Surface SRRM2 as biomarker of prognosis and molecular target for immunotherapy of aml

Abstract

The invention relates to a method for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative or positive prognosis based on the level of SRRM2 expression on the cell surface of blast cells of said subject and the use of the level of SRRM2 expression on the cell surface of blast cells as prognosis marker for AML. The invention further relates to medical use of SRRM2-targeting therapies for treating SRRM2-positive AML patiens.

Description

New International patent applicationApplicant: Zeno Therapeutics (Shenzhen)Our ref.: EXI18775PCTSURFACE SRRM2 AS BIOMARKER OF PROGNOSIS AND MOLECULAR TARGET FOR IMMUNOTHERAPY OF AMLCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority of Chinese patent application 202510277955.X filed on 10 March 2025, the content of which is hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The invention relates to a method for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a relatively negative or positive prognosis based on the level of SRRM2 expression on the cell surface of blast cells of said subject and the use of the level of SRRM2 expression on the cell surface of blast cells as prognostic marker for AML. The invention further relates to medical use of SRRM2 -targeting therapies for treating surface SRRM2-positive AML patiens.BACKGROUND

[0003] Acute myeloid leukemia (AML) is a prevalent hematologic malignancy marked by considerable heterogeneity. Despite recent advancements in therapy, the standard treatment regimen remains the "7 + 3" protocol, which consists of seven days of cytarabine followed by three days of an anthracycline. Unfortunately, the overall clinical outcomes for AML patients remain suboptimal, with long-term survival rates hovering around only 30%. These findings highlight the urgent need for the development of new targeted therapies for both relapsed and refractory cases. A significant barrier to developing new therapies is the scarcity of suitable target antigen that are ideally expressed exclusively in AML blasts while being absent or minimally present in normal hematopoietic stem cells and other healthy tissues. This specificity is crucial to mitigate the risk of serious on-target / off-tumor adverse effects.

[0004] Recent studies have indicated that post-translational phosphorylation of splicing factors contributes to cytarabine resistance in AML. One such splicing factor consistently overexpressed in AML is serine / arginine repetitive matrix protein 2 (SRRM2 / SRm300), an RNA-binding protein rich in serine-arginine (SR) motifs that plays a vital role in the function of nuclear speckles. Although the absolute levels of SRRM2 mRNA do not correlate with clinical parameters in AML, the phosphorylation of SRRM2 has been shown to directly correlate with cytarabine resistance, asreported by Morales et al. They also observed elevated cytoplasmic SRRM2 levels in AML blasts from patients resistant to cytarabine. SRRM2 phosphorylation is essential for its translocation to the cytoplasm, a process also observed in neurons affected by neurodegenerative diseases, resulting in the cytoplasmic accumulation of the protein. Although the molecular function of extra-nuclear SRRM2 remains unclear, these findings underscore its potential role in various diseases, including cancer.SUMMARY OF THE INVENTION

[0005] The invention relates to a method for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative or positive prognosis, comprising determining in vitro the level of SRRM2 expression on the cell surface of blast cells of said subject, wherein (i) a subject having a level of 20% or less SRRM2 expressing blast cells is characterized as being SRRM2-negative, and (ii) a subject having a level of more than 20% SRRM2 expressing blast cells is characterized as being SRRM2-positive, wherein an SRRM2-negative subject has a positive prognosis, whereas an SRRM2-positive subject has a negative prognosis.

[0006] The invention also relates to an in vitro use of the level of SRRM2 expression on the cell surface of blast cells of a subject diagnosed with acute myeloid leukemia (AML) for the evaluation whether said subject will have a negative or positive prognosis, wherein (i) a subject having a level of 20% or less SRRM2 expressing blast cells is characterized as SRRM2-negative and has a positive prognosis, whereas (ii) a subject having a level of more than 20% SRRM2 expressing blast cells is characterized as SRRM2-positive and has a negative prognosis.

[0007] The invention also relates to a use of a binding agent which is capable of specifically binding SRRM2 on the surface of a blast cell for the manufacture of a composition or a kit for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative or positive prognosis.

[0008] The invention also relates to an SRRM2 -targeting therapy for use in treating AML in a subject, wherein the subject is characterized by having a level of more than 20% SRRM2 expressing blast cells.

[0009] The invention also relates to a use of an SRRM2 -targeting therapy for the manufacture of a medicament for treating AML in a subject, wherein the subject is characterized by having a level of more than 20% SRRM2 expressing blast cells.

[0010] The invention also relates to a method of treating AML comprising administering to a subject in need thereof an effective amount of an SRRM2 -targeting therapy, wherein the subject is characterized by having a level of more than 20% SRRM2 expressing blast cells.

[0011] The invention also relates to a method of stratifying and treating a subject diagnosed with AML, comprising evaluating whether said subject will have a negative or positive prognosis according to the method of the invention, and administering to the subject an SRRM2 -targeting therapy, if the subject is characterized as being SRRM2-positive and / or evaluated with a negative prognosis.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1: SRRM2 exposed on the surface of AML cell lines. (A) Flow cytometric analysis of SRRM2 expression in AML cell lines (MOLM-13, MV4-11, SKM-1, and HL-60) revealed binding of the EX-02 antibody to the surface of vital cells (upper panel) and intracellular SRRM2 (lower panel). (B) surface SRRM2 expression in the AML cell line MOLM-13 was confirmed by fluorescence confocal microscope. SRRM2 (green) was localized both on the cell surface of vital cells and intracellularly in permeabilized cells, corroborating results from flow cytometry. Nuclei were counterstained with DAPI (blue).

[0013] Figure 2: SRRM2 is exposed only on the surface of AML blasts but not on normal blood cells (A) Flow cytometric analysis of subgroups of blood cells isolated from the peripheral blood of an AML patients. Upper panel: staining of vital cells with the EX-02 antibody demonstrated that SRRM2 is detectable on the surface of blast cellss, whereas normal blood cells (lymphocytes, monocytes, granulocytes) show only marginal background staining. Lower panel: intracellular staining of the same types of permeabilized cells showed positive intracellular SRRM2 staining of all cell types. (B) same as (A) but with peripheral blood cells derived from a healthy donor. No surface SRRM2 was detectable after EX-02 staining. (C) same staining as in (A) but of bone marrow-derived cells of an AML patients. Again, only blast cells stained surface SRRM2 positive with EX-02 antibody, while all other types of cells showed only intracellular SRRM2 staining. (D) cells isolated from the bone marrow of a patient suffering from iron deficiency anemia (IDA). No surface SRRM2 was detectable on lymphocytes, monocytes, granulocytes or hematopoietic stem cells (HSC). Taken together, surface SRRM2 is exclusively detectable on malignant AML blast cells, but not on normal blood cells.

[0014] Figure 3 Correlation between SRRM2 surface exposure and clinical features in AML patients. (A-B) SRRM2 exposure was analyzed by flow cytometry in de novo vs. secondary AML(A) and various WHO-FAB subtypes (MO, Ml, M2, M4) (B) no significant differences were observed between these groups. (C) SRRM2 surface exposure in patients with standard inductive chemotherapy failure (Not complete response, NCR) was significantly higher than in patients with complete response (CR). (D) SRRM2 surface exposure in adverse and intermediate-risk patients stratified by the 2022 NCCN risk classification remarkably increased comparing to in favorable-risk patients. (E) Relapsed AML patients showed higher SRRM2 surface exposure compared to newly diagnosed (ND) patients. (F) Longitudinal analysis of seven patients with AML revealed that the SRRM2 exposure on surface of AML blast cells at relapse increased considerably. (G-H) The SRRM2 exposure on surface of AML blast cells from ND patients (G) and Relapsed patients (H) greatly increased compared to the other blood cells. (I-J) The complete remission rates (CRR) in AML patients with SRRM2 surface exposure positive (SRRM2Pos) significantly lower than in patients with SRRM2 surface exposure negative (SRRM2Neg), and more SRRM2 surface exposure along with lower CRR. (K-L) Kaplan-Meier analysis showed shorter relapse-free survival (RFS) in SRRM2Pospatients, with high SRRM2 expression linked to reduced RFS. (M-N) SRRM2Pospatients had shorter overall survival (OS) than SRRM2Negpatients. *P<0.05;**P<0.01;***P<0.001.

[0015] Figure 4: SRRM2 expression in non-malignant bone marrow and normal tissues. (A) Flow cytometric analysis of hematopoietic cells from patients with iron-deficiency anemia (IDA) demonstrated minimal surface expression of SRRM2 in lymphocytes, monocytes, neutrophils, and hematopoietic stem cells (HSCs); (B) there was strong nuclear expression of SRRM2 across all cell types, with nearly complete positivity noted. (C) Immunohistochemical analysis revealed no significant surface expression of SRRM2 various normal tissues, including cerebellum, colon, liver, lung, skin, pancreas, heart, kidney, lymph node, muscle and spleen.

[0016] Figure 5: ROC curve analysis for identifying optimal expression level cut-off in predicting response to induction chemotherapy. The ROC curve was used to evaluate SRRM2 expression levels as a predictor of remission following standard induction chemotherapy in ND AML patients. The horizontal axis displayed the false positive rate (1 - specificity), and the vertical axis showed the true positive rate (sensitivity). The area under the curve (AUC) was 0.711, indicating moderate predictive performance of SRRM2 levels for distinguishing between remission and non-remission. The Youden index was 0.37, reflecting the optimal balance of sensitivity and specificity. The cutoff value of 30.14 was determined as the point at which SRRM2 levels most accurately predicted remission.DETAILED DESCRIPTION

[0017] Acute myeloid leukemia (AML) is a life-threatening hematological malignancy that poses significant treatment challenges, characterized by a poor median 5-year survival rate. Despite the approval of several new therapies in recent years, advancements in treatment are often hindered by the scarcity of suitable target molecules for developing specific therapies. Serine / arginine repetitive matrix 2 (SRRM2) is known as a spliceosomal and nuclear speckle protein.

[0018] The inventors of the present application have recently developed a novel SRRM2-specific antibody named EX-02 and demonstrated that this protein was ectopically expressed on the surface of cancer cells from certain solid tumors and malignant blasts from multiple myeloma patients. Notably, in these studies, SRRM2 was predominantly localized within the nucleus of normal blood cells and adjacent normal tissues, suggesting that SRRM2 translocation is cancer specific. This makes surface SRRM2 an attractive target antigen for novel anticancer therapies.

[0019] Now, the inventors of the present application have surprisingly found that SRRM2 is highly expressed on the surface of AML cell lines and primary blasts from AML patients. Conversely, SRRM2 was primarily localized within the nuclei of normal hematopoietic stem cells (HSCs), mature blood cells, and various normal tissues examined. A systematic analysis of patient data revealed a direct correlation between surface SRRM2 levels and key clinical parameters such as genetic mutations, relapse rates, treatment resistance, and overall clinical outcomes. Furthermore, using a human xenograft model of AML, the inventors illustrate the anti-leukemic efficacy of SRRM2-specific CAR-T cells.

