific binding agents are generated in step d) of the aforementioned method of the invention by conventional techniques well known to the skilled artisan based on the design provided by the method, i.e. the envisaged bispecific binding agent shall comprise a first target binding domain that specifically binds to a first target and a second target binding domain that specifically binds to a second target, wherein the first and the second target are biomarker pairs identified in step c) and are different from each other.

The aforementioned method of the present invention can be assisted by automation, e.g., step a) may be carried out by using robotic devices for the measurements, steps b) to d) may be carried out computer-implemented.

The term “bispecific binding agent” as used herein refers to any binding agent that is capable of specifically binding to two targets. Typically, the said binding molecule two separate binding domains wherein each of said binding domains is capable of specifically binding to one of the targets. Suitable binding agents for the envisaged targets, i.e. the biomarkers referred to herein, have been described elsewhere herein in greater detail. Particular envisaged bispecific binding agents in accordance with the present invention are bispecific antibodies as defined elsewhere herein in more detail.

The bispecific binding agent according to the present invention is also preferably envisaged as therapeutically acting agent. The biomarkers referred to herein qualify as targets for anti-cancer therapy and, thus, for the design development of anti-cancer therapeutics based on bispecific binding agents that act in cis, i.e. that bind to two targets present on the same cell. It will be understood that the bispecific binding agents of the invention may act as agonists or antagonists when bound to their targets. Moreover, the bispecific binding agents may be further modified to attract immune cells toward the target cell when bound to the targets and, thus, to elicit an immune response, such as NK cell or cytotoxic T cells. Modifications which can be used in this context are described elsewhere herein for bispecific binding agents being bispecific antibodies. Moreover, the bispecific binding agents may be coupled to drugs.

The present invention, thus, also relates to a bispecific binding agent comprising a first target binding domain that specifically binds to a first target and a second target binding domain that specifically binds to a second target, wherein

(i) the binding agent binds to the first and second target when present on the same cancer cell;

(ii) the first target and the second target are different from each other; and

(iii) the first and the second target are selected from the group consisting of: CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RAB11A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA- DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and J AML.

Preferably, the first and second targets are pairs selected from any one of List A. l, List A.2, List B.3, List B.4, List C.5, List C.6, List D.7, or List D.8, below.

Preferably, said first target of the bispecific binding agent of the invention is CD52. CD52 is known to be an important target for AML therapy. However, by using the bispecific binding agents of the invention, preferably, acting in cis, therapeutic efficacy can be even increased by attacking a further target present on the same cancer cell. Moreover, undesired off-target effects can more efficiently prevented due to the higher specificity of the bispecific binding agent as such. Preferably, said bispecific binding agent is obtainable by the aforementioned method for generating a bispecific binding agent described elsewhere herein in detail.

Preferably, the bispecific binding agent is a bispecific antibody.

In principle, antibodies are particular well suited bispecific binding agents according to the invention. Antibodies, antibody like molecules, antibody fragments and the like have been defined in more detail elsewhere herein. It will be understood that all of these binding agents may be used for generating bispecific antibodies according to the invention. A bispecific antibody is, preferably, an antibody that binds one antigen or epitope with one of two or more binding domains, defined by a first pair of heavy and light chain or of main and shorter/smaller chain, and binds a different antigen or epitope on a second arm, defined by a second pair of heavy and light chain or of main and smaller chain. Such a bispecific antibody has two distinct antigen binding arms with separate specificities and CDR sequences. Typically, a bispecific antibody is monovalent for each antigen. However, bispecific antibodies can also be dimerized or multimerized. A bispecific antibody may be a hybrid antibody molecule, which may have a first binding domain that is defined by a first light chain variable region and a first heavy chain variable region, and a second binding domain that is defined by a second light chain variable region and a second heavy chain variable region.

Bispecific antibodies as referred to herein may, thus, have any natural or synthetic symmetric or non-symmetric (e.g., asymmetric) format. They may be whole antibody- or fragment-based. An overview of suitable formats for bispecific antibodies particularly envisaged in accordance with the present invention are disclosed in Labrijn, A.F., Janmaat, M.L., Reichert, J.M. et al. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov 18, 585-608 (2019). https://doi.org/10.1038/s41573-019-0028-l (in particular, Figure 2).

Methods of making a bispecific antibody molecule are known in the art, e.g. chemical conjugation of two different monoclonal antibodies or, for example, also chemical conjugation of two antibody fragments, for example, of two Fab fragments. Alternatively, bispecific antibody molecules are made by quadroma technology by fusion of the hybridomas producing the parental antibodies. Because of the random assortment of H and L chains, a potential mixture of ten different antibody structures are produced of which only one has the desired binding specificity. The bispecific antibody molecule is typically a monoclonal antibody with respect to each target. Preferably, the antibody is chimeric, humanized or fully human. A bispecific antibody molecule may, for example, be a bispecific tandem single chain Fv, a bispecific Fab2, or a bispecific diabody. Preferably, said bispecific antibody may contain further modifications that enhance or reduce immunological effects. Preferably, the bispecific antibody, thus, contains a modification that enhances or attenuates binding of its Fc domain to its receptor. Preferably, the SDIE mutation may be present in the Fc part of the bispecific antibody of the invention. SDIE mutations are known to mediate markedly enhanced affinity to Fc receptors as well as ADCC. Specifically, the SDIE mutation refers to an amino acid substitution comprising S239D and I332E, wherein the positional numbering is according to the EU index. Other enhanced effector functions for Fc domains are described in US2004013210 or US20060024298. Attenuating Fc receptor modifications are also known in the art. For example, N297G (NG) and D265A, N297G (DANG) and L234A, L235A, P329G (LALA-PG) mutations have been described as reducing FcR binding as well as other effector functions. Also preferably, said bispecific antibody comprises a modification that enhances or attenuates antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) or antibody-dependent cellular phagocytosis (ADCP). In particular, a bispecific antibody as binding agent of the present invention may be modified such that it has an enhanced antibody dependent cellular toxicity (ADCC) activity compared to an unmodified version. The ADCC activity can be measured by well-known assays, such as e.g. CellaTM-TOX assay, GAPDH release assay, which can be obtained from e.g. Promega or Interchim. Thus, the bispecific antibody of the present invention may have an increased ADCC activity when compared to the same but unmodified antibody.

The present invention also relates to a pharmaceutical composition comprising the bispecific binding agent of the invention.

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. 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.

Yet, the present invention relates to a bispecific binding agent according to the invention for use in treating and/or preventing cancer.

The term “cancer” as used herein refers to any disorder or disease caused by abnormal cell growth. Cancer may affect cells in tissues and organs as well as hematopoietic cells. The cancer cells in accordance with the present invention, typically, express at least two targets for the bispecific binding agent, i.e. at least two biomarkers selected from the group consisting of CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RABI 1 A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and JAML. Preferably, the first and second targets are pairs selected from any one of List A.l, List A.2, List B.3, List B.4, List C.5, List C.6, List D.7, or List D.8, below. Preferably, said cancer in accordance with the present invention is leukemia, preferably, myeloid or lymphoid leukemia and, more preferably, a myeloid leukemia. Myeloid leukemia encompasses AML, JMML, JMML-like neoplasms, and JMML associated with neurofibromatosis, Noonan syndrome-associated myeloproliferative disorder, CBL-syndrome, chronic myelogenous leukemia, chronic myelomonocytic leukemia, acute megakaryoblastic leukemia, acute erythroblastic leukemia blastic plasmacytoid dendritic cell neoplasm, hematological malignancies, myeloblast, atypical chronic myeloid leukemia, chronic myelomonocytic leukemia (CMML) or transient myeloproliferative disease. More preferably, said leukemia is acute myeloid leukemia (AML).

The term "treating” as used herein relates to ameliorating and/or curing cancer 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 cancer. 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 cancer 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 cancer. 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.

The present invention also provides for a method of treating and/or prevention AML comprising administering to a subject in need thereof a therapeutically effective amount at least one bispecific binding agent as defined elsewhere herein.

A “therapeutically effective amount” refers to an amount of the at least one bispecific binding agent of the invention which prevents, ameliorates or cures AML 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.

The present invention also contemplates combination therapies using two or more binding agents wherein each of said binding agent binds to a target referred to herein and wherein each of said binding agents binds to a different target. To this end, the two or more binding agents may be administered together as a single preparation or may be administers as separate preparations together or consecutively.

Accordingly, the present invention also relates to a pharmaceutical composition comprising at least two binding agents for use in treating cancer, wherein said each of said at least two binding agents binds to a different target selected from the group consisting of CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RAB11A, SELENOK, HLA- DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and J AML.

Furthermore, the present invention relates to at least two binding agents for use in treating cancer, wherein said each of said at least two binding agents binds to a different target selected from the group consisting of: CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA- DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RAB11A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and JAML.

Yet, the present invention relates to a method a method of treating and/or prevention AML comprising administering to a subject in need thereof a therapeutically effective amount at least two binding agents for use in treating cancer, wherein said each of said at least two binding agents binds to a different target selected from the group consisting of CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RAB11A, SELENOK, HLA- DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and J AML.

Preferably, the first and second targets are pairs selected from any one of List A.l, List A.2, List B.3, List B.4, List C.5, List C.6, List D.7, or List D.8, below.

The following are particular preferred embodiments of the present invention:

Embodiment 1. A method for assessing acute myeloid leukemia (AML) in a subject suspected to suffer therefrom comprising the steps of: a) determining in a sample of said subject the amount of at least one biomarker selected from the group consisting of: CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RABI 1 A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and JAML; b) comparing the said amount of at least one biomarker to a reference; and c) assessing AML based on the comparison.

Embodiment 2. The method of embodiment 1, wherein said assessing comprises (i) diagnosing AML, (ii) determining whether the subject is at risk of worsening of AML or symptoms associated therewith or (iii) determining whether the subject is at risk of death.

Embodiment 3: The method of embodiment 1 or 2, wherein the biological sample is a tissue sample or a body fluid sample.

Embodiment 4: The method of any one of embodiments 1 to 3, wherein the tissue sample is a connective tissue sample, preferably, bone marrow.

Embodiment 5: The method of any one of embodiments 1 to 3, wherein the body fluid sample is a blood sample.

Embodiment 6: The method of any one of embodiments 1 to 5, wherein the subject is a human.

Embodiment 7: The method of embodiment, wherein said human is an adult human, preferably, having an age of at least 17 years, at least 18 years, at least 19 years or at least 20 years, or a juvenile human having an age of less than 17 years. Embodiment 8: The method of any one of embodiment 1 to 7, wherein the reference is derived from at least one subject known to suffer from AML.

Embodiment 9: The method of embodiment 8, wherein the amount for the at least one biomarker determined in step a) which is identical or increased compared to the reference is indicative for a subject suffering from AML whereas an amount for the at least one biomarker determined in step a) which is reduced compared to the reference is indicative for a subject not suffering from AML.

Embodiment 10: The method of any one of embodiments 1 to 7, wherein the reference is derived from at least one subject known not to suffer from AML.

Embodiment 11 : The method of embodiment 10, wherein an amount for the at least one biomarker determined in step a) which is identical or reduced compared to the reference is indicative for a subject not suffering from AML whereas an amount for the at least one biomarker determined in step a) which is increased compared to the reference is indicative for a subject suffering from AML.

Embodiment 12: The method of any one of embodiments 1 to 11 wherein said at least one biomarker is determined by flow cytometry, quantitative PCR (qPCR) or transcriptome sequencing, preferably, bulk RNA-seq or scRNA-seq.