[0020] With other words, the inventors of the present application have found that SRRM2 is highly expressed on the surface of both AML cell lines and patient-derived AML blasts but not on normal blood cells. Notably, it was found that elevated levels of surface SRRM2 correlated with advanced and aggressive disease characteristics, as well as resistance to treatment. Surface SRRM2 also served as an independent prognostic marker for poor outcomes in AML patients. Furthermore, the inventors of the present application demonstrated in a human xenograft model of AML that SRRM2-specific CAR T-cells exhibited significant anti-tumor activity, leading to markedly prolonged survival of treated animals.

[0021] The surprising findings indicate that SRRM2 is prominently exposed on the surface of AML blasts and that surface expression levels directly correlate with critical clinical parameters. This specific ectopic expression on AML cells positions surface SRRM2 as a promising target for innovative treatments like SRRM2-specific CAR-T cell therapy and as a potential biomarker forpatient stratification. Moreover, surface SRRM2 emerges as a promising new biomarker and druggable therapeutic target for the development of novel AML treatments.

[0022] Accordingly, the present invention envisions a method for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative or positive prognosis. Said method comprises determining the level of SRRM2 expression on the cell surface of blast cells of said subject (e.g. obtained from said subject), preferably in vitro. For this, a subject having a level of about 20% or less SRRM2 expressing blast cells is characterized as being SRRM2-negative, whereas a subject having a level of more than about 20% SRRM2 expressing blast cells is characterized as being SRRM2-positive. An SRRM2-negative subject may have a positive prognosis, whereas an SRRM2-positive subject may have a negative prognosis. The method may thus further comprise a step of indicating that the subject has a positive prognosis, if the subject is evaluated as being SRRM2-negative and / or indicating that the subject has a negative prognosis, if the subject is evaluated as SRRM2-positive.

[0023] As used herein, “serine / arginine repetitive matrix protein 2” or “SRRM2” is generally not limited by its origin. However, as used herein, “serine / arginine repetitive matrix protein 2” or “SRRM2” preferably refers to human SRRM2. Human SRRM may have the amino acid sequence as shown in SEQ ID NO: 8 or may be a variant having an amino acid sequence derived from SEQ ID NO: 8. Said variant may comprise a modification of one or more amino acids. Examples of variant include, but are not limited to, protein variants having a sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or even higher sequence identity to SEQ ID NO: 8.

[0024] “Percent (%) sequence identity" with respect to sequences disclosed herein is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are pair-wise identical with the amino acid residues or nucleotides in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. The same is true for nucleotide sequences disclosed herein. For determining sequence identity, uracil (e.g. in RNA) may be considered to be identical to thymine (e.g. in DNA).

[0025] The group of SRRM2- positive subjects may be further subdivided based on the percentage of SRRM2 expressing blast cells. A subject having a level of more than 20% and up to 30% SRRM2 expressing blast cells may be characterized as being SRRM2-positive low. A subject having a level of more than 30% SRRM2 expressing blast cells can be characterized as being SRRM2-positive high.

[0026] A “negative prognosis” of an SSRM2-positive subject can be associated with a higher failure to induction chemotherapy including cytarabine and an anthracycline drug, such as daunorubicin or idarubicin, higher relapse rate, lower complete remission rate (CRR), shorter relapse-free survival (RFS), or shorter overall survival (OS) when compared to SRRM2-negative subjects.

[0027] A “positive prognosis” of an SSRM2-negative subject can be associated with a lower failure to induction chemotherapy including cytarabine and an anthracycline drug, such as daunorubicin or idarubicin, lower relapse rate, higher complete remission rate (CRR), longer relapse-free survival (RFS), longer overall survival (OS) when compared to SRRM2-positive subjects.

[0028] The percentage level of blast cells expressing SRRM2 on their cell surface, as used herein, relates to the percentage of (AML) blast cells expressing SRRM2 on their surface relative to the total number of (AML) blast cells of said subject, which are measured. Here, SRRM2 expression preferably relates to protein expression. Generally, the SRRM2 status of an AML blast cell can be determined using any method known to the skilled person. Determining the percentage of SRRM2-expressing AML blast cells is preferably conducted in vitro on a sample obtained from a subject. A preferred method for determining the percentage of SRRM2-expressing AML blast cells is by flow cytometry (FCM), such as a method essentially described in Example 1, section “flow cytometry”. For example, percentage level of blast cells expressing SSRM2 on their cell surface can essentially be determined as follows:1. Treating peripheral blood from AML patients with erythrocyte lysis buffer and dividing into two tubes for FCM.2. Adding SRRM2 (EX-02) mAb (Eximmium Biotechnologies; Munich, Germany) or an isotype control antibody (cat. No. B355601; Abinvivo, Shanghai, China) and incubating at room temperature (RT) for 40 minutes.3. Washing the cells twice with PBS and then staining with CD34-PE (e.g., cat. no. A07776;Beckman Coulter, Brea, CA), CD117-PE (e.g. cat. no. IM2732; Beckman Coulter), CD33-APC (e.g. cat. no. IA2471; Beckman Coulter), CD45-PC7 (e.g. cat. no. IM3548; Beckman Coulter), and Goat anti-rat IgG Alexa488-coupled antibody (e.g. cat. no. 150165; Abeam) in the dark at RT for 15 minutes.4. Performing the samples with a flow cytometer, such as Cytoflex flow cytometer (Beckman Coulter) and analyzing the results, such as using CytExpert for DxFLEX software.

[0029] The percentage of blast cells expressing SSRM2 on their cell surface is preferably measured in a (biological) sample obtained from subject. The sample may be any sarnie suitable for determining the percentage of blast cells expressing SSRM2 on their cell surface. Such samples include peripheral blood (PB) samples and bone marrow (BM) samples, with PB samples being preferred. In some examples, the sample is a PB sample. In some embodiments, the sample is a BM sample.

[0030] The percentage of blast cells expressing SSRM2 on their cell surface is preferably determined by measuring at least 10,000 total AML blast cells, or determined in a sample of preferably at least 50 pL, more preferably at least 100 pL, of peripheral blood or bone marrow fluid.

[0031] A subject according to the disclosure is one that has been diagnosed with AML. The subject may be newly diagnosed with AML or may be a subject having relapsed AML. The subject may be intended for therapy for treating AML. The subject may also be subject to a therapy for treating AML. The subject may be one that is undergoing or has undergone treatment for AML. In some examples, a method for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative or positive prognosis disclosed herein may be conducted before the subject receives AML treatment.

[0032] Expression of SRRM2 on the cell surface of a blast cell can be determined using a binding agent that can bind to SRRM2. Hence, according to the the methods of the disclosure, SRRM2 expression on the cell surface of blast cells can determined with a binding agent that binds to SRRM2 on the cell surface of SRRM2 expressing cells. Such a binding agent may comprise an antibody or antigen-binding fragment therofs such as an antibody that is capable of specifically binding to (human) SSRM2.

[0033] As set forth above, the present invention comprises the finding that SRRM2 can be detected on the cell surface of AML blast cells. Accordingly, it is particularly preferred that the binding agent which binds to human SRRM2 binds to human SRRM2 on the the surface of a blast cell. It is also particularly preferred that the binding agent binds to non-permeabilised cells.“Permeabilization of cells” refers to the break up of the cell membrane by electrical, mechanical or chemical means, i.e. the cell membrane becomes permeable. However, contrary thereto, the cells bound by the binding agent according to the methods of the present disclosure preferably have an intact cell membrane, on which SRRM2 is expressed, i.e. these cells are non-permeabilised. It is equally envisaged that the SRRM2-specific binding agent used in the context of the present dislcosure binds to human SRRM2 on the surface of a living cell. A “living cell” is a structural and functional unit comprising at least an intact cell membrane, a nucleus, and the cytoplasm, that controls the substances that go into and out of the cell and is able to function independently. Accordingly, a blast cell disclosed herein is preferably a non-permeabilized cell. Accordingly, a blast cell disclosed herein is preferably a living and / or intact blast cell.

[0034] The term “surface” or specifically “cell surface” as used herein means the cell membrane. The cell membrane, which may also be known as plasma membrane, is the thin membrane that surrounds every living cell, delimiting the cell from the environment around it. Enclosed by this cell membrane are the cell’s constituents, often large, water-soluble, highly charged molecules such as proteins, nucleic acids, carbohydrates, and substances involved in cellular metabolism. The cell membrane, therefore, has at least two functions: first, to be a barrier keeping the constituents of the cell in and unwanted substances out and, second, to be a gate allowing transport into the cell of essential nutrients and removal of waste products from the cell. ER-derived vesicles may also participate in the building or formation of the cell membrane.

[0035] A binding agent, such as an antibody that binds to human SRRM2, is preferably one that is capable of binding to an SRRM2 polypeptide extracellularly. In other words, an antibody as described herein may be capable of binding to SRRM2 when it is outside the cell, e.g., when SRRM2 is on the surface of the cell. Accordingly, the antibody, which binds to human SRRM2 is preferably not an intracellular antibody, which is also called an intrabody. An “intrabody” (from intracellular and antibody) is an antibody that works within the cell to bind to an intracellular protein. This requires the expression of the antibody within the target cell, which can be accomplished, e.g. in transgenic animals or by gene therapy. As a result, intrabodies are antibodies that have been modified for intracellular localization and include antibodies, which are produced in prokaryotes or other non-target cells. The term “intrabody” can apply to several types of protein targeting: the antibody may remain in the cytoplasm, or it may have a nuclear localization signal, or it may undergo co-translational translocation across the membrane into the lumen of the endoplasmic reticulum, provided that it is retained in that compartment through a KDEL sequence.

[0036] The term "antibody" generally refers to a proteinaceous binding molecule with immunoglobulin-like functions. Typical examples of an antibody are, but are not limited to, immunoglobulins, as well as derivatives or functional fragments thereof which still retain the binding specificity. Techniques for the production of antibodies are well known in the art. The term "antibody" also includes immunoglobulins (Ig's) of different classes (i.e. IgA, IgG, IgM, IgD, IgE, IgY etc.) and subclasses (such as IgGl, lgG2 etc.), even if recombinantly produced in foreign hosts using techniques known to those skilled in the art. Illustrative examples of an antibody are full length immunoglobulins, Fab fragments, F(ab')2, Fv fragments, single-chain Fv fragments (scFv), diabodies or domain antibodies. Domain antibodies may be single-domain antibodies, single variable domain antibodies or immunoglobulin single variable domain having only one variable domain, which may be VH or VL, that specifically bind an antigen or epitope independently of other V regions or domains. The definition of the term "antibody" also includes embodiments such as chimeric antibodies, humanized antibodies, monovalent antibodies, polyvalent antibodies, low-molecular antibodies, a diabody or a scFv. The term “antibody” may also include fragments of antibodies, preferably antigen-binding fragments of an antibody.

[0037] The term “diabody” as used herein and in the context of the present invention may refer to a bivalent antibody fragment constructed by gene fusion. The diabody is a dimer comprising two polypeptide chains. Usually, each of the polypeptide chains constituting the dimer comprises heavy and light chain variable regions linked via a linker on the same chain. The linker in the diabody is generally too short to allow paring between heavy and light chain variable regions on the same chain. Specifically, the number of amino acid residues constituting the linker is, for example, approximately 5 residues. Therefore, heavy and light chain variable regions encoded on the same polypeptide chain cannot together form a single chain variable region fragment. Instead, they form a dimer by pairing with another single chain variable region fragment. As a result, the diabody has two antigen-binding sites.

[0038] A “low-molecular antibody” as used herein, encompasses an antibody fragment deficient in a portion of the whole antibody (e.g. whole IgG). Such partial deficiency of the antibody molecule is accepted as long as the resultant antibody fragment is capable of binding to its target, e.g., SRRM2. It is preferred that a low-molecular antibody should contain one or both of heavy chain variable (VH) and light chain variable (VL) regions. It is also preferred that a low-molecular antibody should contain CDRs. The number of CDRs contained in the low-molecular antibody is not particularly limited and is preferably at least 6 CDRs: heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3.

[0039] An immunoglobulin as used herein preferably refers to be a tetrameric glycosylated protein composed of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each.

[0040] The terms "Fab", "Fab region", "Fab portion" or “Fab fragment" are understood to define a polypeptide that includes a VH, a CHI, a VL, and a CL immunoglobulin domain. Fab may refer to this region in isolation, or this region in the context of an antibody molecule according to the invention, as well as a full-length immunoglobulin or immunoglobulin fragment. Typically, a Fab region contains an entire light chain of an antibody. A Fab region can be taken to define “an arm” of an immunoglobulin molecule. It contains the epitope-binding portion of that Ig. The Fab region of a naturally occurring immunoglobulin can be obtained as a proteolytic fragment by a partial papain-digestion. A "F(ab')2 portion" is the proteolytic fragment of a partially pepsin-digested immunoglobulin. A "Fab1portion" is the product resulting from reducing the disulfide bonds of an F(ab')2 portion. As used herein the terms "Fab", "Fab region", "Fab portion" or “Fab fragment" may further include a hinge region that defines the C-terminal end of the antibody arm (cf. above). This hinge region corresponds to the hinge region found C-terminally of the CHI domain within a full-length immunoglobulin at which the arms of the antibody molecule can be taken to define a Y. The term hinge region is used in the art because immunoglobulin has some flexibility at this region.

[0041] An “Fv” or “Fv fragment” consists of only the VL and VH domains of a “single arm” of an immunoglobulin. Thus an "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and binding site. A “two-chain” Fv fragment consists of a dimer of one heavy-and one light-chain variable domain in a tight, non-covalent association. A single-chain Fv species (scFv) includes a VH and a VL domain of an immunoglobulin, with these domains being present in a single polypeptide chain in which they are covalently linked to each other by a flexible peptide linker. Typically, in a scFv fragment the variable domains of the light and heavy chain associate in a dimeric structure analogous to that in a two-chain Fv species. In single chain Fv fragments, it is possible to either have the variable domain of the light chain arranged at the N-terminus of the single polypeptide chain, followed by the linker and the variable domain of the heavy chain arranged at the C-terminus of the polypeptide chain or vice versa, having the variable domain of the heavy chain arranged on the N-terminus and the variable domain of the light chain at the C-terminus with the peptide linker arranged in between. The peptide linker can be any flexible linker known in the art, for example, made from glycine and serine residues. It is also possible to additionally stabilize the domain association between the VH and the VL domain by introducingdisulfide bonds into conserved framework regions (see Reiter et al. Stabilization of the Fv fragments in recombinant immunotoxins by disulfide bonds engineered into conserved framework regions, Biochemistry 1994, 33, 6551-5459). Such scFv fragments are also known as disulfide-stabilized scFv fragments (ds-scFv).

[0042] The term "Fc region" or “Fc fragment” is used herein to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. The Fc part mediates the effector function of antibodies, e.g. the activation of the complement system and of Fc-receptor bearing immune effector cells, such as NK cells. In human IgG molecules, the Fc region is generated by papain cleavage N-terminal to Cys226. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody molecule, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody molecule. Accordingly, a composition of intact antibodies may include antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native-sequence Fc regions for use in the antibodies of the invention include mammalian, e.g. human or murine, IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. The Fc region contains two or three constant domains, depending on the class of the antibody. In embodiments where the immunoglobulin is an IgG the Fc region has a CH2 and a CH3 domain.

[0043] The term "variable" refers to the portions of the immunoglobulin domains that exhibit variability in their sequence and that are involved in determining the specificity and binding affinity of a particular antibody (i.e., the "variable domain(s)"). Variability is not evenly distributed throughout the variable domains of antibodies; it is concentrated in sub-domains of each of the heavy and light chain variable regions. These sub-domains are called "hypervariable regions”, "HVR," or "HV," or "complementarity determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domains are called the "framework" regions (FR). The variable domains of naturally occurring heavy and light chains each include four FR regions, largely adopting a P-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the P -sheet structure. The hypervariable regions in each chain are held together in close proximity by the FR and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site (see Kabat etal., see below). Generally, naturally occurring immunoglobulins include six CDRs (see below); three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). In naturally occurring immunoglobulins, H3 and L3 display the most diversity of the six CDRs, and H3 in particular is believed to play a unique role in conferring fine specificity to immunoglobulins. Immunoglobulins naturally devoid of light chains, however, include three CDRs that are in the VHH region. The constant domains are not directly involved in antigen binding, but exhibit various effector functions, such as, for example, antibody-dependent, cell-mediated cytotoxicity and complement activation.

[0044] An antibody which binds to human SRRM2 according to the present disclosure can have various formats. It needs to be capable of binding to the SRRM2 protein. Said antibody is not particularly limited by its origin, type, shape. In some examples, said antibody may have a cytotoxic activity. Specifically, antibody which binds to human SRRM2 can be a non-human animal-derived antibody (e.g., a mouse, rat, or camel antibody), a human-derived antibody, a chimeric antibody, or a humanized antibody. An antibody which binds to human SRRM2 according to the present disclosure can be a polyclonal or a monoclonal antibody and is preferably a monoclonal antibody.

[0045] An antibody which binds to human SRRM2 can be obtained as a polyclonal or monoclonal antibody using means known in the art. For exampole, such an antibody may be a mammal-derived monoclonal antibody. The mammal-derived monoclonal antibody encompasses, for example, those produced by hybridomas and those produced by hosts transformed with expression vectors containing an antibody gene by a genetic engineering approach.

[0046] Chimeric antibodies refer to antibodies comprising variable and constant regions of different origins ligated with each other. For example, mouse-human heterogeneous chimeric antibodies are antibodies comprising the heavy and light chain variable regions of a mouse antibody and the heavy and light chain constant regions of a human antibody. Mouse antibody variable region-encoding DNAs are ligated with human antibody constant region-encoding DNAs, and the ligation products can be incorporated into expression vectors to prepare chimeric antibodyexpressing recombinant vectors. Cells transformed with these vectors (recombinant cells) can be cultured for the expression of the DNA insert to obtain the chimeric antibodies produced during the culture.

[0047] In general, the chimeric antibodies may comprise non-human animal-derived antibody variable regions and human antibody-derived constant regions. By contrast, humanized antibodiescomprise non-human animal-derived antibody complementarity-determining regions (CDRs), human antibody-derived framework regions (FRs), and human antibody-derived constant regions. Humanized antibodies are sometimes also called reshaped human antibodies. Specifically, for example, humanized antibodies may comprise non-human animal (e.g., mouse) antibody CDRs grafted in human antibodies. Humanized antibodies are for example useful as therapeutic agents, owing to their reduced antigenicity in the human body.

[0048] An antibody according to the present disclosure may be an isolated antibody molecule. The term "isolated antibody molecule" as used herein refers to an antibody molecule that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are matter that would interfere with uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments the antibody molecule is purified to greater than 95% by weight of antibody as determined by the Lowry method, such as more than 99% by weight. In some examples, the antibody molecule is purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator. In some examples, the antibody is purified to homogeneity as judged by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. An isolated antibody molecule may be present within foreign host cells with one or more component(s) of the antibody's natural environment not being present. Typically, an isolated antibody is prepared by at least one purification step.

[0049] A binding agent, such as an antibody, used in the methods of the disclosure may comprise a detectable label, e.g., they can be conjugated to a detectable lable. The term “detectable label” generally refers to any appropriate chemical substance or enzyme, which directly or indirectly generates a detectable compound or signal in a chemical, physical, optical, or enzymatic reaction. For example, a fluorescent or radioactive label can be conjugated to the antibody to generate fluorescence or X-rays as detectable signal. Alkaline phosphatase, horseradish peroxidase and P-galactosidase are examples of enzyme labels (and at the same time optical labels), which catalyze the formation of chromogenic reaction products. The detectable label refers to detectable entities that can be used for the detection of the target of interest such as in microscopy, immunohistochemistry, flow cytometry, or in vivo application, such as in vivo imaging. Preferably, the label does not negatively affect the characteristics of the binding agent, such as antibody, to which the label is conjugated. There are many types of detectable labels, including a fluorescent label, a chromophore label, an isotope label, a metal label, and a radioactive label. Non-exhaustiveexamples for a suitable chromophore label are alkaline phosphatase or peroxidase exposed to TMB (3, 3', 5, 5' tetramethylbenzidine), DAB (3, 3', 4, 4' diaminobenzidine), and 4CN (4-chloro-l-naphthol). ABTS (2,2'-azino-di [3-ethyl- benzthiazoline] sulfonate), OPD (o-phenylenediamine), and to BCIP / NBT (5-bromo- 4-chloro-3-indolyl-phosphate / nitroblue tetrazolium). Non-exhaustive examples for isotope labels are 13C, 15N, 19F, 27A1, 11B, 1271 or different Lanthanides isotopes. Non-exhaustive examples for a metal label are Au, Pd, Pb, Pt Ag, Hg and Os. The label may be a direct label, i.e. a label that is directly detectable. Preferably, the detectable label can be a fluorescent label. Examples of fluorescent labels include, but are not limited to, phycoerythrin, allophycocyanin (APC), Brilliant Violet 421, Alexa Fluor 488, coumarin or rhodamines to name only a few. Detection of a fluorescent label means detection of emitted light upon excitation of the fluorescent label. Non-exhaustive examples for suitable fluorescent labels are “green” emitters (Atto488, Alexa488, Cy2, etc.), “orange” emitters (Atto542, alexa555, Cy3, etc.), “Red-far-Red” emitters (Alexa633, Atto 647N, Cy5, etc.), infrared emitters (Atto700, LiCor IRDye700, LiCor IRDye800, etc.), ultra-violet absorbing fluorescent dyes (Atto390 or Alexa405). A fluorescent label may also be a fluorescent protein, such as GFP, eGFP, YFP, RFP, CFP, BFP, mCherry, or near-infrared fluorescent proteins.

[0050] An antibody which binds to human SRRM2 according to the present disclosure may be modified with, such as conjugated with, various molecules such as polyethylene glycol (PEG). Further, the antibody, which binds to human SRRM2 may also be modified with, such as conjugated with, a cytotoxic substance such as a chemotherapeutic agent, a toxic peptide, a radioactive chemical, or the like having a cytotoxic activity.