Embodiment 13: A device adopted for carrying out the method of any one of embodiments 1 to 12 comprising:

(a) an analysing unit comprising a binding agent for at least one biomarker selected from the group consisting of CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA- DRA, CD 164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RAB11A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA- DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and JAML, wherein binding of the binding agent to the at least one biomarker when present in a sample of a subject can be detected; and

(b) an evaluation unit comprising a processor and a database comprising a stored reference for said at least one biomarker, wherein said evaluation unit is adapted for providing the assessment of AML.

Embodiment 14: A kit for assessing AML in a subject comprising at least one detection agent and instructions to carry out the method of any one of embodiments 1 to 12, wherein the at least one detection agent is capable of specifically detecting a biomarker selected from the group consisting of CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RABI 1 A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and J AML.

Embodiment 15: At least one biomarker or a detection agent for said at least one biomarker for use in assessing AML in a subject, wherein said at least one biomarker is selected from the group consisting of CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RAB11A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and J AML.

Embodiment 16: Use of least one biomarker or a detection agent for the said at least one biomarker for assessing AML in a sample of a subject, wherein said at least one biomarker is selected from the group consisting of CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RAB11A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and JAML.

Embodiment 17: A method for generating a bispecific binding agent comprising the steps of: a) determining the abundance of a plurality of biomarkers present in each single cell of (i) leukemic cells, preferably, AML cells, and control cells and (ii) leukemic stem cells, preferably, AML stem cells, and control cells; b) identifying among the plurality of biomarkers those which are present above a predetermined threshold on (i) leukemic cells, preferably, AML cells, but not control cells and (ii) leukemic stem cells, preferably, AML stem cells, but not control cells; c) identifying among the biomarkers present above the pre-defined threshold biomarker pairs which are present in one single cell; and d) generating a bispecific binding agent comprising a first target binding domain that specifically binds to a first target and a second target binding domain that specifically binds to a second target, wherein the first and the second target are biomarker pairs identified in step c) and are different from each other.

Embodiment 18: A bispecific binding agent comprising a first target binding domain that specifically binds to a first target and a second target binding domain that specifically binds to a second target, wherein

(i) the binding agent binds to the first and second target when present on the same cancer cell; (ii) the first target and the second target are different from each other; and

(iii) the first and the second target are selected from the group consisting of CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RAB11A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and JAML.

Embodiment 19: The bispecific binding agent of embodiment 18, wherein said first target is CD52.

Embodiment 20: The bispecific binding agent of embodiment 18 or 19, wherein said binding agent is obtainable by the method of embodiment 17.

Embodiment 21 : The bispecific binding agent of any one of embodiments 18 to 20, wherein said bispecific binding agent is a bispecific antibody.

Embodiment 22: The bispecific binding agent of embodiment 21, wherein said antibody contains a modification that enhances or attenuates binding of its Fc domain to its receptor.

Embodiment 23: The bispecific binding agent of embodiment 21 or 22, wherein said antibody comprises a modification that enhances or attenuates antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP).

Embodiment 24: A pharmaceutical composition comprising the bispecific binding agent of any one of embodiments 18 to 23.

Embodiment 25: A bispecific binding agent of any one of embodiments 18 to 23 for use in treating and/or preventing cancer.

Embodiment 26: The bispecific antibody for use of embodiment 25, wherein said cancer is leukemia and, preferably, myeloid leukemia.

Embodiment 27: The bispecific antibody for use of embodiment 25 or 26, wherein said leukemia is acute myeloid leukemia (AML).

All references cited throughout the specification are herewith incorporated by reference with respect to the specifically mentioned disclosure or in their entireties. FIGURES

Figure 1: Analysis of the clinical relevance of JMML stem cell surface markers in AML. A Univariate hazard ratio for overall survival (OS) on TCGA-LAML Bulk-RNA data of 43 detected JMML genes. B Workflow for creating a predictive score based on JMML genes. C Coefficients of JMML score. D Kaplan-Meier estimates of TCGA-LAML for OS. The dataset was used as the training cohort to develop the score. E Univariate hazard ratio for OS of JMML- Score on TCGA-LAML. F Multivariate hazard ratios for OS on TCGA-LAML considering age, risk stratification, LSC-17 score (Ng et al., 2016), and JMML score. Patients who did not have full annotation were removed before testing. G Kaplan-Meier estimates of BEAT-AML cohort for OS. The dataset was used as an independent validation cohort for the score. H Univariate hazard ratio for OS of JMML-Score on BEAT-AML. I Multivariate hazard ratios for OS on BEAT-AML considering age, ELN risk stratification (Dbhner et al., 2017), cohort, LSC-17 score, and JMML score. Patients who did not have full annotation were removed before testing.

Figure 2: Selection of candidates for therapies targeting immature AML cells. Percent of immature AML cells expressing JMML HSC marker genes across scRNA-seq datasets.

Figure 3: Workflow for gene-pair detection for the development of bispecific therapies. Venn diagram displays gene pairs that had a positive correlation in all three correlation methods in at least one dataset. Number under the Venn diagram displays the number of total gene pairs with at least one positive correlation in one dataset.

Figure 4: JMML stem cell score identifies high-risk AML independent of classical subtypes, enabling stratification of AML patients. Gene expression data was obtained from BEAT-AML cohort (Bottomly et al., 2022) and ordered according to their JMML score. The LSC-17 score and European Leukemia Net (ELN) categories were applied as described previously (Ng et al., 2016; Dbhner et al., 2022).

Figure 5: scRNA-seq profiling reveals co-expression of JMML HSC markers in AML LSCs. The LSC-17 score (Ng et al., 2016) together with GPR56 and CD34 expression patterns enable the identification of AML LSCs based on single-cell transcriptomes.

Figure 6: Proof-of-concept showing surface marker expression on protein level. Spectral flow cytometry analysis revealing protein co-expression of JMML LSC markers on immature AML cells containing AML LSCs. Hematopoietic markers: CD34 (HSPCs and LSCs), CD 164 (HSPCs), CD38 (immature hematopoietic & immune cells), CD45 (leukocytes), CD3 + CD 19 (lymphocytes), CD14 (myeloid cells). AML LSC markers: CD34, GPR56. JMML LSC markers: CD 164, CD69, CD96, CD74, CD52, CD47, HCST, FCMR, HLA-DR.

EXAMPLES

The Examples shall merely illustrate the invention. They shall not limit the scope thereof whatsoever.

Example 1: Materials and methods

Prognostic value of single JMML HSC genes in AML

The normalized bulk RNA seq data and clinical annotation for TCGA-LAML ("Genomic and Epigenomic Landscapes of Adult De Novo Acute Myeloid Leukemia," 2013) were obtained from https://portal.gdc.cancer.gov/projects/TCGA-LAML and BEAT-AML (Bottomly et al., 2022; Tyner et al., 2018) from https://biodev.github.io/BeatAML2/. The prognostic value of individual genes was tested with a univariate Cox hazards model for each gene on TCGA- LAML for overall survival (OS).

Generation of a JMML HSC-based score to stratify AML

10 genes that had a significant prognostic value determined by univariate analysis were subjected to a 10 times cross-validated lasso model for OS. 7 genes were determined to be noncorrelating and subjected to multivariate Cox regression for OS on TCGA-LAML. The coefficients of the model were extracted to construct the final score. To test the relevance of this prognostic score Kaplan-Meier estimates and univariate and multivariate Cox hazards regressions including common parameters for AML risk stratification were calculated for TCGA-LAML and BEAT-AML respectively.

Expression of JMML HSC genes in immature AML cells

Two publicly available AML single-cell RNA sequencing (scRNA-seq) datasets were obtained for this analysis: The dataset referred to as Clonetracer (Beneyto-Calabuig et al., 2023) from https://figshare.eom/articles/dataset/Seurat_CloneTracer_Cohort/20291628 and the dataset referred to as Lasry (Lasry et al., 2023) from https://singlecell.broadinstitute.org/single_cell/study/SCP1987. To determine immature leukemic cells both datasets were projected onto a healthy bone marrow single-cell reference to determine for each cell their closest healthy counterpart (Triana et al., 2020). Datasets were subsetted on leukemic cells using their existing annotation and immature cells after projection. The Lasry dataset was further subdivided into AML samples from adult or pediatric patients. For these datasets, the percentages of cells expressing any transcripts for each of the 44 detected genes were determined.

Co-expression analysis of JMML HSC candidate genes in immature AML cells

The three scRNA-seq datasets were subjected to correlation testing for 39 genes expressed in at least 10% of cells in at least 1 of the datasets. Correlations were tested either with (1) Pearson correlation on normalized counts, (2) with CSCORE (Su et al., 2023), or (3) Magic (Van Dijk et al., 2018). Correlations were also tested on immature cells of healthy donors and healthy cells in AML patients for each dataset, respectively. Gene pairs were considered as positively correlating if the determined correlation coefficient was positive and the fdr-value was below 0.05.

Example 2: Differentially expressed JMML Biomarkers in stem cells

In a multi-omics analysis of JMML stem cells, 44 genes encoding for cell surface markers were identified to be differentially expressed in stem cells across JMML risk groups.

To test the clinical relevance of JMML stem cell surface markers in AML, the inventors examined the prognostic value of each gene in a univariate analysis (Figure l.A). Out of 44 JMML stem cell markers tested, 15 genes appeared as adverse (Hazard ratio >1.1) and 5 genes as favorable (Hazard ratio <0.9) risk factors, revealing a prognostic value of the JMML stem cell signature in AML. Out of the 10 genes determined as having a significant impact on overall survival, 7 were found to be non-correlating. These genes were extracted to develop a multivariate risk stratification score (Figure l.B-C), which significantly stratified AML patients in two independent cohorts (TCGA and BEAT) (Figure l.D-I). This JMML score performed better than the established LSC-17 score, confirming the JMML stem cell gene list as valid in AML and the validity of such a cross-entity approach in general.

Example 3: JMML Biomarkers in AML

To test, whether JMML stem cell surface markers can potentially serve as therapeutic targets in AML, the inventors tested which of these genes are expressed in at least 10% of immature cells in various AML scRNA-seq datasets. Out of 44 genes, 39 were found to be expressed in immature cells of AML patient samples in at least one of the datasets (Figure 2). Those candidate genes were considered as potential therapeutic targets in AML: CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RABI 1 A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and J AML.

Example 4; Identification of Biomarker pairs for bi specific AML therapy

To identify gene pairs, which function as therapeutic targets for bispecific therapeutic approaches, the inventors identified gene pairs that are significantly co-expressed in immature AML cells. To analyze the co-expression of those genes, three different correlation algorithms were applied and overall, eight different layers of confidence were defined by the use of various overlaps and unions across analyses, which are summarized in Figure 3.

A. Correlation of pairs in leukemic cells

A.1. Union of correlations

List of gene pairs across all AML datasets filtered for pairs that had a significant positive correlation in the leukemic cells for at least one correlation algorithm in one dataset. 633 gene pairs were found:

List A.1:

CD74 HLA-DRA, CD74 CD164, CD74 CD52, CD74 HCST, CD74 CD34, CD74 HSPA5, CD74 MGST1, CD74 CD47, CD74 TFPI, CD74 IGHM, CD74 SELL, CD74 CD82, CD74 CD69, CD74 LST1, CD74 NDFIP1, CD74 ZFITM3, CD74 RALA, CD74 RAB11A, CD74 SELENOK, CD74 HLA-DRB5, CD74 RAMP1, CD74 HLA-DQA1, CD74 CD96, CD74 SLC5A3, CD74 VAMP5, CD74 CALCRL, CD74 LPAR6, CD74 NINJ1, CD74 LTB, CD74 CD7, CD74 SLC2A5, CD74 FCMR, CD74 DLK1, CD74 CLEC7A, CD74 JAML, CD74 CLEC9A, HLA-DRA CD52, HLA-DRA HCST, HLA-DRA CD34, HLA-DRA HSPA5, HLA-DRA MGST1, HLA-DRA CD47, HLA-DRA TFPI, HLA- DRA IGHM, HLA-DRA SELL, HLA-DRA CD82, HLA-DRA CD69, HLA-DRA LST1, HLA-DRA NDFIPl, HLA-DRA IFITM3, HLA-DRA RALA, HLA-DRA RAB11A, HLA- DRA SELENOK, HLA-DRA HLA-DRB5, HLA-DRA RAMP1, HLA-DRA HLA-DQA1, HLA-DRA CD96, HLA-DRA SLC5A3, HLA-DRA VAMP5, HLA-DRA CALCRL, HLA- DRA LPAR6, HLA-DRA NINJ1, HLA-DRA LTB, HLA-DRA CD7, HLA-DRA SLC2A5, HLA-DRA FCMR, HLA-DRA DLKl, HLA-DRA CLEC7A, HLA-DRA JAML, HLA- DRA CLEC9A, CD164 CD52, CD164 HCST, CD164 CD34, CD164 HSPA5, CD164 MGST1, CD164 CD47, CD164 TFPI, CD164 IGHM, CD164 SELL, CD164 CD82, CD164 CD69, CD164 LST1, CD164 NDFIP1, CD164 RALA, CD164 SELENOK, CD164 HLA-DRB5, CD164 RAMP1, CD164 IGLL1, CD164 CD96, CD164 SLC5A3, CD164 CALCRL, CD164 LPAR6, CD164 NINJ1, CD164 LTB, CD164 CD7, CD164 SLC2A5, CD164 EREG, CD164 FCMR, CD164 CLEC7A, CD164 JAML, CD164 CLEC9A, CD52 HCST, CD52 CD34, CD52 MGST1, CD52 CD47, CD52 TFPI, CD52 IGHM, CD52 SELL, CD52 CD82, CD52 CD69, CD52 LST1, CD52 NDFIP1, CD52 IFITM3, CD52 RALA, CD52 RAB11A, CD52 SELENOK, CD52 HLA-DRB5, CD52 RAMP1, CD52 IGLL1, CD52 HLA-DQA1, CD52 CD96, CD52 VAMP5, CD52 CALCRL, CD52 NINJ1, CD52 LTB, CD52 CD7, CD52 SLC2A5, CD52 EREG, CD52 FCMR, CD52 DLK1, CD52 CLEC7A, CD52 JAML, CD52 CLEC9A, HCST CD34, HCST HSPA5, HCST MGST1, HCST CD47, HCST TFPI, HCST IGHM, HCST SELL, HCST CD82, HCST CD69, HCST LST1, HCST NDFIPl, HCST IFITM3, HCST RALA, HCST RAB11A, HCST SELENOK, HCST HLA-DRB5, HCST RAMP1, HCST IGLL1, HCST HLA-DQA1, HCST CD96, HCST SLC5A3, HCST VAMP5, HCST CALCRL, HCST LPAR6, HCST NINJ1, HCST LTB, HCST CD7, HCST SLC2A5, HCST FCMR, HCST DLKl, HCST CLEC7A, HCST JAML, HCST CLEC9A, CD34 HSPA5, CD34 MGST1, CD34 CD47, CD34 TFPI, CD34 IGHM, CD34 SELL, CD34 CD82, CD34 CD69, CD34 LST1, CD34 NDFIP1, CD34 IFITM3, CD34 RALA, CD34 RAB11A, CD34 SELENOK, CD34 HLA-DRB5, CD34 RAMP1, CD34 IGLL1, CD34 HLA-DQA1, CD34 CD96, CD34 SLC5A3, CD34 VAMP5, CD34 CALCRL, CD34 LPAR6, CD34 LTB, CD34 CD7, CD34 SLC2A5, CD34 EREG, CD34 FCMR, CD34 DLK1, CD34 CLEC7A, CD34 JAML, CD34 CLEC9A, HSPA5 MGST1, HSPA5 CD47, HSPA5 SELL, HSPA5 CD69, HSPA5 LST1, HSPA5 NDFIP1, HSPA5 IFITM3, HSPA5 RAB11A, HSPA5 SELENOK, HSPA5 HLA- DRB5, HSPA5 IGLL1, HSPA5 HLA-DQA1, HSPA5 CD96, HSPA5 VAMP5, HSPA5 LPAR6, HSPA5 NINJ1, HSPA5 LTB, HSPA5 CD7, HSPA5 SLC2A5, HSPA5 EREG, HSPA5 DLK1, HSPA5 CLEC7A, HSPA5 JAML, HSPA5 CLEC9A, MGST1 IGHM, MGST1 SELL, MGST1 CD69, MGST1 LST1, MGST1 NDFIP1, MGST1 IFITM3, MGST1 RALA, MGST1 RAB11A, MGST1 SELENOK, MGST1 HLA- DRB5, MGST1 RAMP1, MGST1 IGLL1, MGST1 HLA-DQA1, MGST1 CD96, MGST1 VAMP5, MGST1 NINJ1, MGST1 CD7, MGST1 SLC2A5, MGST1 EREG, MGST1 DLK1, MGST1 CLEC7A, MGST1 CLEC9A, CD47 TFPI, CD47 IGHM, CD47 SELL, CD47 CD82, CD47 CD69, CD47 LST1, CD47 NDFIP1, CD47 IFITM3, CD47 RALA, CD47 RAB11A, CD47 SELENOK, CD47 HLA-DRB5, CD47 RAMP1, CD47 IGLL1, CD47 HLA-DQA1, CD47 CD96, CD47 SLC5A3, CD47 VAMP5, CD47 CALCRL, CD47 LPAR6, CD47 LTB, CD47 CD7, CD47 SLC2A5, CD47 FCMR, CD47 CLEC7A, CD47 JAML, CD47 CLEC9A, TFPI IGHM, TFPI SELL, TFPI CD82, TFPI CD69, TFPI LST1, TFPI NDFIPl, TFPI IFITM3, TFPI RALA, TFPI SELENOK, TFPI HLA-DRB5, TFPI RAMP1, TFPI HLA-DQA1, TFPI CD96, TFPI SLC5A3, TFPI VAMP5, TFPI CALCRL, TFPI LPAR6, TFPI NINJ1, TFPI LTB, TFPI CD7, TFPI SLC2A5, TFPI EREG, TFPI FCMR, TFPI DLKl, TFPI CLEC7A, TFPI JAML, TFPI CLEC9A, IGHM SELL, IGHM CD82, IGHM CD69, IGHM LST1, IGHM NDFIPl, IGHM IFITM3, IGHM RALA, IGHM SELENOK, IGHM HLA-DRB5, IGHM RAMP1, IGHM HLA-DQA1, IGHM CD96, IGHM SLC5A3, IGHM VAMP5, IGHM CALCRL, IGHM LPAR6, IGHM NINJ1, IGHM LTB, IGHM CD7, IGHM SLC2A5, IGHM FCMR, IGHM CLEC7A, IGHM JAML, IGHM CLEC9A, SELL CD82, SELL CD69, SELL LST1, SELL NDFIPl, SELL IFITM3, SELL RALA, SELL RAB11A, SELL SELENOK, SELL HLA-DRB5, SELL RAMP1, SELL IGLL1, SELL HLA-DQA1, SELL CD96, SELL SLC5A3, SELL VAMP5, SELL CALCRL, SELL LPAR6, SELL NINJ1, SELL LTB, SELL CD7, SELL SLC2A5, SELL FCMR, SELL JAML, SELL CLEC9A, CD82 CD69, CD82 LST1, CD82 NDFIP1, CD82 IFITM3, CD82 RALA, CD82 RAB11A, CD82 SELENOK, CD82 HLA-DRB5, CD82 RAMP1, CD82 IGLL1, CD82 VAMP5, CD82 CALCRL, CD82 NINJ1, CD82 LTB, CD82 CD7, CD82 SLC2A5, CD82 EREG, CD82 DLK1, CD82 CLEC7A, CD82 CLEC9A, CD69 LST1, CD69 NDFIP1, CD69 RALA, CD69 RAB11A, CD69 SELENOK, CD69 HLA-DRB5, CD69 RAMP1, CD69 IGLL1, CD69 HLA-DQA1, CD69 CD96, CD69 SLC5A3, CD69 CALCRL, CD69 LPAR6, CD69 NINJ1, CD69 CD7, CD69 SLC2A5, CD69 EREG, CD69 FCMR, CD69 DLK1, CD69 CLEC7A, CD69 JAML, LST1 NDFIPl, LST1 IFITM3, LST1 RAB11A, LST1 SELENOK, LST1 HLA-DRB5, LST1 RAMP1, LST1 IGLL1, LST1 HLA-DQA1, LST1 CD96, LST1 SLC5A3, LST1 VAMP5, LST1 CALCRL, LST1 LPAR6, LST1 NINJ1, LST1 LTB, LST1 CD7, LST1 SLC2A5, LST1 FCMR, LST1 DLK1, LST1 CLEC7A, LST1 JAML, LST1 CLEC9A, NDFIPl IFITM3, NDFIPI RALA, NDFIP1 RAB11A, NDFIPl SELENOK, NDFIP1 HLA-DRB5, NDFIPI RAMPI, NDFIPI IGLLI, NDFIP1 HLA-DQA1, NDFIP1 SLC5A3, NDFIP1 VAMP5, NDFIPl CALCRL, NDFIP1 NINJ1, NDFIP1 LTB, NDFIP1 CD7, NDFIPl SLC2A5, NDFIP1 EREG, NDFIPI FCMR, NDFIP1 DLK1, NDFIP1 CLEC7A, NDFIPI JAML, NDFIP1 CLEC9A, IFITM3 RALA, IFITM3 RAB11 A, IFITM3 SELENOK, IFITM3 HLA-DRB5, IFITM3 RAMP1, IFITM3 IGLL1, IFITM3 HLA-DQA1, IFITM3 CD96, IFITM3 SLC5A3, IFITM3 VAMP5, IFITM3 CALCRL, IFITM3 NINJ1, IFITM3 LTB, IFITM3 SLC2A5, IFITM3 EREG, IFITM3 FCMR, IFITM3 DLK1, IFITM3 CLEC7A, IFITM3 JAML, IFITM3 CLEC9A, RALA RAB11A, RALA SELENOK, RALA HLA-DRB5, RALA RAMP1, RALA IGLL1, RALA HLA-DQA1, RALA CD96, RALA SLC5A3, RALA VAMP5, RALA CALCRL, RALA LPAR6, RALA NINJ1, RALA LTB, RALA CD7, RALA SLC2A5, RALA EREG, RALA FCMR, RALA DLKl, RALA CLEC7A, RABI 1A SELENOK, RAB11A HLA- DRB5, RAB11A RAMP1, RAB11A IGLL1, RABI 1A HLA-DQA1, RAB11A CD96, RAB11A SLC5A3, RABI 1A VAMP5, RABI 1A CALCRL, RAB11A LTB, RAB 11 A CD7, RAB 11 A SLC2 A5 , RAB 11 A EREG, RAB 11 A DLK 1 , RAB 11 A CLEC7 A, RAB11A JAML, RABI 1A CLEC9A, SELENOK HLA-DRB5, SELENOK RAMP1,