[0051] A binding agent, such as an antibody or antibody molecule / fragment is said to “specifically” bind to an antigen, such as SRRM2, when it recognizes its target antigen within a complex mixture of proteins and / or macromolecules. Typically, the binding agent, such as antibody, is capable of specifically interacting with and / or binding to its target but does not essentially bind to another (preferably non-related) epitope or antigen. Binding agents, such as antibodies, are said to "bind to the same epitope" if the antibodies cross-compete so that only one binding agent can bind to the epitope at a given point of time, i.e. one binding agent prevents the binding or modulating effect of the other. A binding agent, such as an antibody or antibody molecule / fragment that specifically binds to a certain target, can however be cross-reactive with structures that are similar, such as with closely related variants of the target it specifically binds to.

[0052] Typically, binding that is considered specific may also have a high affinity, e.g. when the binding affinity is higher than 10'6M (in terms of KD). In particular, the binding affinity may be about 10'8to 10'11M (KD), or of about 10'9to 10'11M or even higher. If necessary, nonspecific binding of a binding site can be reduced without substantially affecting specific binding by varying the binding conditions.

[0053] The term "epitope", also known as the “antigenic determinant”, refers to the portion of an antigen to which an antibody specifically binds, thereby forming a complex. Thus, the term "epitope" includes any molecule or protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. The binding site(s) (paratope) of an antibody molecule described herein may specifically bind to / interact with conformational or continuous epitopes, which are unique for the target structure. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three-dimensional structural characteristics, and / or specific charge characteristics. With regard to polypeptide antigens a conformational or discontinuous epitope is characterized by the presence of two or more discrete amino acid residues, separated in the primary sequence, but assembling to a consistent structure on the surface of the molecule when the polypeptide folds into the native protein / antigen (Sela, M., Science (1969) 166, 1365-1374; Laver, W.G., et al. Cell (1990) 61, 553-556). The two or more discrete amino acid residues contributing to the epitope may be present on separate sections of one or more polypeptide chain(s). These residues come together on the surface of the molecule when the polypeptide chain(s) fold(s) into a three-dimensional structure to constitute the epitope. Generally, epitopes can be linear in nature or can be a discontinuous epitope. Thus, as used herein, the term "conformational epitope" refers to a discontinuous epitope formed by a spatial relationship between amino acids of an antigen other than an unbroken series of amino acids. The term “epitope” also includes an antigenic determinant of a hapten, which is known as a small molecule that can serve as an antigen by displaying one or more immunologically recognized epitopes upon binding to larger matter such as a larger molecule e.g. a protein.

[0054] The methods of the disclosure envisions that any suitable SMMR2 binding agent can be used for determining the level of SRRM2 expression on a cell surface. Particularly preferred are SMMR2 binding agents of the present disclosure, such an antibody that binds to human SRRM2 of the present disclosure.

[0055] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 9, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 10, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 11, and a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 12, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 13, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 14, or an antibody, which binds to the same epitope. Optionally, such an antibody may comprise a heavy chain variable region having an amino acid sequence and a light chain variable region having an amino acid sequence that have at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or that are identical to the amino acid sequences set forth SEQ ID NOs: 1 and 2.

[0056] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 15, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 16, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 17, and a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 18, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 19, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 20, or an antibody, which binds to the same epitope. Optionally, such an antibody may comprise a heavy chain variable region having an amino acid sequence and a light chain variable region having an amino acid sequence that have at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or that are identical to the amino acid sequences set forth SEQ ID NOs: 3 and 4.

[0057] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 21, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 22, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 23, and a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 24, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 25, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 26, or an antibody, which binds to the same epitope. Optionally, such an antibody may comprise a heavy chain variable region having an amino acidsequence and a light chain variable region having an amino acid sequence that have at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or that are identical to the amino acid sequences set forth SEQ ID NOs: 5 and 6.

[0058] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 21, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 22, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 23, and a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 27, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 28, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 29, or an antibody, which binds to the same epitope. Optionally, such an antibody may comprise a heavy chain variable region having an amino acid sequence and a light chain variable region having an amino acid sequence that have at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or that are identical to the amino acid sequences set forth SEQ ID NOs: 5 and 7.

[0059] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence with at least 85% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region having an amino acid sequence with at least 85% identity to the amino acid sequence shown in SEQ ID NO: 2 or an antibody, which binds to the same epitope.

[0060] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence with at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region having an amino acid sequence with at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2 or an antibody, which binds to the same epitope.

[0061] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence with at least 95% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region having an amino acid sequence with at least 95% identity to the amino acid sequence shown in SEQ ID NO: 2 or an antibody, which binds to the same epitope.

[0062] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequencewith at least 99% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region having an amino acid sequence with at least 99% identity to the amino acid sequence shown in SEQ ID NO: 2 or an antibody, which binds to the same epitope.

[0063] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region having an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 2 or an antibody.

[0064] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody that binds to the same epitope as an antibody comprising a heavy chain variable region having an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region having an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 2.

[0065] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence with at least 85% identity to the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region having an amino acid sequence with at least 85% identity to the amino acid sequence shown in SEQ ID NO: 4 or an antibody, which binds to the same epitope.

[0066] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence with at least 90% identity to the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region having an amino acid sequence with at least 90% identity to the amino acid sequence shown in SEQ ID NO: 4 or an antibody, which binds to the same epitope.

[0067] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence with at least 95% identity to the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region having an amino acid sequence with at least 95% identity to the amino acid sequence shown in SEQ ID NO: 4 or an antibody, which binds to the same epitope.

[0068] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence with at least 99% identity to the amino acid sequence shown in SEQ ID NO: 3 and alight chain variable region having an amino acid sequence with at least 99% identity to the amino acid sequence shown in SEQ ID NO: 4 or an antibody, which binds to the same epitope.

[0069] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region having an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 4 or an antibody.

[0070] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody that binds to the same epitope as an antibody comprising a heavy chain variable region having an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region having an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 4.

[0071] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence with at least 85% identity to the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region having an amino acid sequence with at least 85% identity to the amino acid sequence shown in SEQ ID NO: 6 or an antibody, which binds to the same epitope.

[0072] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence with at least 90% identity to the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region having an amino acid sequence with at least 90% identity to the amino acid sequence shown in SEQ ID NO: 6 or an antibody, which binds to the same epitope.

[0073] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence with at least 95% identity to the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region having an amino acid sequence with at least 95% identity to the amino acid sequence shown in SEQ ID NO: 6 or an antibody, which binds to the same epitope.

[0074] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence with at least 99% identity to the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region having an amino acid sequence with at least 99% identity to the amino acid sequence shown in SEQ ID NO: 6 or an antibody, which binds to the same epitope.

[0075] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region having an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 6 or an antibody.

[0076] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody that binds to the same epitope as an antibody comprising a heavy chain variable region having an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region having an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 6.

[0077] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence with at least 85% identity to the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region having an amino acid sequence with at least 85% identity to the amino acid sequence shown in SEQ ID NO: 7 or an antibody, which binds to the same epitope.

[0078] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence with at least 90% identity to the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region having an amino acid sequence with at least 90% identity to the amino acid sequence shown in SEQ ID NO: 7 or an antibody, which binds to the same epitope.

[0079] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence with at least 95% identity to the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region having an amino acid sequence with at least 95% identity to the amino acid sequence shown in SEQ ID NO: 7 or an antibody, which binds to the same epitope.

[0080] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequence with at least 99% identity to the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region having an amino acid sequence with at least 99% identity to the amino acid sequence shown in SEQ ID NO: 7 or an antibody, which binds to the same epitope.

[0081] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody comprising a heavy chain variable region having an amino acid sequencethat is identical to the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region having an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 7 or an antibody.

[0082] In some examples, an antibody that binds to human SRRM2 according to the present disclosure, is an antibody that binds to the same epitope as an antibody comprising a heavy chain variable region having an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region having an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 7.

[0083] An antibody that binds to human SRRM2 according to the present disclosure is preferably one that is less than 20 % cross-reactive with SRRM2-related proteins. This means preferably that said antibody, which binds to human SRRM2, is less than 20 % cross-reactive with other serine / arginine repetitive matrix proteins such as SRRM1, SRRM3, SRRM4 or SRRM5. More preferably, said antibody, which binds to human SRRM2 is less than 15 % cross-reactive with SRRM2-related proteins, e.g. SRRM1, SRRM3, SRRM4 or SRRM5. Even more preferably, said antibody, which binds to human SRRM2, is less than 10 % cross-reactive with SRRM2-related proteins, e.g. SRRM1, SRRM3, SRRM4 or SRRM5. Even more preferably, said antibody, which binds to human SRRM2, is less than 5 % cross-reactive with SRRM2-related proteins, e.g. SRRM1, SRRM3, SRRM4 or SRRM5. Even more preferably, said antibody, which binds to human SRRM2, is less than 3 % cross-reactive with SRRM2-related proteins, e.g. SRRM1, SRRM3, SRRM4 or SRRM5. Even more preferably, said antibody, which binds to human SRRM2, is less than 2 % cross-reactive with SRRM2-related proteins, e.g. SRRM1, SRRM3, SRRM4 or SRRM5. Even more preferably, said antibody, which binds to human SRRM2, is less than 1 % cross-reactive with SRRM2-related proteins, e.g. SRRM1, SRRM3, SRRM4 or SRRM5.

[0084] An antibody, which binds to human SRRM2 according to the present disclosure may include antibodies comprising the substitution, deletion, addition, and / or insertion of one or more amino acids, which may be prepared or occur naturally. Examples of a method for introducing a mutation in the polypeptide include site-directed mutagenesis (Hashimoto-Gotoh, T. et al., 1995) (Zoller, MJ, and Smith, M., 1983) (Kramer, W. et al., 1984) (Kramer W, and Fritz HJ, 1987) (Kunkel, TA, 1985) (Kunkel, 1988). This is one of the methods well known by those skilled in the art for preparing a polypeptide functionally equivalent to a certain polypeptide. Those skilled in the art can appropriately introduce a mutation in the antibody of the present disclosure and thereby prepare an antibody functionally equivalent to such antibody. Moreover, amino acid mutations may occur in the natural world. Such an antibody that has an amino acid sequence derived fromthe amino acid sequence of the antibody of the present disclosure comprising the mutation of one or more amino acids, is functionally equivalent or a variant to the antibody and is also encompassed by the antibody of the present disclosure. The number of amino acids mutated in such a variant is usually within 50 amino acids, preferably within 30 amino acids, more preferably within 10 amino acids (e.g. within 5 amino acids).

[0085] For amino acid residues to be mutated, it is preferred that this mutation should be performed conservatively between amino acids having the same side chain property. For example, the following classification based on the properties of amino acid side chains has been established: hydrophobic amino acids (A, I, L, M, F, P, W, Y, and V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, and T), amino acids having an aliphatic side chain (G, A, V, L, I, and P), amino acids having a hydroxyl group-containing side chain (S, T, and Y), amino acids having a sulphur atom-containing side chain (C and M), amino acids having a side chain containing carboxylic acid and amide (D, N, E, and Q), amino acids having a base-containing side chain (R, K, and H), and amino acids having an aromatic group-containing side chain (H, F, Y, and W) (all symbols within the parentheses represent single letter codes of amino acids).

[0086] A polypeptide having an amino acid sequence modified from a certain amino acid sequence by the deletion and / or addition of one or more amino acid residue(s) and / or the substitution thereof by other amino acids is already known to maintain the biological activity of the original polypeptide. Specifically, when amino acids in an amino acid sequence constituting a certain polypeptide are substituted by amino acids classified in the same group thereas, it is generally said that the polypeptide is likely to maintain its activity.