A.2. Overlap of correlations

List of gene pairs across all AML datasets filtered for pairs that had a significant positive correlation in the leukemic cells for all correlation algorithms in all three datasets. 45 gene pairs were found:

List A.2: CD74 HLA-DRA, CD74 CD52, CD74 HCST, CD74 CD34, CD74 IGHM, CD74 LST1, CD74 HLA-DQA1, CD74 LTB, CD74 SLC2A5, CD74 FCMR, HLA-DRA CD52, HLA- DRA HCST, HLA-DRA CD34, HLA-DRA IFITM3, HLA-DRA HLA-DRB5, HLA- DRA HLA-DQAl, HLA-DRA VAMP5, HLA-DRA LTB, CD164 CD47, CD52 IFITM3, CD52 RALA, CD52 HLA-DQA1, CD52 VAMP5, HCST CD34, HCST CD47, HCST LST1, HCST HLA-DQA1, HCST CD96, HCST LTB, HCST FCMR, CD34 LST1, CD34 HLA-DQA1, MGST1 IFITM3, CD47 SELL, CD47 LST1, CD47 SLC5A3, TFPI LPAR6, IGHM SELL, IGHM FCMR, LST1 LTB, LST1 FCMR, IFITM3 VAMP5, HLA-DQA1 VAMP5, LTB FCMR, SLC2A5 FCMR

B. Correlation pairs in leukemic cells that do not correlate in healthy donors

B.3. Union of correlations

List of gene pairs across all AML datasets filtered for pairs that had a significant positive correlation in the leukemic cells for at least one correlation algorithms in at least one of the datasets and no correlation in the healthy donors from the same dataset with the same correlation algorithm.592 gene pairs were found:

List B.3:

CD74 HLA-DRA, CD74 CD164, CD74 CD52, CD74 HCST, CD74 CD34, CD74 HSPA5, CD74 MGST1, CD74 CD47, CD74 TFPI, CD74 SELL, CD74 CD82, CD74 CD69, CD74 LST1, CD74 NDFIP1, CD74 IFITM3, CD74 RALA, CD74 RAB11A, CD74 SELENOK, CD74 HLA-DRB5, CD74 RAMP1, CD74 HLA-DQA1, CD74 CD96, CD74 SLC5A3, CD74 VAMP5, CD74 CALCRL, CD74 LPAR6, CD74 NINJ1, CD74 LTB, CD74 CD7, CD74 SLC2A5, CD74 FCMR, CD74 DLK1, CD74 CLEC7A, CD74 JAML, CD74 CLEC9A, HLA-DRA CD52, HLA-DRA HCST, HLA-DRA CD34, HLA-DRA HSPA5, HLA-DRA MGST1, HLA-DRA CD47, HLA-DRA TFPI, HLA- DRA IGHM, HLA-DRA SELL, HLA-DRA CD82, HLA-DRA CD69, HLA-DRA LST1, HLA-DRA NDFIPl, HLA-DRA IFITM3, HLA-DRA RALA, HLA-DRA RAB11A, HLA- DRA SELENOK, HLA-DRA HLA-DRB5, HLA-DRA RAMP1, HLA-DRA HLA-DQA1, HLA-DRA CD96, HLA-DRA SLC5A3, HLA-DRA VAMP5, HLA-DRA CALCRL, HLA- DRA LPAR6, HLA-DRA NINJ1, HLA-DRA LTB, HLA-DRA CD7, HLA-DRA SLC2A5, HLA-DRA FCMR, HLA-DRA DLKl, HLA-DRA CLEC7A, HLA-DRA JAML, HLA- DRA CLEC9A, CD164 CD52, CD164 HCST, CD164 CD34, CD164 HSPA5, CD164 MGST1, CD164 CD47, CD164 TFPI, CD164 IGHM, CD164 SELL, CD164 CD82, CD164 CD69, CD164 LST1, CD164 NDFIP1, CD164 SELENOK, CD164 HLA-DRB5, CD164 RAMP1, CD164 IGLL1, CD164 CD96, CD164 SLC5A3, CD164 LPAR6, CD164 NINJ1, CD164 LTB, CD164 CD7, CD164 SLC2A5, CD164 FCMR, CD164 CLEC7A, CD164 JAML, CD52 HCST, CD52 CD34, CD52 MGST1, CD52 CD47, CD52 TFPI, CD52 SELL, CD52 CD82, CD52 CD69, CD52 LST1, CD52 NDFIP1, CD52 IFITM3, CD52 RALA, CD52 RAB11A, CD52 SELENOK, CD52 HLA-DRB5, CD52 RAMP1, CD52 IGLL1, CD52 HLA-DQA1, CD52 CD96, CD52 VAMP5, CD52 CALCRL, CD52 NINJ1, CD52 LTB, CD52 CD7, CD52 SLC2A5, CD52 EREG, CD52 DLK1, CD52 CLEC7A, CD52 CLEC9A, HCST CD34, HCST HSPA5, HCST MGST1, HCST CD47, HCST TFPI, HCST SELL, HCST CD82, HCST CD69, HCST LST1, HCST NDFIPl, HCST IFITM3, HCST RALA, HCST RAB11A, HCST SELENOK, HCST HLA-DRB5, HCST RAMP1, HCST HLA- DQA1, HCST CD96, HCST SLC5A3, HCST VAMP5, HCST CALCRL, HCST LPAR6, HCST NINJ1, HCST LTB, HCST CD7, HCST SLC2A5, HCST FCMR, HCST DLKl, HCST CLEC7A, HCST JAML, HCST CLEC9A, CD34 HSPA5, CD34 MGST1, CD34 CD47, CD34 TFPI, CD34 IGHM, CD34 SELL, CD34 CD82, CD34 CD69, CD34 LST1, CD34 NDFIP1, CD34 IFITM3, CD34 RALA, CD34 RAB11A, CD34 SELENOK, CD34 HLA-DRB5, CD34 RAMP1, CD34 IGLL1, CD34 HLA-DQA1, CD34 CD96, CD34 SLC5A3, CD34 VAMP5, CD34 CALCRL, CD34 LPAR6, CD34 LTB, CD34 CD7, CD34 SLC2A5, CD34 FCMR, CD34 DLK1, CD34 CLEC7A, CD34 CLEC9A, HSPA5 MGST1, HSPA5 CD47, HSPA5 SELL, HSPA5 LST1, HSPA5 NDFIP1, HSPA5 IFITM3, HSPA5 SELENOK, HSPA5 HLA-DRB5, HSPA5 IGLL1, HSPA5 HLA-DQA1, HSPA5 CD96, HSPA5 VAMP5, HSPA5 LPAR6, HSPA5 NINJ1, HSPA5 LTB, HSPA5 CD7, HSPA5 SLC2A5, HSPA5 EREG, HSPA5 DLK1, HSPA5 CLEC7A, HSPA5 JAML, HSPA5 CLEC9A, MGST1 IGHM, MGST1 SELL, MGST1 CD69, MGST1 LST1, MGST1 NDFIP1, MGST1 IFITM3, MGST1 RALA, MGST1 RAB11A, MGST1 SELENOK, MGST1 HLA-DRB5, MGST1 RAMP1, MGST1 IGLL1, MGST1 HLA-DQA1, MGST1 CD96, MGST1 VAMP5, MGST1 NINJ1, MGST1 CD7, MGST1 SLC2A5, MGST1 EREG, MGST1 DLK1, MGST1 CLEC9A, CD47 TFPI, CD47 IGHM, CD47 SELL, CD47 CD82, CD47 CD69, CD47 LST1, CD47 NDFIP1, CD47 IFITM3, CD47 RALA, CD47 HLA- DRB5, CD47 RAMP1, CD47 IGLL1, CD47 HLA-DQA1, CD47 CD96, CD47 SLC5A3, CD47 VAMP5, CD47 CALCRL, CD47 LPAR6, CD47 LTB, CD47 CD7, CD47 SLC2A5, CD47 FCMR, CD47 CLEC7A, CD47 JAML, CD47 CLEC9A, TFPI IGHM, TFPI SELL, TFPI CD82, TFPI CD69, TFPI LST1, TFPI NDFIPl, TFPI IFITM3, TFPI RALA, TFPI SELENOK, TFPI HLA-DRB5, TFPI RAMP1, TFPI HLA-DQA1, TFPI CD96, TFPI SLC5A3, TFPI VAMP5, TFPI CALCRL, TFPI LPAR6, TFPI NINJ1, TFPI LTB, TFPI CD7, TFPI SLC2A5, TFPI FCMR, TFPI CLEC7A, TFPI JAML, IGHM SELL, IGHM CD82, IGHM CD69, IGHM LST1, IGHM NDFIPl, IGHM IFITM3, IGHM RALA, IGHM SELENOK, IGHM HLA-DRB5, IGHM RAMP1, IGHM HLA- DQA1, IGHM CD96, IGHM SLC5A3, IGHM VAMP5, IGHM CALCRL, IGHM LPAR6, IGHM NINJ1, IGHM CD7, IGHM SLC2A5, IGHM FCMR, IGHM CLEC7A, IGHM JAML, IGHM CLEC9A, SELL CD82, SELL CD69, SELL LST1, SELL NDFIPl, SELL IFITM3, SELL RALA, SELL RAB11A, SELL SELENOK, SELL HLA-DRB5, SELL RAMP1, SELL HLA-DQA1, SELL CD96, SELL SLC5A3, SELL VAMP5, SELL CALCRL, SELL LPAR6, SELL NINJ1, SELL LTB, SELL CD7, SELL SLC2A5, SELL FCMR, SELL CLEC9A, CD82 CD69, CD82 LST1, CD82 NDFIP1, CD82 IFITM3, CD82 RALA, CD82 RAB11A, CD82 SELENOK, CD82 HLA-DRB5, CD82 RAMP1, CD82 IGLL1, CD82 VAMP5, CD82 CALCRL, CD82 NINJ1, CD82 LTB, CD82 CD7, CD82 EREG, CD82 DLK1, CD82 CLEC7A, CD82 CLEC9A, CD69 LST1, CD69 NDFIP1, CD69 RALA, CD69 RAB11A, CD69 SELENOK, CD69 HLA-DRB5, CD69 RAMP1, CD69 HLA-DQA1, CD69 SLC5A3, CD69 CALCRL, CD69 LPAR6, CD69 NINJ1, CD69 CD7, CD69 SLC2A5, CD69 EREG, CD69 FCMR, CD69 DLK1, CD69 CLEC7A, CD69 JAML, LST1 NDFIP1, LST1 IFITM3, LST1 RAB11A, LST1 SELENOK, LST1 HLA-DRB5, LST1 RAMP1, LST1 IGLL1, LST1 HLA-DQA1, LST1 CD96, LST1 SLC5A3, LST1 VAMP5, LST1 CALCRL, LST1 LPAR6, LST1 NINJ1, LST1 LTB, LST1 CD7, LST1 SLC2A5, LST1 FCMR, LST1 DLK1, LST1 CLEC7A, LST1 JAML, LST1 CLEC9A, NDFIP1 IFITM3, NDFIP1 RALA, NDFIPI RABllA, NDFIP1 HLA-DRB5, NDFIPI RAMPI, NDFIP1 IGLL1, NDFIP1 HLA-DQA1, NDFIP1 SLC5A3, NDFIP1 VAMP5, NDFIP1 NINJ1, NDFIP1 LTB, NDFIP1 CD7, NDFIP1_SLC2A5, NDFIP1 EREG, NDFIP1 FCMR, NDFIPI DLKl, NDFIP1 CLEC7A, NDFIPI JAML, NDFIP1 CLEC9A, IFITM3 RALA, IFITM3 RAB11A, IFITM3 SELENOK, IFITM3 HLA-DRB5, IFITM3 RAMP1, IFITM3 IGLL1, IFITM3 HLA-DQA1, IFITM3 CD96, IFITM3 SLC5A3, IFITM3 VAMP5, IFITM3 CALCRL, IFITM3 NINJ1, IFITM3 LTB, IFITM3 SLC2A5, IFITM3 FCMR, IFITM3 DLK1, IFITM3 CLEC7A, IFITM3 JAML, IFITM3 CLEC9A, RALA RAB11A, RALA SELENOK, RALA HLA-DRB5, RALA RAMP1, RALA IGLL1, RALA HLA- DQA1, RALA CD96, RALA SLC5A3, RALA VAMP5, RALA CALCRL, RALA LPAR6, RALA NINJ1, RALA LTB, RALA CD7, RALA SLC2A5, RALA FCMR, RALA DLKl, RALA CLEC7A, RABI 1A SELENOK, RABI 1A HLA-DRB5, RABI 1A RAMP1, RAB11A IGLL1, RABI 1A HLA-DQA1, RAB11A CD96, RABI 1A VAMP5, RABI 1A CALCRL, RABI 1A LTB, RABI 1A CD7, RABI 1A DLK1, RABI 1A CLEC7A, RAB11A JAML, RABI 1A CLEC9A, SELENOK HLA-DRB5, SELENOK RAMP1, SELENOK IGLL1, SELENOK HLA-DQA1, SELENOK CD96, SELENOK VAMP5, SELENOK CALCRL, SELENOK NINJ1, SELENOK LTB, SELENOK CD7, SELENOK EREG, SELENOK CLEC7A, HLA-DRB5 IGLL1, HLA-DRB5 HLA-DQA1, HLA-DRB5 CD96, HLA-DRB5 SLC5A3, HLA-DRB5 VAMP5, HLA-DRB5 NINJ1, HLA-DRB5 LTB, HLA-DRB5 CD7, HLA-DRB5 SLC2A5, HLA-DRB5 EREG, HLA- DRB5 FCMR, HLA-DRB5 DLK1, HLA-DRB5 JAML, HLA-DRB5 CLEC9A, RAMP1 HLA-DQA1, RAMP1 SLC5A3, RAMP1 VAMP5, RAMP1 CALCRL, RAMP1 LPAR6, RAMP1 NINJ1, RAMP1 LTB, RAMP1 SLC2A5, RAMPI EREG, RAMP1 FCMR, RAMP1 DLK1, RAMP1 CLEC7A, RAMP1 JAML, RAMP1 CLEC9A, IGLL1 HLA-DQA1, IGLL1 CD96, IGLL1 VAMP5, IGLL1 LPAR6, IGLL1 NINJ1, IGLL1 CD7, IGLL1 EREG, IGLL1 DLK1, IGLL1 CLEC9A, HLA-DQA1 CD96, HLA- DQA1_SLC5A3, HLA-DQA1 VAMP5, HLA-DQA1 LPAR6, HLA-DQA1 LTB, HLA- DQA1 CD7, HLA-DQA1 SLC2A5, HLA-DQA1 FCMR, HLA-DQA1 DLK1, HLA- DQA1 JAML, HLA-DQA1 CLEC9A, CD96 VAMP5, CD96 LPAR6, CD96 LTB, CD96 CD7, CD96 SLC2A5, CD96 EREG, CD96 DLK1, CD96 JAML, SLC5A3 VAMP5, SLC5A3 CALCRL, SLC5A3 LPAR6, SLC5A3 LTB, SLC5A3 CD7, SLC5A3 SLC2A5, SLC5A3 FCMR, SLC5A3 DLK1, SLC5A3 CLEC7A, SLC5A3 JAML, VAMP5 CALCRL, VAMP5 LPAR6, VAMP5 NINJ1, VAMP5 LTB, VAMP5 SLC2A5, VAMP5 EREG, VAMP5 FCMR, VAMP5 DLK1, VAMP5 JAML, VAMP5 CLEC9A, CALCRL LPAR6, CALCRL LTB, CALCRL CD7, CALCRL SLC2A5, CALCRL FCMR, CALCRL DLKl, CALCRL CLEC7A, CALCRL JAML, CALCRL CLEC9A, LPAR6 LTB, LPAR6 CD7, LPAR6 SLC2A5, LPAR6 EREG, LPAR6 FCMR, LPAR6 CLEC7A, LPAR6 JAML, LPAR6 CLEC9A, NINJ1 LTB, NINJ1 SLC2A5, NINJ1 EREG, NINJ1 DLK1, NINJ1 CLEC7A, NINJ1 JAML, LTB CD7, LTB SLC2A5, LTB FCMR, LTB DLKl, LTB CLEC7A, LTB JAML, LTB CLEC9A, CD7 SLC2A5, CD7 EREG, CD7 FCMR, CD7 CLEC7A, CD7 JAML, CD7 CLEC9A, SLC2A5 FCMR, SLC2A5 DLK1, SLC2A5 CLEC7A, SLC2A5 JAML, SLC2A5 CLEC9A, EREG DLKl, EREG CLEC7A, FCMR CLEC7A, FCMR JAML, FCMR CLEC9A, DLK1 CLEC7A, DLK1 CLEC9A, CLEC7A JAML, CLEC7A CLEC9A, JAML CLEC9A

B.4. Overlap of correlations

List of gene pairs across all AML datasets filtered for pairs that had a significant positive correlation in the leukemic cells for all correlation algorithms in all datasets and no correlation in the healthy donors from all datasets with all algorithms.8 gene pairs were found:

List B.4:

HLA-DRA HCST, HLA-DRA LTB, CD164 CD47, CD52 RALA, CD52 VAMP5, HCST HLA-DQA1, CD34 HLA-DQA1, TFPI LPAR6

C. Correlation pairs in leukemic cells that do not correlate in healthy cells from leukemia patients

C.5. Union of correlations

List of gene pairs across all AML datasets filtered for pairs that had a significant positive correlation in the leukemic cells for at least one correlation algorithm in at least one of the datasets and no correlation in the healthy cells of AML patients from the same dataset with the same correlation algorithm.596 gene pairs were found:

List C.5:

CD74_CD164, CD74 CD52, CD74 HCST, CD74 CD34, CD74 HSPA5, CD74 MGST1, CD74 CD47, CD74 TFPI, CD74 IGHM, CD74 SELL, CD74 CD82, CD74 CD69, CD74 LST1, CD74 NDFIP1, CD74 IFITM3, CD74 RALA, CD74 RAB11A, CD74 SELENOK, CD74 HLA-DRB5, CD74 RAMP1, CD74 CD96, CD74 SLC5A3, CD74 VAMP5, CD74 CALCRL, CD74 LPAR6, CD74 NINJ1, CD74 LTB, CD74 CD7, CD74 SLC2A5, CD74 FCMR, CD74 CLEC7A, CD74 JAML, CD74 CLEC9A, HLA- DRA CD52, HLA-DRA HCST, HLA-DRA CD34, HLA-DRA MGST1, HLA-DRA CD47, HLA-DRA TFPI, HLA-DRA IGHM, HLA-DRA SELL, HLA-DRA CD82, HLA- DRA CD69, HLA-DRA LST1, HLA-DRA NDFIPl, HLA-DRA IFITM3, HLA- DRA RALA, HLA-DRA RAB11 A, HLA-DRA SELENOK, HLA-DRA HLA-DRB5, HLA- DRA RAMPl, HLA-DRA HLA-DQA1, HLA-DRA CD96, HLA-DRA SLC5A3, HLA- DRA VAMP5, HLA-DRA CALCRL, HLA-DRA LPAR6, HLA-DRA NINJ1, HLA- DRA LTB, HLA-DRA CD7, HLA-DRA SLC2A5, HLA-DRA FCMR, HLA- DRA CLEC7A, HLA-DRA JAML, HLA-DRA CLEC9A, CD164 CD52, CD164 HCST, CD164 CD34, CD164 HSPA5, CD164 MGST1, CD164 CD47, CD164 TFPI, CD164 IGHM, CD164 SELL, CD164 CD82, CD164 CD69, CD164 LST1,