[0087] In some examples, a method for the evaluation wheter a subject diagnosed with AML will have a negative or positive prognosis of the disclosure is a method that does not have the immediate prupose to iagnose and / or treat a disease. In some examples, a method for the evaluation wheter a subject diagnosed with AML will have a negative or positive prognosis of the disclosure is not diagnostic method practiced on the human body.

[0088] According to the methods of the disclosure, an SRRM2-positive subject may be amenable for an SRRM2 -targeting therapy. Such therapy may include a therapy with an antibody, antibody construct, or antibody fragment binding to SRRM2 on the cell surface of SRRM2 expressing cells. The methods of the present disclosure may therefore comprise evaluating whether a subject diagnosed with AML will have a negative or positive prognosis of the disclosure, and selecting said subject for an SRRM2 -targeting therapy, if the subject has a negative prognosis and / or if thesubject is characterized as being SRRM2- negative. Accordingly, a method for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative or positive prognosis disclosed herein may be conducted before a patient receives an SRRM2 -targeting therapy.

[0089] The present invention also envisions a method of stratifying and treating subject diagnosed with AML. Said method comprises conducting a method for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative or positive prognosis of the present disclsoure, and administering to the subject an SRRM2 -targeting therapy, if the subject is evaluated with a negative prognosis and / or as SRRM2-positive.

[0090] The present invention also relates to an SRRM2 -targeting therapy for use in treating AML in a subject, wherein the subject is characterized by having a level of more than 20% SRRM2 expressing blast cells. The determination of the level of SRRM2 expressing blast cells of the subject can be conducted by any suitable method, preferably by a method of the present disclosure, such as a a method for the evaluation whether a subject diagnosed with AML will have a negative or positive prognosis of the present disclosure. However, it is envisioned that the step of determining the level of SRRM2 expressing blast cells may be conducted prior to treating the subject. Said step of determining may not be part of the use in treating AML in a subject.

[0091] The present invention also relates to the use of an SRRM2-targeting therapy for the manufacture of a medicament for treating AML in a subject, wherein the subject is characterized by having a level of more than 20% SRRM2 expressing blast cells. The determination of the level of SRRM2 expressing blast cells of the subject can be conducted by any suitable method, preferably by a method of the present disclosure, such as a method for the evaluation whether a subject diagnosed with AML will have a negative or positive prognosis of the present disclosure. However, it is envisioned that the step of determining the level of SRRM2 expressing blast cells may be conducted prior to manufacturing the medicament. Said step of determining may not be a step of the manufacturing of the medicament. However, the (intended) use of the medicament can be limited to specific patient group of subjects that are characterized by having a level of more than 20% SRRM2 expressing blast cells.

[0092] The present invention also relates to a method of treating AML comprising administering to a subject in need thereof an effective amount of an SRRM2 -targeting therapy, wherein the subject is characterized by having a level of more than 20% SRRM2 expressing blast cells. The determination of the level of SRRM2 expressing blast cells of the subject can be conducted by anysuitable method, preferably by a method of the present disclosure, such as a method for the evaluation whether a subject diagnosed with AML will have a negative or positive prognosis of the present disclosure. The step of determining the level of SRRM2 expressing blast cells may be part of the method of treating AML. Alternatively, the step of determining the level of SRRM2 expressing blast cells may not be part of the method of treating AML.

[0093] The term “therapeutically effective amount” refers to an amount of the SRRM2 -targeting therapy according to the present disclosure or drug effective to “treat” a disease, such as AML in the patient. Specifically, in the case of cancer, the therapeutically effective amount of the antibody / pharmaceutical composition / drug can reduce the number of cancer cells; reduce the tumour size; inhibit or stop cancer cell infiltration into peripheral organs; inhibit and stop tumour metastasis; inhibit and stop tumour growth; relieve to some extent one or more of the symptoms associated with the cancer, or a combination of such effects on cancer cells. To the extent the SRRM2 -targeting therapy prevents the growth and / or kills existing cancer cells, it can be referred to as cytostatic and / or cytotoxic.

[0094] Terms such as “treating” or “treatment” or “to treat” refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. A subject is successfully “treated” according to the methods of the present invention or with the pharmaceutical composition or antibody, which binds to human SRRM2 according to the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumour size; inhibition of or an absence of cancer cell infiltration into peripheral organs including the spread of cancer into soft tissue and bone; inhibition of or an absence of tumour metastasis; inhibition or an absence of tumour growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; and improvement in quality of life.

[0095] A “subject” or “patient” in the context of the present invention is a human being. For example, the patient may be a patient being suspected of having a disease or clinical condition associated with blood cancer or being diagnosed with such a disease or clinical condition.

[0096] An SRRM2 -targeting therapy as used herein, may include any therapy, e.g. a therapeutic agent that is capable of binding SRRM2 and / or pharmaceutical composition comprising saidtherapeutic agent. Said pharmaceutical composition may comprise said therapeutic agent, and, further, a pharmaceutically acceptable carrier, diluent or excipient. The term “pharmaceutically acceptable carrier, diluent or excipient” as used in the context of the present invention, may comprise any pharmaceutically acceptable carrier, diluent or excipient for a pharmaceutical composition known by the person skilled in the art. It will be understood that such therapeutic agent or pharmaceutical composition as described herein may be mixed with carriers or diluents, which will not interfere with the intended purpose of the present invention. For example, such a carrier as used within the present invention may be a carrier protein, such as bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH). A carrier may also be e.g., be selected from the group consisting of water, aqueous saline solution, aqueous buffer solution, cell culture medium and combinations of the foregoing carriers.

[0097] An SRRM2-targeting therapy may include an antibody or antigen-binding fragment thereof, such as an antibody that binds to human SRRM2 or an antigen-binding fragment thereof. Preferably such SRRM2 -targeting therapy comprises an an antibody that binds to human SRRM2 or an antigen-binding fragment thereof of the present disclosure.

[0098] An SRRM2 -targeting therapy may comprise antibody that binds to human SRRM2, preferably of the present disclosure, that has antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Thus, examples of the cytotoxic activity according to the present invention can include ADCC and / or CDC activities. In the context of the present disclosure, ADCC activity means the activity of damaging target cells through the binding of Fey receptor-bearing cells (immunocytes, etc.) via the Fey receptors to the Fc domains of antibodies specifically attached to the cell surface antigens of the target cells. On the other hand, CDC activity means a cytotoxic activity mediated by the complement system. Whether or not the antibody has an ADCC activity or has a CDC activity can be determined by a method known in the art.

[0099] Thus, an antibody, which binds to human SRRM2 of the present disclosure may have activities such as an ADCC activity and as such, may be useful as a pharmaceutical drug, preferably, an anti-cancer agent, wherein the cancer is a blood cancer, preferably acute myeloid leukemia (AML).

[0100] An SRRM2 -targeting therapy may also comprise an antibody that binds to human SRRM2, preferably of the disclosure, that is conjugated with a cytotoxic substance. For example, the antibody, which binds to human SRRM2 may be conjugated with a cytotoxic substance, such as achemotherapeutic agent, a toxic peptide, or a radioactive chemical. Such a modified antibody (hereinafter, referred to as an antibody conjugate) can be obtained by chemically modifying the obtained antibody. A method for the antibody modification has already been established in the art.

[0101] Examples of the chemotherapeutic agent whose cytotoxic activity functions through the conjugation to the antibody that binds SRRM2 can include the following chemotherapeutic agents: azaribine, anastrozole, azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan, camptothecin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin, irinotecan, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin, dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin glucuronide, epirubicin, ethinyl estradiol, estramustine, etoposide, etoposide glucuronide, floxuridine, fhidarabine, fhitamide, fluorouracil, fhioxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide, leucovorin, lomustine, mechlorethamine, medroxyprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, phenylbutyrate, prednisone, procarbazine, paclitaxel, pentostatin, semustine, streptozocin, tamoxifen, taxanes, taxol, testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vinblastine, vinorelbine, and / or vincristine.

[0102] The chemotherapeutic agent is preferably a low-molecular chemotherapeutic agent. The low-molecular chemotherapeutic agent is unlikely to interfere with the antibody even after its conjugation to the antibody. The low-molecular chemotherapeutic agent usually has a molecular weight of 100 to 2000, preferably 200 to 1000. All of the chemotherapeutic agents exemplified above are low-molecular chemotherapeutic agents. These chemotherapeutic agents encompass prodrugs that are converted in vivo to active chemotherapeutic agents. The prodrug activation may be an enzymatic conversion or a non-enzymatic conversion.

[0103] Examples of toxic peptides are snake venom peptides comprising Three-Finger Toxins (3FTxs), a disintegrin, a Kunitz-type inhibitor, a natriuretic peptide, or a Sarafotoxin. Further examples are a trypsin inhibitor, an islanditoxin, a pallotoxin, or an amatoxin.

[0104] Examples of a radioactive chemical are chemicals having a cytotoxic radionuclide, such radionuclide could be for example iodine-131, indium-ill, yttrium-90, lutetium- 177, actinium-225, gallium-68, or bismuth-213. The antibody coupled to the cytotoxic substance could be used for delivering the cytotoxic substance specifically to the target cell which may reduce unwanted side effects.

[0105] An SRRM2-targeting therapy may also include a bispecific antibody, wherein said bispecific antibody preferably comprises a moiety, such as an antibody or antigen-binding fragment thereof, that binds to human SRRM2. Preferalby such SRRM2 -targeting therapy comprises an an antibody that binds to human SRRM2 or an antigen-binding fragment thereof of the disclosure.

[0106] As used herein, the term "bispecific construct" refers to an antibody which is bispecific, i.e., it comprises at least a first binding domain and a second binding domain, wherein the first binding domain binds to one antigen or target, and the second binding domain binds to another antigen or target.

[0107] A bispecific antibody construct of the disclosure may be a bispecific single chain antibody. Generally, a single-chain antibody further comprises a polypeptide linker between the VH and VL domains which enables it to form the desired structure which would allow for antigen binding. Single chain antibodies are discussed in detail by Plueckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds. Springer- Verlag, New York, pp. 269-315 (1994). Various methods of generating single chain antibodies are known, including those described in U.S. Pat. Nos. 4,694,778 and 5,260,203; International Patent Application Publication No. WO 88 / 01649; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038- 1041. In specific embodiments, single-chain antibodies can also be human, and / or humanized and / or synthetic. The term “bi-scFv” or “ta-scFv” (tandem scFv) as used herein refers to two scFv that are fused together. Such a bi-scFv or ta-scFv may comprise a linker between the two scFv moieties. Generally, the arrangement of the VH and VL domains on the polypeptide chain within each of the scFv may be in any order. This means that the “bi-scFv” of “ta-scFv” can be arranged in the order VH(1)-VL(1)-VH(2)-VL(2), VL(1)-VH(1)-VH(2)-VL(2), VH(1)-VL(1)-VL(2)-VH(2), or VL(1)-VH(1)-VL(2)-VH(2), where (1) and (2) stand for the first and second scFv, respectively. A bispecific single chain antibody preferably is or comprises a “bi-scFv” or “ta-scFv”.