CD164 NDFIP1, CD164 RALA, CD164 HLA-DRB5, CD164 RAMP1, CD164 SLC5A3, CD164 CALCRL, CD164 LPAR6, CD164 NINJ1, CD164 LTB, CD164 CD7, CD164 SLC2A5, CD164 EREG, CD164 FCMR, CD164 CLEC7A, CD164 JAML, CD164 CLEC9A, CD52 HCST, CD52 CD34, CD52 MGST1, CD52 CD47, CD52 TFPI, CD52 IGHM, CD52 SELL, CD52 CD82, CD52 LST1, CD52 NDFIP1, CD52 IFITM3, CD52 RALA, CD52 RAB11A, CD52 SELENOK, CD52 HLA-DRB5, CD52 RAMP1, CD52 IGLL1, CD52 HLA-DQA1, CD52 CD96, CD52 VAMP5, CD52 CALCRL, CD52 LTB, CD52 CD7, CD52 SLC2A5, CD52 FCMR, CD52 DLK1, CD52 CLEC7A, CD52 JAML, CD52 CLEC9A, HCST CD34, HCST HSPA5, HCST CD47, HCST TFPI, HCST IGHM, HCST SELL, HCST CD82, HCST CD69, HCST LST1, HCST NDFIPl, HCST IFITM3, HCST RALA, HCST RAB11A, HCST SELENOK, HCST HLA-DRB5, HCST IGLL1, HCST HLA-DQA1, HCST CD96, HCST SLC5A3, HCST VAMP5, HCST CALCRL, HCST LPAR6, HCST NINJ1, HCST LTB, HCST CD7, HCST SLC2A5, HCST FCMR, HCST DLKl, HCST CLEC7A, HCST JAML, HCST CLEC9A, CD34 MGST1, CD34 CD47, CD34 TFPI, CD34 IGHM, CD34 SELL, CD34 CD82, CD34 CD69, CD34 LST1, CD34 NDFIP1, CD34 IFITM3, CD34 RALA, CD34 RAB11A, CD34 SELENOK, CD34 HLA-DRB5, CD34 RAMP1, CD34 IGLL1, CD34 HLA-DQA1, CD34 CD96, CD34 SLC5A3, CD34 VAMP5, CD34 CALCRL, CD34 LPAR6, CD34 LTB, CD34 CD7, CD34 SLC2A5, CD34 FCMR, CD34 DLK1, CD34 CLEC7A, CD34 JAML, CD34 CLEC9A, HSPA5 MGST1, HSPA5 CD47, HSPA5 SELL, HSPA5 CD69, HSPA5 LST1, HSPA5 IFITM3, HSPA5 SELENOK, HSPA5 HLA-DRB5, HSPA5 IGLL1, HSPA5 HLA-DQA1, HSPA5 CD96, HSPA5 VAMP5, HSPA5 NINJ1, HSPA5 LTB, HSPA5 CD7, HSPA5 SLC2A5, HSPA5 EREG, HSPA5 DLK1, HSPA5 CLEC7A, HSPA5 JAML, HSPA5 CLEC9A, MGST1 IGHM, MGST1 SELL, MGST1 CD69, MGST1 LST1, MGST1 NDFIP1, MGST1 IFITM3, MGST1 RALA, MGST1 RAB11A, MGST1 SELENOK, MGST1 HLA-DRB5, MGST1 RAMP1, MGST1 IGLL1, MGST1 HLA-DQA1, MGST1 CD96, MGST1 VAMP5, MGST1 NINJ1, MGST1 SLC2A5, MGST1 EREG, MGST1 DLK1, MGST1 CLEC7A, MGST1 CLEC9A, CD47 TFPI, CD47 IGHM, CD47 SELL, CD47 CD82, CD47 CD69, CD47 LST1, CD47 NDFIP1, CD47 IFITM3, CD47 RALA, CD47 RAB11A, CD47 SELENOK, CD47 HLA-DRB5, CD47 RAMP1, CD47 HLA-DQA1, CD47 CD96, CD47 SLC5A3, CD47 VAMP5, CD47 CALCRL, CD47 LPAR6, CD47 LTB, CD47 CD7, CD47 SLC2A5, CD47 FCMR, CD47 CLEC7A, CD47 JAML, CD47 CLEC9A, TFPI IGHM, TFPI SELL, TFPI CD82, TFPI CD69, TFPI LST1, TFPI NDFIPl, TFPI RALA, TFPI SELENOK, TFPI HLA-DRB5, TFPI RAMP1, TFPI HLA-DQA1, TFPI CD96, TFPI SLC5A3, TFPI VAMP5, TFPI CALCRL, TFPI LPAR6, TFPI NINJ1, TFPI LTB, TFPI CD7, TFPI SLC2A5, TFPI FCMR, TFPI CLEC7A, TFPI JAML, IGHM SELL, IGHM CD82, IGHM CD69, IGHM LST1, IGHM NDFIPl, IGHM IFITM3, IGHM RALA, IGHM HLA- DRB5, IGHM RAMP1, IGHM HLA-DQA1, IGHM CD96, IGHM SLC5A3, IGHM CALCRL, IGHM LPAR6, IGHM NINJ1, IGHM LTB, IGHM CD7, IGHM SLC2A5, IGHM FCMR, IGHM CLEC7A, IGHM JAML, IGHM CLEC9A, SELL CD82, SELL CD69, SELL LST1, SELL NDFIPl, SELL IFITM3, SELL RALA, SELL RAB11A, SELL SELENOK, SELL HLA-DRB5, SELL RAMP1, SELL HLA- DQA1, SELL CD96, SELL SLC5A3, SELL VAMP5, SELL CALCRL, SELL LPAR6, SELL NINJ1, SELL LTB, SELL CD7, SELL SLC2A5, SELL FCMR, SELL JAML, SELL CLEC9A, CD82 CD69, CD82 LST1, CD82 NDFIP1, CD82 IFITM3, CD82 RALA, CD82 RAB11A, CD82 SELENOK, CD82 HLA-DRB5, CD82 RAMP1, CD82 IGLL1, CD82 VAMP5, CD82 CALCRL, CD82 NINJ1, CD82 LTB, CD82 CD7, CD82 EREG, CD82 DLK1, CD82 CLEC7A, CD82 CLEC9A, CD69 LST1, CD69 NDFIP1, CD69 RALA, CD69 RAB11A, CD69 SELENOK, CD69 HLA-DRB5, CD69 RAMP1, CD69 IGLL1, CD69 HLA-DQA1, CD69 CD96, CD69 SLC5A3, CD69 CALCRL, CD69 LPAR6, CD69 NINJ1, CD69 CD7, CD69 SLC2A5, CD69 EREG, CD69 FCMR, CD69 DLK1, CD69 CLEC7A, CD69 JAML, LST1 NDFIP1, LST1 IFITM3, LST1 RAB11A, LST1 SELENOK, LST1 HLA-DRB5, LST1 RAMP1, LST1 HLA-DQA1, LST1 CD96, LST1 SLC5A3, LST1 VAMP5, LST1 CALCRL, LST1 LPAR6, LST1 NINJ1, LST1 LTB, LST1 CD7, LST1 SLC2A5, LST1 FCMR, LST1 DLK1, LST1 CLEC7A, LST1 JAML, LST1 CLEC9A, NDFIPl IFITM3, NDFIP1 RALA, NDFIPI RABllA, NDFIPl SELENOK, NDFIP1 HLA-DRB5, NDFIPI RAMPI, NDFIP1 IGLL1, NDFIP1 HLA-DQA1, NDFZP1 SLC5A3, NDFIP1 VAMP5, NDFIP1 CALCRL, NDFIP1 NINJ1, NDFIP1 LTB, NDFIP1 CD7, NDFZP1 SLC2A5, NDFIP1 EREG, NDFIP1 FCMR, NDFIP1 DLK1, NDFIP1 CLEC7A, NDFIP1 JAML, NDFIP1 CLEC9A, IFITM3 RALA, IFITM3 RAB11A, IFITM3 SELENOK, IFITM3 HLA- DRB5, IFITM3 RAMP1, IFITM3 IGLL1, IFITM3 HLA-DQA1, IFITM3 CD96, IFITM3 SLC5A3, IFITM3 VAMP5, IFITM3 CALCRL, IFITM3 NINJ1, IFITM3 LTB, IFITM3 SLC2A5, IFITM3 EREG, IFITM3 FCMR, IFITM3 DLK1, IFITM3 CLEC7A, IFITM3 JAML, IFITM3 CLEC9A, RALA RAB11A, RALA SELENOK, RALA HLA- DRB5, RALA RAMP1, RALA IGLL1, RALA HLA-DQA1, RALA CD96, RALA SLC5A3, RALA VAMP5, RALA CALCRL, RALA LPAR6, RALA NINJ1, RALA LTB, RALA CD7, RALA SLC2A5, RALA EREG, RALA DLKl, RAB11A SELENOK, RABI 1A HLA-DRB5, RABI 1A RAMP1, RABI 1A IGLL1, RAB11A HLA-DQA1, RAB11A CD96, RABI 1A SLC5A3, RABI 1A VAMP5, RAB11A CALCRL, RAB11A LTB, RAB11A EREG, RAB11A DLK1, RAB11A CLEC7A, RABI 1 A JAML, RAB11A CLEC9A, SELENOK HLA-DRB5, SELENOK RAMP1, SELENOK IGLL1, SELENOK HLA-DQA1, SELENOK CD96, SELENOK VAMP5, SELENOK CALCRL, SELENOK NINJ1, SELENOK LTB, SELENOK CD7, SELENOK EREG, SELENOK DLKl, SELENOK CLEC7A, HLA- DRB5 IGLL1, HLA-DRB5 HLA-DQA1, HLA-DRB5 CD96, HLA-DRB5 SLC5A3, HLA- DRB5 VAMP5, HLA-DRB5 NINJ1, HLA-DRB5 LTB, HLA-DRB5 CD7, HLA- DRB5 SLC2A5, HLA-DRB5 EREG, HLA-DRB5 FCMR, HLA-DRB5 DLK1, HLA- DRB5 JAML, HLA-DRB5 CLEC9A, RAMP1 HLA-DQA1, RAMP1 SLC5A3, RAMP1 VAMP5, RAMP1 CALCRL, RAMP1 LPAR6, RAMP1 NINJ1, RAMP1 LTB, RAMP1 SLC2A5, RAMPI EREG, RAMP1 FCMR, RAMP1 DLK1, RAMP1 CLEC7A, RAMP1 JAML, RAMP1 CLEC9A, IGLL1 HLA-DQA1, IGLL1 CD96, IGLL1 VAMP5, IGLL1 LPAR6, IGLL1 NINJ1, IGLL1 CD7, IGLL1 EREG, IGLL1 DLK1, IGLL1 CLEC9A, HLA-DQA1 CD96, HLA-DQA1 SLC5A3, HLA-DQA1 VAMP5, HLA- DQA1 CALCRL, HLA-DQA1 LPAR6, HLA-DQA1 LTB, HLA-DQA1 CD7, HLA- DQA1_SLC2A5, HLA-DQA1 FCMR, HLA-DQA1 DLK1, HLA-DQA1 JAML, HLA- DQA1 CLEC9A, CD96 VAMP5, CD96 LPAR6, CD96 LTB, CD96 CD7, CD96 SLC2A5, CD96 EREG, CD96 FCMR, CD96 DLK1, CD96 JAML, SLC5A3 VAMP5, SLC5A3 CALCRL, SLC5A3 LPAR6, SLC5A3 LTB, SLC5A3 CD7, SLC5A3 SLC2A5, SLC5A3 FCMR, SLC5A3 DLK1, SLC5A3 CLEC7A, SLC5A3 JAML, VAMP5 CALCRL, VAMP5 LPAR6, VAMP5 NINJ1, VAMP5 LTB, VAMP5 SLC2A5, VAMP5 EREG, VAMP5 FCMR, VAMP5 DLK1, VAMP5 JAML, VAMP5 CLEC9A, CALCRL LPAR6, CALCRL LTB, CALCRL CD7, CALCRL SLC2A5, CALCRL FCMR, CALCRL DLKl, CALCRL CLEC7A, CALCRL JAML, CALCRL CLEC9A, LPAR6 LTB, LPAR6 CD7, LPAR6 SLC2A5, LPAR6 EREG, LPAR6 FCMR, LPAR6 CLEC7A, LPAR6 JAML, LPAR6 CLEC9A, NINJ1 LTB, NINJ1 SLC2A5, NINJ1 EREG, NINJ1 FCMR, NINJ1 DLK1, NINJ1 JAML, LTB CD7, LTB SLC2A5, LTB FCMR, LTB DLKl, LTB CLEC7A, LTB JAML, LTB CLEC9A, CD7 SLC2A5, CD7 EREG, CD7 FCMR, CD7 CLEC7A, CD7 JAML, CD7 CLEC9A, SLC2A5 EREG, SLC2A5 FCMR, SLC2A5 DLK1, SLC2A5 CLEC7A, SLC2A5 JAML, SLC2A5 CLEC9A, EREG DLKl, EREG CLEC7A, FCMR CLEC7A, FCMR JAML, FCMR CLEC9A, DLK1 CLEC7A, DLK1 CLEC9A, CLEC7A JAML, CLEC7A CLEC9A, JAML CLEC9A

C.6. Overlap of correlations

List of gene pairs across all AML datasets filtered for pairs that had a significant positive correlation in the leukemic cells for all correlation algorithms in all datasets and no correlation in the healthy cells from AML patients from all datasets with all algorithms.6 gene pairs were found:

List C.6:

CD74 LTB, CD74 SLC2A5, HLA-DRA LTB, CD52 RALA, IGHM SELL,

SLC2A5 FCMR

D. Overlap from all datasets and correlations

D.7. Union of correlations

List of gene pairs across all AML datasets filtered for pairs that had a significant positive correlation in the leukemic cells for at least one algorithm in at least one dataset and no correlation in healthy donors and healthy cells of AML patients in the same datasets with the same algorithms.559 gene pairs were found:

List D.7:

CD74 CD164, CD74 CD52, CD74 HCST, CD74 CD34, CD74 HSPA5, CD74 MGST1, CD74 CD47, CD74 TFPI, CD74 SELL, CD74 CD82, CD74 CD69, CD74 LST1, CD74 NDFIP1, CD74 IFITM3, CD74 RALA, CD74 RAB11A, CD74 SELENOK, CD74 RAMP1, CD74 CD96, CD74 SLC5A3, CD74 VAMP5, CD74 CALCRL, CD74 LPAR6, CD74 NINJ1, CD74 LTB, CD74 CD7, CD74 SLC2A5, CD74 FCMR, CD74 CLEC7A, CD74 JAML, CD74 CLEC9A, HLA-DRA CD52, HLA-DRA HCST, HLA-DRA CD34, HLA-DRA MGST1, HLA-DRA CD47, HLA-DRA TFPI, HLA- DRA IGHM, HLA-DRA SELL, HLA-DRA CD82, HLA-DRA CD69, HLA-DRA LST1, HLA-DRA NDFIPl, HLA-DRA IFITM3, HLA-DRA RALA, HLA-DRA RAB11A, HLA- DRA SELENOK, HLA-DRA HLA-DRB5, HLA-DRA RAMPl, HLA-DRA CD96, HLA- DRA SLC5A3, HLA-DRA VAMP5, HLA-DRA CALCRL, HLA-DRA LPAR6, HLA- DRA NINJl, HLA-DRA LTB, HLA-DRA CD7, HLA-DRA SLC2A5, HLA-DRA FCMR, HLA-DRA CLEC7A, HLA-DRA JAML, HLA-DRA CLEC9A, CD164 CD52, CD164 HCST, CD164_CD34, CD164 HSPA5, CD164 MGST1, CD164 CD47, CD164 TFPI, CD164 IGHM, CD164 SELL, CD164 CD82, CD164 CD69, CD164 LST1, CD164 NDFIP1, CD164 HLA-DRB5, CD164 RAMP1, CD164 SLC5A3, CD164 LPAR6, CD164 NINJ1, CD164 LTB, CD164 CD7, CD164 SLC2A5, CD164 FCMR, CD164 CLEC7A, CD164 JAML, CD52 HCST, CD52 CD34, CD52 MGST1, CD52 CD47, CD52 TFPI, CD52 SELL, CD52 CD82, CD52 LST1, CD52 NDFIP1, CD52 IFITM3, CD52 RALA, CD52 RAB11A, CD52 SELENOK, CD52 HLA-DRB5, CD52 RAMP1, CD52 IGLL1, CD52 HLA-DQA1, CD52 CD96, CD52 VAMP5, CD52 CALCRL, CD52 LTB, CD52 CD7, CD52 SLC2A5, CD52 DLK1, CD52 CLEC7A, CD52 CLEC9A, HCST CD34, HCST HSPA5, HCST CD47, HCST TFPI, HCST SELL, HCST CD82, HCST CD69, HCST LST1, HCST NDFIPl, HCST IFITM3, HCST RALA, HCST RAB11A, HCST SELENOK, HCST HLA-DRB5, HCST HLA-DQA1, HCST CD96, HCST SLC5A3, HCST VAMP5, HCST CALCRL, HCST LPAR6, HCST NINJ1, HCST LTB, HCST CD7, HCST SLC2A5, HCST FCMR, HCST DLKl, HCST CLEC7A, HCST JAML, HCST CLEC9A, CD34 MGST1, CD34 CD47, CD34 TFPI, CD34 IGHM, CD34 SELL, CD34 CD82, CD34 CD69, CD34 LST1, CD34 NDFIP1, CD34 IFITM3, CD34 RALA, CD34 RAB11A, CD34 SELENOK, CD34 HLA-DRB5, CD34 RAMP1, CD34 IGLL1, CD34 HLA-DQA1, CD34 CD96, CD34 SLC5A3, CD34 VAMP5, CD34 CALCRL, CD34 LPAR6, CD34 LTB, CD34 CD7, CD34 SLC2A5, CD34 FCMR, CD34 DLK1, CD34 CLEC7A, CD34 CLEC9A, HSPA5 MGST1, HSPA5 CD47, HSPA5 SELL, HSPA5 LST1, HSPA5 IFITM3, HSPA5 SELENOK, HSPA5 HLA-DRB5, HSPA5 IGLL1, HSPA5 HLA-DQA1, HSPA5 CD96, HSPA5 VAMP5, HSPA5 NINJ1, HSPA5 LTB, HSPA5 CD7, HSPA5 SLC2A5, HSPA5 EREG, HSPA5 DLK1, HSPA5 CLEC7A, HSPA5 JAML, HSPA5 CLEC9A, MGST1 IGHM, MGST1 SELL, MGST1 CD69, MGST1 LST1, MGST1 NDFIP1, MGST1 IFITM3, MGST1 RALA, MGST1 RAB11A, MGST1 SELENOK, MGST1 HLA-DRB5, MGST1 RAMP1, MGST1 IGLL1, MGST1 HLA-DQA1, MGST1 CD96, MGST1 VAMP5, MGST1 NINJ1, MGST1 SLC2A5, MGST1 EREG, MGST1 DLK1, MGST1 CLEC9A, CD47 TFPI, CD47 IGHM, CD47 SELL, CD47 CD82, CD47 CD69, CD47 LST1, CD47 NDFIP1, CD47 IFITM3, CD47 RALA, CD47 HLA-DRB5, CD47 RAMP1, CD47 HLA-DQA1, CD47 CD96, CD47 SLC5A3, CD47 VAMP5, CD47 CALCRL, CD47 LPAR6, CD47 LTB, CD47 CD7, CD47 SLC2A5, CD47 FCMR, CD47 CLEC7A, CD47 JAML, CD47 CLEC9A, TFPI IGHM, TFPI SELL, TFPI CD82, TFPI CD69, TFPI LST1, TFPI NDFIPl, TFPI RALA, TFPI SELENOK, TFPI HLA-DRB5, TFPI RAMP1, TFPI HLA-DQA1, TFPI CD96, TFPI SLC5A3, TFPI VAMP5, TFPI CALCRL, TFPI LPAR6, TFPI NINJ1, TFPI LTB, TFPI CD7, TFPI SLC2A5, TFPI FCMR, TFPI CLEC7A, TFPI JAML, IGHM SELL, IGHM CD82, IGHM CD69, IGHM LST1, IGHM NDFIPl, IGHM IFITM3, IGHM RALA, IGHM HLA-DRB5, IGHM RAMP1, IGHM HLA-DQA1, IGHM CD96, IGHM SLC5A3, IGHM CALCRL, IGHM LPAR6, IGHM NINJ1, IGHM CD7, IGHM SLC2A5, IGHM FCMR, IGHM CLEC7A, IGHM JAML, IGHM CLEC9A, SELL CD82, SELL CD69, SELL LST1, SELL NDFIPl, SELL IFITM3, SELL RALA, SELL RAB11A, SELL SELENOK, SELL HLA-DRB5, SELL RAMP1, SELL HLA-DQA1, SELL CD96, SELL SLC5A3, SELL VAMP5, SELL CALCRL, SELL LPAR6, SELL NINJ1, SELL LTB, SELL CD7, SELL SLC2A5, SELL FCMR, SELL CLEC9A, CD82 CD69, CD82 LST1, CD82 NDFIP1, CD82 IFITM3, CD82 RALA, CD82 RAB11A, CD82 SELENOK, CD82 HLA-DRB5, CD82 RAMP1, CD82 IGLL1, CD82 VAMP5, CD82 CALCRL, CD82 NINJ1, CD82 LTB, CD82 CD7, CD82 EREG, CD82 DLK1, CD82 CLEC7A, CD82 CLEC9A, CD69 LST1, CD69 NDFIP1, CD69 RALA, CD69 RAB11A, CD69 SELENOK, CD69 HLA-DRB5, CD69 RAMP1, CD69 HLA-DQA1, CD69 SLC5A3, CD69 CALCRL, CD69 LPAR6, CD69 NINJ1, CD69 CD7, CD69 SLC2A5, CD69 EREG, CD69 FCMR, CD69 DLK1, CD69 CLEC7A, CD69 JAML, LST1 NDFIP1, LST1 IFITM3, LST1 RAB11A, LST1 SELENOK, LST1 RAMP1, LST1 HLA-DQA1, LST1 CD96, LST1 SLC5A3, LST1 VAMP5, LST1 CALCRL, LST1 LPAR6, LST1 NINJ1, LST1 LTB, LST1 CD7, LST1 SLC2A5, LST1 FCMR, LST1 DLK1, LST1 CLEC7A, LST1 JAML, LST1 CLEC9A, NDFIP1 IFITM3, NDFIPI RALA, NDFIPI RABI 1A, NDFIPI HLA- DRB5, NDFIPI RAMPI, NDFIPI IGLLI, NDFIP1 HLA-DQA1, NDFIP1 SLC5A3, NDFIP1 VAMP5, NDFIP1 NINJ1, NDFIP1 LTB, NDFIP1 CD7, NDFIP1_SLC2A5, NDFIP1 EREG, NDFIPI FCMR, NDFIPI DLKl, NDFIP1_CLEC7A, NDFIP1 JAML, NDFZP1 CLEC9A, IFITM3 RALA, IFITM3 RAB11A, IFITM3 SELENOK, IFITM3 HLA- DRB5, IFITM3 RAMP1, IFITM3 IGLL1, IFITM3 HLA-DQA1, IFITM3 CD96, IFITM3 SLC5A3, IFITM3 CALCRL, IFITM3 NINJ1, IFITM3 LTB, IFITM3 SLC2A5, IFITM3 FCMR, IFITM3 DLK1, IFITM3 CLEC7A, IFITM3 JAML, IFITM3 CLEC9A, RALA RAB11A, RALA SELENOK, RALA HLA-DRB5, RALA RAMP1, RALA IGLL1, RALA HLA-DQA1, RALA CD96, RALA SLC5A3, RALA VAMP5, RALA CALCRL, RALA LPAR6, RALA NINJ1, RALA LTB, RALA CD7, RALA SLC2A5, RALA DLKl, RAB11A SELENOK, RABI 1A HLA-DRB5, RABI 1A RAMP1, RABI 1A IGLL1, RAB11A HLA-DQA1, RAB11A CD96, RABI 1A VAMP5, RABI 1A CALCRL, RAB11A LTB, RAB11A DLK1, RAB11A CLEC7A, RAB11A JAML, RAB11A CLEC9A, SELENOK HLA-DRB5, SELENOK RAMP1, SELENOK IGLL1, SELENOK HLA-DQA1, SELENOK CD96, SELENOK VAMP5, SELENOK CALCRL, SELENOK NINJ1, SELENOK LTB, SELENOK CD7, SELENOK EREG, SELENOK CLEC7A, HLA-DRB5 IGLL1, HLA-DRB5 HLA-DQA1, HLA-DRB5 CD96, HLA-DRB5 SLC5A3, HLA-DRB5 VAMP5, HLA-DRB5 NINJ1, HLA-DRB5 LTB, HLA- DRB5 CD7, HLA-DRB5 SLC2A5, HLA-DRB5 EREG, HLA-DRB5 FCMR, HLA- DRB5 DLK1, HLA-DRB5 JAML, HLA-DRB5 CLEC9A, RAMP1 HLA-DQA1, RAMP1 SLC5A3, RAMP1 VAMP5, RAMP1 CALCRL, RAMP1 LPAR6, RAMP1 NINJ1, RAMP1 LTB, RAMP1 SLC2A5, RAMPI EREG, RAMP1 FCMR, RAMP1 DLK1, RAMP1 CLEC7A, RAMP1 JAML, RAMP1 CLEC9A, IGLL1 HLA-DQA1, IGLL1 VAMP5, IGLL1 NINJ1, IGLL1 CD7, IGLLI EREG, IGLL1 DLK1, IGLL1 CLEC9A, HLA-DQA1 CD96, HLA-DQA1 SLC5A3, HLA-DQA1 VAMP5, HLA- DQA1 LPAR6, HLA-DQA1 LTB, HLA-DQA1 CD7, HLA-DQA1 SLC2A5, HLA- DQA1 FCMR, HLA-DQA1 DLK1, HLA-DQA1 JAML, HLA-DQA1 CLEC9A, CD96 VAMP5, CD96 LTB, CD96 CD7, CD96 SLC2A5, CD96 EREG, CD96 DLK1, CD96 JAML, SLC5A3 VAMP5, SLC5A3 CALCRL, SLC5A3 LPAR6, SLC5A3 LTB, SLC5A3 CD7, SLC5A3 SLC2A5, SLC5A3 FCMR, SLC5A3 DLK1, SLC5A3 CLEC7A, SLC5A3 JAML, VAMP5 CALCRL, VAMP5 LPAR6, VAMP5 NINJ1, VAMP5 LTB, VAMP5 SLC2A5, VAMP5 EREG, VAMP5 FCMR, VAMP5 DLK1, VAMP5 JAML, VAMP5 CLEC9A, CALCRL LPAR6, CALCRL LTB, CALCRL CD7, CALCRL SLC2A5, CALCRL FCMR, CALCRL DLKl, CALCRL CLEC7A, CALCRL JAML, CALCRL CLEC9A, LPAR6 LTB, LPAR6 CD7, LPAR6 SLC2A5, LPAR6 EREG, LPAR6 FCMR, LPAR6 CLEC7A, LPAR6 JAML, LPAR6 CLEC9A, NINJ1 LTB, NINJ1 SLC2A5, NINJ1 EREG, NINJ1 DLK1, NINJI JAML, LTB CD7, LTB SLC2A5, LTB FCMR, LTB DLKl, LTB CLEC7A, LTB JAML, LTB CLEC9A, CD7 SLC2A5, CD7 EREG, CD7 FCMR, CD7 CLEC7A, CD7 JAML, CD7 CLEC9A, SLC2A5 FCMR, SLC2A5 DLK1, SLC2A5 CLEC7A, SLC2A5 JAML, SLC2A5 CLEC9A, EREG DLKl, EREG CLEC7A, FCMR CLEC7A, FCMR JAML, FCMR CLEC9A, DLK1 CLEC7A, DLK1 CLEC9A, CLEC7A JAML, CLEC7A CLEC9A, JAML CLEC9A