[0108] In some examples, a bispecific antibody may be a BiTE. A “bispecific T cell engager” or “BiTE” as used herein, relates to bispecific antibody constructs, that are capable of recruiting T cells to target cells, such as cancer cells, such as AML cells. Typically, BiTE molecules are fusion proteins comprising two single-chain variable fragments (scFvs) of different antibodies. Typically, one binding domain is capable of binding to a target on the surface of a T cell, while another binding domain is capable of binding to a to a tumor antigen or a tumor-associated antigen, e.g. SRRM2. A BiTE is preferably a bispecific single chain antibody.

[0109] In some examples, a bispecific antibody construct of the disclosure may comprise a first binding domain that is capable of specifically binding to a first target that is an immune-regulatory antigen on the surface of an immune effector cell, and a second binding domain that is capable of specifically binding to an SRRM2.

[0110] The term "binding domain" as used herein characterizes a domain which is capable of specifically binding to / interacting with / recognizing a given target epitope or a given target site on the target molecules (antigens), e.g. SRRM2.[OHl] The term “immune effector cell” as used herein may refer to any leukocyte or precursor involved e.g. in defending the body against cancer, diseases induced by infectious agents, foreign materials or autoimmune reactions. For example, the immune effector cell can be a T cell or NK cell.

[0112] T cells or T lymphocytes can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. They are called T cells because they mature in the thymus (although some also mature in the tonsils). There are several subsets of T cells, each with a distinct function.

[0113] Cytotoxic T cells (TC cells, or CTLs) destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T cells since they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells. Through IL- 10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevents autoimmune diseases.

[0114] Natural killer (NK) cells are CD56+CD3- large granular lymphocytes that can kill virally infected and transformed cells, and constitute a critical cellular subset of the innate immune system (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676). Unlike cytotoxic CD8+ T lymphocytes, NK cells launch cytotoxicity against tumor cells without the requirement for prior sensitization and can also eradicate MHC-I-negative cells (Nami-Mancinelli E, et al. Int Immunol 2011 23:427-431).

[0115] The term “immune-regulatory antigen” as used herein relates to an antigen which is preferably a receptor. Said antigen or preferably receptor is capable of receiving and / or transducing signals, and its engagement is considered to influence the quality and intensity of the innate immune cell response. Such antigens include inhibitory receptors, activating receptors, adhesion molecules and co-stimulatory molecules. Such “immune-regulatory antigen” includes but is notlimited to CD3, CD 16, NKG2A, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 (CD226), SLAMF7 (CD319), CD244 (2B4), 0X40, CD47, SIRPa, CD89, CD96, CD137, CD160, TIGIT, nectin-4, PD-1, PD-L1, LAG-3, CTLA-4, TIM-3, KIR2DL1-5, KIR3DL1-3, KIR2DS1-5, and KIR3DS1. A preferred immune-regulatory antigen is CD3.

[0116] An SRRM2-targeting therapy may also include cell therapy, such as an immunce cell comprising an antigen receptor that is capable of specifically binding to SRRM2 on the cell surface of SRRM2 expressing cells. Said immune cell is preferably an immune effector cell, preferably a T cell or NK cell, such as a cytotoxic T cell and / or a CD8+ T cell.

[0117] In some examples, such immune effector cells, such as T cells or NK cells, may be engineered. Such engineered cells may express one or more recombinant receptor(s). Among the receptors are antigen receptors and receptors containing one or more component thereof. The recombinant receptors may include chimeric receptors, such as those containing ligand-binding domains or binding fragments thereof and intracellular signaling domains or regions, functional non-TCR antigen receptors, chimeric antigen receptors (CARs), T cell receptors (TCRs), such as recombinant or transgenic TCRs, chimeric autoantibody receptor (CAAR) and components of any of the foregoing. The recombinant receptor, such as a CAR, generally includes the extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some aspects via linkers and / or transmembrane domain(s). In some examples, the engineered cells express two or more receptors that contain different components, domains or regions. In some examples, two or more receptors allows spatial or temporal regulation or control of specificity, activity, antigen (or ligand) binding, function and / or expression of the recombinant receptors.

[0118] In some examples, chimeric antigen receptors, contain one or more domains that combine a ligand-binding domain (e.g. antibody or antibody fragment) that provides specificity for a desired antigen (e.g., tumor antigen) with intracellular signaling domains. The intracellular signaling domain is preferably an activating intracellular domain portion, such as a T cell activating domain, providing a primary activation signal. In some examples, the intracellular signaling domain contains or additionally contains a costimulatory signaling domain to facilitate effector functions. Chimeric receptors when genetically engineered into immune cells can preferably modulate T cell activity, and, in some cases, can modulate T cell differentiation or homeostasis, thereby resulting in genetically engineered cells with improved longevity, survival and / or persistence in vivo, such as for use in adoptive cell therapy methods

[0119] A chimeric receptor, such as CARs, may generally include an extracellular antigen binding domain, such as a portion of an antibody molecule, generally a variable heavy (VH) chain region and / or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment.

[0120] The antigen receptor may comprise an intracellular domain linked directly or indirectly to the extracellular domain. In some examples, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. For example, the intracellular signaling domain may comprise an IT AM. For example, the antigen recognition domain (e.g. extracellular domain) generally can be linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and / or signal via another cell surface receptor. In some examples, the CAR comprises a transmembrane domain linked or fused between the extracellular domain (e.g. a scFv) and intracellular signaling domain. Thus, in some examples, the antigen-binding component (e.g., antibody or fragment or variant thereof) is linked to one or more transmembrane and intracellular signaling domains.

[0121] The antigen receptor, e.g. CAR, may generally include at least one intracellular signaling component or components. In some examples, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon. In some examples, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta. In some examples, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain.

[0122] The chimeric antigen receptor may also contain an intracellular domain of a T cell costimulatory molecule. For example, the CAR may include a signaling domain and / or transmembrane portion of a costimulatory receptor, such as CD28, 4- IBB, 0X40, DAP 10, and ICOS. In some examples, the same CAR includes both the activating and costimulatory components. The chimeric antigen receptor may contain an intracellular domain derived from a T cell costimulatory molecule or a functional variant thereof, such as between the transmembrane domain and intracellular signaling domain. The T cell costimulatory molecule may be selected from the group consistign of CD28 and 4-1BB, preferably CD28.

[0123] The transmembrane domain can generally be derived either from a natural or from a synthetic source. Where the source is natural, the domain can be derived from any membranebound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. For example, the transmembrane domain may comprise a transmembrane portion of CD28.

[0124] In some examples, an antibody which binds to human SRRM2, preferably an antibody which binds to human SRRM2 of the disclosure, may be modified to form a chimeric antigen receptor (CAR), expressed by an immune cell(s), preferably T cells, NK cells, NK-T cells, or macrophages, most preferably T cells (CAR T cells). Accordingly, the extracellular region of a CAR may comprise the light and heavy chain variable regions of an antibody that recognizes human SRRM2, preferably an antibody which binds to human SRRM2 of the disclosure. The light and heavy chain variable regions may be formed into a single-chain variable fragment (scFv). A CAR so described may result in T cell activation upon SRRM2 binding.

[0125] In some examples, an antibody which binds to human SRRM2 of the disclosure may be modified to form a chimeric antigen receptor (CAR) expressed by an immune cell(s), including, but not limited, to T cells, NK cells, NK-T cells, or macrophages, wherein said immune cells may be derived from induced pluripotent stem cells (iPSCs).

[0126] In some examples, the antibody which binds to human SRRM2 of the disclosure may be modified to form a chimeric antigen receptor (CAR), expressed by an immune cell(s), including but not limited to T cells, NK cells, NK-T cells, or macrophages, wherein said immune cells are immune cells in which p2-microglobulin has been inactivated.

[0127] Further SRRM2-specific CARs and CAR-T cells are described in PCT / EP2024 / 075361, which is incorporated herewith by reference in its entirety.

[0128] The present invention also relates to an in vitro use of the level of SRRM2 expression on the cell surface of blast cells of a subject diagnosed with acute myeloid leukemia (AML) for the evaluation whether said subject will have a negative or positive prognosis, wherein (i) a subject having a level of 20% or less SRRM2 expressing blast cells is characterized as SRRM2-negative and has a positive prognosis, whereas (ii) a subject having a level of more than 20% SRRM2 expressing blast cells is characterized as SRRM2-positive and has a negative prognosis.

[0129] The use of the level of SRRM2 expression on the cell surface of blast cells of a subject diagnosed with acute myeloid leukemia (AML) may further comprise sub-classifying a subject having a level of more than 20% SRRM2 expressing blast cells. Accordingly, an SRRM2-positive subject that has a level of more than 20% and up to 30% SRRM2 expressing blast cells can be characterized as being SRRM2-positive low. An SRRM2-positive subject that has a level of more than 30% SRRM2 expressing blast cells can be characterized as being SRRM2-positive high.

[0130] In some examples, immediate purpose of the in vitro use of the level of SRRM2 expression on the cell surface of blast cells of a subject diagnosed with acute myeloid leukemia (AML) is not to diagnose and / or treat diseases.

[0131] The use of the level of SRRM2 expression on the cell surface of blast cells of a subject diagnosed with acute myeloid leukemia (AML) can be in any method, including any method for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative or positive prognosis. Accordingly, any feature disclosed herein in the context of a method for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative or positive prognosis, also applies to the in vitro use of the level of SRRM2 expression on the cell surface of blast cells of a subject diagnosed with acute myeloid leukemia (AML) of the disclosure.

[0132] The present invention also relates to the use of a binding agent which is capable of specifically binding SRRM2 on the surface of a blast cell for the manufacture of a composition or a kit for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative or positive prognosis. Generally, said binding agent can be any binding agent disclosed herein. Such binding agent may be or comprise an antibody and / or an antigen-binding fragment thereof, which is capable of specifically binding to SRRM2, such as an antibody that binds to human SRRM2 of the present disclosure or antigen-binding fragment thereof.

[0133] The use of the binding agent which is capable of specifically binding SRRM2 on the surface of a blast cell for the manufacture of a composition or a kit for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative or positive prognosis can be for the use in any method for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative or positive prognosis of the disclosure. Accordingly, any feature disclosed herein in the context of a method for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative or positive prognosis, also applies to the use of the binding agent which is capable of specifically binding SRRM2 on the surface of a blastcell for the manufacture of a composition or a kit for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative or positive prognosis of the disclosure.

[0134] The description of the sequences, shown in the sequence listing and as used in the context of the present invention, is as follows:

[0135] SEQ ID NO: 1 shows the amino acid sequence of the VH-region of antibody 13F11.

[0136] SEQ ID NO: 2 shows the amino acid sequence of the VL-region of antibody 13F11.

[0137] SEQ ID NO: 3 shows the amino acid sequence of the VH-region of antibody 23 A7.

[0138] SEQ ID NO: 4 shows the amino acid sequence of the VL-region of antibody 23 A7.

[0139] SEQ ID NO: 5 shows the amino acid sequence of the VH-region of antibody 18A4 and of antibody 18A4-2.

[0140] SEQ ID NO: 6 shows the amino acid sequence of the VL-region of antibody 18A4.

[0141] SEQ ID NO: 7 shows the amino acid sequence of the VL-region of antibody 18A4-2.

[0142] SEQ ID NO: 8 shows the amino acid sequence of SRRM2 as shown in UniProt database entry Q9UQ35, version 2 of 6 March 2007.

[0143] SEQ ID NO: 9 shows the amino acid sequence of the VH-CDR1 of antibody 13F11.

[0144] SEQ ID NO: 10 shows the amino acid sequence of the VH-CDR2 of antibody 13F11.

[0145] SEQ ID NO: 11 shows the amino acid sequence of the VH-CDR3 of antibody 13F11.

[0146] SEQ ID NO: 12 shows the amino acid sequence of the VL-CDR1 of antibody 13F11.

[0147] SEQ ID NO: 13 shows the amino acid sequence of the VL-CDR2 of antibody 13F11.

[0148] SEQ ID NO: 14 shows the amino acid sequence of the VL-CDR3 of antibody 13F11.

[0149] SEQ ID NO: 15 shows the amino acid sequence of the VH-CDR1 of antibody 23 A7.

[0150] SEQ ID NO: 16 shows the amino acid sequence of the VH-CDR2 of antibody 23 A7.

[0151] SEQ ID NO: 17 shows the amino acid sequence of the VH-CDR3 of antibody 23 A7.

[0152] SEQ ID NO: 18 shows the amino acid sequence of the VL-CDR1 of antibody 23 A7.

[0153] SEQ ID NO: 19 shows the amino acid sequence of the VL-CDR2 of antibody 23 A7.

[0154] SEQ ID NO: 20 shows the amino acid sequence of the VH-CDR3 of antibody 23 A7.

[0155] SEQ ID NO: 21 shows the amino acid sequence of the VH-CDR1 of antibody 18A4 and of antibody 18A4-2.

[0156] SEQ ID NO: 22 shows the amino acid sequence of the VH-CDR2 of antibody 18A4 and of antibody 18A4-2.

[0157] SEQ ID NO: 23 shows the amino acid sequence of the VH-CDR3 of antibody 18A4 and of antibody 18A4-2.

[0158] SEQ ID NO: 24 shows the amino acid sequence of the VL-CDR1 of antibody 18A4.

[0159] SEQ ID NO: 25 shows the amino acid sequence of the VL-CDR2 of antibody 18A4.

[0160] SEQ ID NO: 26 shows the amino acid sequence of the VL-CDR3 of antibody 18A4.

[0161] SEQ ID NO: 27 shows the amino acid sequence of the VL-region of antibody 18A4-2.

[0162] SEQ ID NO: 28 shows the amino acid sequence of the VL-CDR1 of antibody 18A4-2.

[0163] SEQ ID NO: 29 shows the amino acid sequence of the VL-CDR2 of antibody 18A4-2.

[0164] An overview of the SEQ ID NOs and detailed sequences, as used in the context of the present invention, is given in the following Table 1 (in case of conflict between the sequences shown in Table 1 and the sequences of the sequence listing, which has to be filed for formal reasons, the sequences of Table 1 supercede the sequences of the sequence listing):

[0165] Table 1:Sequence (CDRs are underlined in the order CDR 1, SEQ ID NO : Designation2 , 3 starting from left upper line) EVQLVESGGGLVQPGRSLKLSCAASRFTFSNYDMAWVRQAPTKGLEWVA1 13F11 VH SISPSNVNTYYRDSVKGRFTVSRDNAKSSLYLQMDSLRSEDTATYYCAR LVHSFWYFDFWGPGTMVTVSS D I VMT QT P S S QAVS AGE KVTMRC KSSQSLLYSKNKKNYLAW YQQ K P GQ S2 13F11 VL PKLLIYWASTRESGVPDRFLGSGSGTDFTLTISSVQAEDLAVYYCQQYY KFPPTFGGGTKLELKRA EVQLVESGGDLVQPGRSLKLSCAASGFTFSNYDMAWVRQAPTQGLEWVA3 23A7 VH SISGSGGTTYYRDSVKGRFTVSRDNAKSTLYLQMDGLRSEDTATYFCAR HPPSFWYFDFWGPGSMVTVS S D I VMT QT P S S QAVS AGE KVTMS CKSGQSLLFNKNKKDYLAWYQ RK P GQ S4 23A7 VL PKLLIYWASTRESGVPDRFIGGGSGTDFTLTISSVQAEDLAVYYCQQYY RFPPTFGHGTKLELKRA EVQLVESGGGLVQPGRSLKLSCAASGFTFSNYDMAWVRQAPTKGLEWVA18 A4 VH5 SISPSGGSIYYRDSVKGRFTVS RNNAKS S LYLQMDS LRS EDTAT YYCAR andl8A4-2 VHLTTYFWYFDFWGPGTMVTVS S DIQMTQSPSFLSASVGDRVTINCKASQNSNKYLNWYQQKLGEAPKLLIY6 18A4 VL NTNNLQTGIPSRFSGSGSGTDYTLTISSLQPEDVATYFCLQHSSRMDTFGAGTKLELKRAD I VMT QT P S S QAVS AGE KVTMS C K SSQSLLYSENKKNYLAW YQQ K P GQ S 18A4-2 VL PKLLIYWASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLAVYYCQQYY KFPPTFGSGTKLEIK MYNGIGLPTPRGSGTNGYVQRNLSLVRGRRGERPDYKGEEELRRLEAAL VKRPNPDILDHERKRRVELRCLELEEMMEEQGYEEQQIQEKVATFRLML LEKDVNPGGKEETPGQRPAVTETHQLAELNEKKNERLRAAFGISDSYVD GSSFDPQRRAREAKQPAPEPPKPYSLVRESSSSRSPTPKQKKKKKKKDR GRRSESSSPRRERKKSSKKKKHRSESESKKRKHRSPTPKSKRKSKDKKR KRS RSTT PARKS RRAHRST SADSAS S S DT S RS RS RSAAAKTHTTALAGR SPSPASGRRGEGDAPFSEPGTTSTQRPSSPETATKQPSSPYEDKDKDKK EKSATRPSPSPERSSTGPEPPAPTPLLAERHGGSPQPLATTPLSQEPVN PPSEASPTRDRSPPKSPEKLPQSSSSESSPPSPQPTKVSRHASSSPESP KPAPAPGSHREISSSPTSKNRSHGRAKRDKSHSHTPSRRMGRSRSPATA KRGRSRSRTPTKRGHSRSRSPQWRRSRSAQRWGRSRSPQRRGRSRSPQR P GWS RS RNTQRRGRS RS ARRGRS H S RS PAT RGRS RS RT PARRGRS RS RT PARRRS RS RT PT RRRS RS RT PARRGRS RS RT PARRRS RT RS P VRRRS RS RS PARRS GRS RS RT PARRGRS RS RT PARRGRS RS RT PARRS GRS RS RT P ARRGRSRSRTPRRGRSRSRSLVRRGRSHSRTPQRRGRSGSSSERKNKSR TSQRRSRSNSSPEMKKSRISSRRSRSLSSPRSKAKSRLSLRRSLSGSSP CPKQKSQTPPRRSRSGSSQPKAKSRTPPRRSRSSSSPPPKQKSKTPSRQ SHSSSSPHPKVKSGTPPRQGSITSPQANEQSVTPQRRSCFESSPDPELK SRTPSRHSCSGSSPPRVKSSTPPRQSPSRSSSPQPKVKAIISPRQRSHS GSSSPSPSRVTSRTTPRRSRSVSPCSNVESRLLPRYSHSGSSSPDTKVK PETPPRQSHSGSISPYPKVKAQTPPGPSLSGSKSPCPQEKSKDSLVQSC PGSLSLCAGVKSSTPPGESYFGVSSLQLKGQSQTSPDHRSDTSSPEVRQ SHSESPSLQSKSQTSPKGGRSRSSSPVTELASRSPIRQDRGEFSASPML KSGMSPEQSRFQSDSSSYPTVDSNSLLGQSRLETAESKEKMALPPQEDA TASPPRQKDKFSPFPVQDRPESSLVFKDTLRTPPRERSGAGSSPETKEQ NSALPTSSQDEELMEWEKSEEPAGQILSHLSSELKEMSTSNFESSPEV EERPAVSLTLDQSQSQASLEAVEVPSMASSWGGPHFSPEHKELSNSPLR ENSFGSPLEFRNSGPLGTEMNTGFSSEVKEDLNGPFLNQLETDPSLDMK SRRM2 EQSTRSSGHSSSELSPDAVEKAGMSSNQSISSPVLDAVPRTPSRERSSS ASSPEMKDGLPRTPSRRSRSGSSPGLRDGSGTPSRHSLSGSSPGMKDIP RTPSRGRSECDSSPEPKALPQTPRPRSRSPSSPELNNKCLTPQRERSGS ESSVDQKTVARTPLGQRSRSGSSQELDVKPSASPQERSESDSSPDSKAK TRTPLRQRSRSGSSPEVDSKSRLSPRRSRSGSSPEVKDKPRAAPRAQSG SDSSPEPKAPAPRALPRRSRSGSSSKGRGPSPEGSSSTESSPEHPPKSR TARRGSRSSPEPKTKSRTPPRRRSSRSSPELTRKARLSRRSRSASSSPE TRSRTPPRHRRSPSVSSPEPAEKSRSSRRRRSASSPRTKTTSRRGRSPS PKPRGLQRSRSRSRREKTRTTRRRDRSGSSQSTSRRRQRSRSRSRVTRR RRGGS GYHS RS PARQES S RTSS RRRRGRS RT P PT S RKRS RS RT S PAPWK RS RS RAS PATHRRS RS RT P L I S RRRS RS RT S P VS RRRS RS RT S VT RRRS RS RAS P VS RRRS RS RT P PVT RRRS RS RT PTT RRRS RS RT P PVT RRRS RS RT P PVT RRRS RS RT S P I T RRRS RS RT S PVT RRRS RS RT S PVT RRRS RS R TSPVTRRRSRSRTPPAIRRRSRSRTPLLPRKRSRSRSPLAIRRRSRSRT P RTARGKRS LT RS P PAI RRRS AS GS S S DRS RS AT P PAT RNH S GS RT P P V ALNSSRMSCFSRPSMSPTPLDRCRSPGMLEPLGSSRTPMSVLQQAGGSM MDGPGPRIPDHQRTSVPENHAQSRIALALTAISLGTARPPPSMSAAGLA ARMS Q VP AP VP LMS L RT AP AAN LAS RI P AAS AAAMN LAS ART P Al P T AV N LAD S RT P AAAAAMN LAS P RT AVAP S AVN LAD P RT P TAP AVN LAGART P AALAAL SLTGSGTPP T AAN Y P S S S RT P QAP AS AN L VG P RS AHAT AP VN I AGS RTAAALAPAS LT S ARMAPAL S GANLT S P RVP L S AYERVS GRT S P P L LDRARSRTPPSAPSQSRMTSERAPSPSSRMGQAPSQSLLPPAQDQPRSP VPSAFSDQSRCLIAQTTPVAGSQSLSSGAVATTTSSAGDHNGMLSVPAP GVPHSDVGEPPASTGAQQPSALAALQPAKERRSSSSSSSSSSSSSSSSS SSSSSSSSGSSSSDSEGSSLPVQPEVALKRVPSPTPAPKEAVREGRPPE PTPAKRKRRSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS SSPSPAKPGPQALPKPASPKKPPPGERRSRSPRKPIDSLRDSRSLSYSP VERRRPSPQPSPRDQQSSSSERGSRRGQRGDSRSPSHKRRRETPSPRPM RHRSSRSP9 13F11 VH- NYDMACDR110 13F11 VH- SIS P SNVNT YYRDS VKGCDR211 13F11 VH- LVHSFWYFDFCDR312 13F11 VL- KSSQSLLYSKNKKNYLACDR113 13F11 VL- WAS T RESCDR214 13F11 VL- QQYYKFPPTCDR315 23A7 VH-CDR1 NYDMA16 23A7 VH-CDR2 SISGSGGTTYYRDSVKG17 23A7 VH-CDR3 HPPSFWYFDF18 23A7 VL-CDR1 KSGQSLLFNKNKKDYLA19 23A7 VL-CDR2 WAS T RES20 23A7 VL-CDR3 QQYYRFPPT21 18A4 VH-CDR1 NYDMAand 18A4-2VH-CDR122 18A4 VH-CDR2 SISPSGGSIYYRDSVKGand 18A4-2VH-CDR223 18A4 VH-CDR3 LTTYFWYFDFand 18A4-2VH-CDR124 18A4 VL-CDR1 KASQNSNKYLN25 18A4 VL-CDR2 NTNNLQT26 18A4 VL-CDR3 LQHSSRMDT27 18A4-2 VL- KSSQSLLYSENKKNYLACDR128 18A4-2 VL- WAS T RESCDR229 18A4-2 VL- QQYYKFPPTCDR3