D.8. Overlap of correlations

List of gene pairs across all AML datasets filtered for pairs that had a significant positive correlation in the leukemic cells for all algorithms in all datasets an no correlation in healthy donors and healthy cells of AML patients in all datasets with all algorithms.2 gene pairs were found:

List D.8:

HLA-DRA LTB, CD52 RALA

In summary, a novel cross-entity score was identified to determine risk in AML, which is applicable to quick and economic risk stratification methods such as FACS or gene expression analyses. Moreover, novel targets of immature AML cells for bispecific or combinatorial therapeutic strategies in AML were identified. CITED LITERATURE

Dohner, H., Weisdorf, D. J. & Bloomfield, C. D. Acute Myeloid Leukemia. N Engl J Med 373, 1136-1152 (2015). https://doi.org:10.1056/NEJMral406184

Goldman, J. M. & Melo, J. V. Chronic myeloid leukemia— advances in biology and new approaches to treatment. N Engl J Med 349, 1451-1464 (2003). https://doi.org: 10.1056/NEJMra020777

Pui, C. H., Relling, M. V. & Downing, J. R. Acute lymphoblastic leukemia. N Engl J Med 350, 1535-1548 (2004). https://doi.org: 10.1056/NEJMra023001

Siegel, R. L., Miller, K. D. & Jemal, A. Cancer Statistics, 2017. CA Cancer J Clin 67, 7-30 (2017). https://doi.org: 10.3322/caac.21387

Dores, G. M., Devesa, S. S., Curtis, R. E., Linet, M. S. & Morton, L. M. Acute leukemia incidence and patient survival among children and adults in the United States, 2001-2007. Blood 119, 34-43 (2012). https://doi.org: 10.1182/blood-2011-04-347872

Sant, M. et al. Incidence of hematologic malignancies in Europe by morphologic subtype: results of the HAEMACARE project. Blood 116, 3724-3734 (2010). https://doi.org: 10.1182/blood-2010-05-282632

Khoury, J.D., Solary, E., Abla, O. et al., The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Myeloid and Histiocytic/Dendritic Neoplasms" Leukemia. 36 (7): 1703-1719 (2022). doi:10.1038/s41375-022-01613-l. PMC 9252913. PMID 35732831.

Labrijn, A.F., Janmaat, M.L., Reichert, J.M. et al. Bispecific antibodies: a mechanistic review ofthe pipeline. Nat Rev Drug Di scov 18, 585-608 (2019). https://doi.org/10.1038/s41573-019- 0028-1

US2004013210

US20060024298

Ng, S.W.L. et al., A 17-gene sternness score for rapid determination of risk in acute leukaemia, Nature, Dec 15;540(7633):433-437 https://doi.org/10.1038/nature20598(2016). https://doi.org/10.1038/nature20598 Dohner H. et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood 129, 424-47 (2017). https://doi.org/10.1182/blood- 2016-08-733196

Alaggio, R. et al. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms. Leukemia 36, 1720-1748 (2022). https ://doi. org: 10.1038/s41375-022-01620-2

Arber, D. A. et al. International Consensus Classification of Myeloid Neoplasms and Acute Leukemias: integrating morphologic, clinical, and genomic data. Blood 140, 1200-1228 (2022). https://doi.org: 10.1182/blood.2022015850

Bottomly, D. et al. Integrative analysis of drug response and clinical outcome in acute myeloid leukemia. Cancer Cell 40, 850-864.e859 (2022). https://doi.org: 10.1016/j.ccell.2022.07.002

Tyner, J. W. et al. Functional genomic landscape of acute myeloid leukaemia. Nature 562, 526- 531 (2018). https://doi.org: 10.1038/s41586-018-0623-z

Beneyto-Calabuig, S. et al. Clonally resolved single-cell multi-omics identifies routes of cellular differentiation in acute myeloid leukemia. Cell Stem Cell, 30(5), 706-721. e708 (2023). https ://doi. org/ 10.1016/j . stem.2023.04.001

Lasry, Aet al. An inflammatory state remodels the immune microenvironment and improves risk stratification in acute myeloid leukemia. Nat Cancer, 4(1), 27-42 (2023). https ://doi. org/10.1038/s43018-022-00480-0

Su, C. et al. Cell-type-specific co-expression inference from single cell RNA-sequencing data. Nature Communications, 14, 4846 (2023) https://doi.org/10.1038/s41467-023-40503-7

Van Dijk, D et al. Recovering Gene Interactions from Single-Cell Data Using Data Diffusion. Cell, 174(3), 716-729. e727 (2018). https://doi.Org/10.1016/j.cell.2018.05.061

Triana, S. et al. Single-cell proteo-genomic reference maps of the hematopoietic system enable the purification and massive profiling of precisely defined cell states. Nature Immunology 22, 1577-1589 (2021). https://doi.org: 10.1038/s41590-021-01059-0

Dohner H. et al. Diagnosis and management of AML in adults: 2022 recommendations from an international expert panel on behalf of the ELN. Blood (2022) 140(12): 1345-1377.

Claims

1. A method for assessing acute myeloid leukemia (AML) in a subject suspected to suffer therefrom comprising the steps of: a) determining in a sample of said subject the amount of at least one biomarker selected from the group consisting of CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RABI 1 A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA-DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and JAML; b) comparing the said amount of the at least one biomarker to a reference; and c) assessing AML based on the comparison.

2. The method of claim 1, wherein said assessing comprises (i) diagnosing AML, (ii) determining whether the subject is at risk of worsening of AML or symptoms associated therewith or (iii) determining whether the subject is at risk of death.

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

4. The method of any one of claim 1 to 3, wherein the reference is derived from at least one subject known to suffer from AML.

5. The method of claim 4, wherein the amount determined for the at least one biomarker in step a) which is identical or increased compared to the reference is indicative for a subject suffering from AML whereas an amount for the at least one biomarker determined in step a) which is reduced compared to the reference is indicative for a subject not suffering from AML.

6. The method of any one of claims 1 to 3, wherein the reference is derived from at least one subject known not to suffer from AML.

7. The method of claim 6, wherein an amount for the at least one biomarker determined in step a) which is identical or reduced compared to the reference is indicative for a subject not suffering from AML whereas an amount for the at least one biomarker determined in step a) which is increased compared to the reference is indicative for a subject suffering from AML.

8. A method for generating a bispecific binding agent comprising the steps of: a) determining the abundance of a plurality of biomarkers present in each single cell of (i) leukemic cells, preferably, AML cells, and control cells and (ii) leukemic stem cells, preferably, AML stem cells, and control cells; b) identifying among the plurality of biomarkers those which are present above a pre-determined threshold on (i) leukemic cells, preferably, AML cells, but not control cells and (ii) leukemic stem cells, preferably, AML stem cells, but not control cells; c) identifying among the biomarkers present above the pre-defined threshold biomarker pairs which are present in one single cell; and d) generating a bispecific binding agent comprising a first target binding domain that specifically binds to a first target and a second target binding domain that specifically binds to a second target, wherein the first and the second target are biomarker pairs identified in step c) and are different from each other.

9. A bispecific binding agent comprising a first target binding domain that specifically binds to a first target and a second target binding domain that specifically binds to a second target, wherein

(ii) the first target and the second target are different from each other; and

(iii) the first and the second target are selected from the group consisting of CLEC7A, CLEC9A, HCST, LST1, LTB, IFITM3, CD74, HLA-DRA, CD 164, CD52, CD34, HSPA5, MGST1, CD47, TFPI, IGHM, SELL, CD82, CD69, NDFIP1, RALA, RAB11A, SELENOK, HLA-DRB5, RAMP1, IGLL1, HLA- DQA1, CD96, SLC5A3, VAMP5, CALCRL, LPAR6, NINJ1, CD7, SLC2A5, EREG, FCMR, DLK1, and JAML.

10. The bispecific binding agent of claim 9, wherein said bispecific binding agent is obtainable by the method of claim 8.

11. The bispecific binding agent of claim 9 or 10, wherein said bispecific binding agent is a bispecific antibody.

12. A pharmaceutical composition comprising the bispecific binding agent of any one of claim 9 to 11.

13. A bispecific binding agent of any one of claims 9 to 11 for use in treating and/or preventing cancer.

14. The bispecific binding agent for use of claim 13, wherein said cancer is leukemia and, preferably, myeloid leukemia.

15. The bispecific binding agent for use of claim 13 or 14, wherein said leukemia is acute myeloid leukemia (AML).