[0166] The fo lowing abbreviations were used in the context of the present invention: VH = variable heavy chain; VH = variable heavy chain; VL = variable light chain; CDR = complementarity determining region; VH-CDR = CDR of a variable heavy region; VL-CDR = CDR of a variable light region; CDRs can be determined by using the Kabat algorithm, e.g., http : / / aby si s . org / aby si s / .

[0167] It must be noted that as used herein, the singular forms "a", "an" and "the" include plural references and vice versa unless the context clearly indicates otherwise.

[0168] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series.

[0169] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0170] The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0171] The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It includes, however, also the concrete number, e.g., about 20 includes 20.

[0172] Throughout this specification and the claims, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”.

[0173] When used herein “consisting of' excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of' does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0174] In each instance herein any of the terms "comprising", "consisting essentially of' and "consisting of' may be replaced with either of the other two terms. For example, when disclosure uses the term “comprising”, the disclosure also encompasses the replacement of the term “comprising” with the terms “consisting essentially of’ as well as “consisting of’ and vice versa. E.g., the term "comprising" is meant to provide explicit support also for its replacement with "consisting essentially of' and / or "consisting of', the term "consisting essentially of' is meant to provide explicit support also for its replacement with "comprising" and / or "consisting of', and the term "consisting of' is meant to provide explicit support also for its replacement with "consisting essentially of' and "comprising". The possibility to replace terms with each other is not to be understood that these terms are synonymous. For the avoidance of doubt, the term "comprising", "consisting essentially of' or "consisting of' that is explicitly recited in the respective context is the preferred term, while its replacement with any one of the other two terms is less preferred.EXAMPLESExample 1: Materials and methods

[0175] Cell lines

[0176] MOLM-13 (myelodysplasia-related AML) and HL-60 (promyelocytic leukemia) were purchased from Nanjing Saihongrui Biotechnology Co. Ltd. The MV4-11 (biphenotypic B-myelomonocytic leukemia, FLT3-ITD positive) was purchased from Wuhan Warner Bio Co. Ltd. and the SKM-1 (an AML-MDS cell line, transformation of myelodysplastic syndromes to acute myeloid leukemia) from Hefei Zhihong Tuoda Biotechnology Co. Ltd. HL-60 and MV4-11 cells were cultured in IMDM medium (Cytiva, SH30228.01), supplemented with 10% fetal bovine serum (FBS, Lonsera, S711-001S). Cells were maintained in RPMI 1640 (Cytiva, SH30255.01) with 10% FBS and incubated at 37 °C in a humidified atmosphere containing 5% CO2.

[0177] Patients

[0178] In this study, 76 patients were enrolledwith acute myeloid leukemia (AML) admitted to the Second Affiliated Hospital of Anhui Medical University from January 2022 to March 2024. This cohort included 52 newly diagnosed (ND) and 24 relapsed AML cases. Normal controls were selected from patients who underwent bone marrow aspiration for diagnostic purposes and were subsequently confirmed to be free of hematologic malignancies, including iron deficiency anemia (IDA). The use of human materials for this research was approved by the Ethics Committee of the Second Affiliated Hospital of Anhui Medical University (Approval No. YJ-YX2019-015). The diagnosis of AML adhered to the 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Myeloid and Histiocytic / Dendritic Neoplasms, which incorporates molecular, cytogenetic, and clinical parameters to ensure precise disease classification. Patient cytogenetic stratification and clinical management followed the 2022 NCCN guidelines. Peripheral blood (PB) and bone marrow (BM) samples were collected and immediately used for analysis of surface SRRM2 expression using flow cytometry (FCM). Comprehensive patient information is available in the supplementary materials.

[0179] Flow cytometry

[0180] Peripheral blood from AML patients was treated with erythrocyte lysis buffer and divided into two tubes for FCM. SRRM2 (EX-02) mAb (Eximmium Biotechnologies; Munich, Germany) or an isotype control antibody (cat. No. B355601; Abinvivo, Shanghai, China) were added and incubated at room temperature (RT) for 40 minutes. The cells were washed twice with PBS and then stained with CD34-PE (cat. no. A07776; Beckman Coulter, Brea, CA), CD117-PE (cat. no.IM2732; Beckman Coulter), CD33-APC (cat. no. IA2471; Beckman Coulter), CD45-PC7 (cat. no. IM3548; Beckman Coulter), and Goat anti-rat IgG Alexa488-coupled antibody (cat. no. 150165; Abeam) in the dark at RT for 15 minutes. Samples were performed using a Cytoflex flow cytometer (Beckman Coulter) and results were analyzed using CytExpert for DxFLEX software.

[0181] For intracellular staining, 6xl06PBMCs were fixed in 100 pL fixative (cat. no. A07803; Beckman Coulter) at RT for 15 minutes. Then, 4 mL of PBS were added, cells were pelleted by centrifugation, and the supernatant was discarded. Permeabilization was performed by adding 100 pL of Reagent 2 for 5 minutes, followed by gentle agitation. Cells were incubated with EX-02 or an isotype antibody for 30 minutes, washed twice in PBS, and then incubated with an Alexa488-coupled secondary antibody (cat. no. 150165; Abeam) in the dark for 15 minutes. After a final PBS wash, cells were analyzed by flow cytometry.

[0182] Preparation of conditioned medium and culture of RAJI cells for SRRM2 expression analysis

[0183] To investigate the effect of SRRM2 expression on RAJI cells, MOLM-13 cells with high surface expression of SRRM2 were used to prepare conditioned medium. MOLM-13 cells were cultured in RPML1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. After reaching 70-80% confluence, the culture supernatant was collected, centrifuged at 300 * g for 5 minutes to remove cell debris, and filtered through a 0.22 pm filter to obtain the conditioned medium. RAJI cells were seeded in 6-well plates at a density of 1 x 106cells per well and cultured in the conditioned medium derived from MOLM-13 cells for 24, 48, and 72 hours. RAJI cells cultured in standard RPML1640 medium with 10% FBS served as the control group. After the indicated incubation periods, RAJI cells were harvested, washed twice with PBS and analyzed by FCM.

[0184] Immunofluorescence

[0185] The cells were grown on glass coverslips and fixed with 4% paraformaldehyde (Biosharp, BL539A). Cells were blocked in 2% normal donkey serum (Solarbio, SL050) for 30 minutes at RT. Cells were incubated with anti-SRRM2 antibodies (EX02) dissolved in PBST (Servicebio, G2157-1L) overnight at 4 °C. After washing thrice with PBST, cells were incubated with Goat Anti-Rat IgG H&L / AF488 (Abeam, ab 150165) antibodies for 2 hours at RT. The nuclei were stained using Antifade Mounting Medium with DAPI (P0130; Beyotime, Shanghai, China). Images were captured using a fluorescence microscope (Axio Scope Al, Zeiss, Oberkochen, Germany).

[0186] Immunohi stochemi stry

[0187] Bone marrow smears from AML (n=2) and IDA (n=2) patients utilized in this study which were obtained from our research institution were prepared following standard protocols. The normal tissue specimens used in this study were obtained from Shanghai Outdo Biotech Company (Shanghai, China; Cat no. HOrgC120PG04; Lot no. XT19-008). The project, titled Development and Application of Multi-Organ Tissue Chip Products, was reviewed and approved by the Ethics Committee of Shanghai Outdo Biotech Company, ensuring compliance with ethical standards. Smears were deparaffinized in xylene, rehydrated through graded ethanol solutions (100% twice for 5 minutes each, 95%, 80%, and 70% each for 5 minutes), and rinsed in distilled water for 5 minutes. Rehydration was performed in PBS containing 1% normal donkey serum (DSA; Solarbio, Beijing, China) for 5 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 5 min. Antigen retrieval was achieved by heating the smears in citrate buffer (1.8 mM citric acid, 8.2 mM sodium citrate) at 95 °C for 20 minutes. Non-specific binding was blocked with PBS / 5% DSA for 1 h. Smears were then incubated with EX-02 (1:200) at 4 °C overnight and then incubated with an HRP-labeled anti-rat IgG H&L antibody (Abeam, Shanghai, China; ab7097; 1:200) at RT for 1 h. Detection was performed using a DAB substrate kit (No. SD3102; CELNOVTE, Suzhou, China) until optimal staining was achieved. Counterstaining was done with hematoxylin (Biosharp, Hefei, China; #71025010). The smears were mounted with neutral balsam and examined under a light microscope, and images were captured with a microscopic imaging system.

[0188] Construction of EX-02 CARs and CAR-T cells

[0189] A humanized single-chain variable fragment (scFv) of the original rat-derived EX-02 antibody was cloned in frame into a lentiviral expression vector carrying a 2nd generation CAR backbone with 4-1BB / CD137 and CD3-(^ transactivation domains and checked for integrity by sequencing. Lentiviral vectors were produced by co-transfecting HEK293 cells with VSV-G envelope; GAG-Pol packaging factors, and Rev plasmids. Supernatants were harvested 72h later, filtered, concentrated by centrifugation, tested on HEK293 cells to calculate the titer of infectious virus, and finally cryopreserved at -80°C until use.

[0190] Peripheral blood mononuclear cells (PBMCs) were isolated from the PB of healthy volunteers by Ficoll separation. The cells were seeded at a density of 2* 106cells / ml and stimulated using CD3 / CD28 monoclonal antibody (mAb) (100 lU / ml or 300 lU / ml, Miltenyi, Bergisch-Gladbach, Germany) for 24 hour. T cells were transduced with LVs at a multiplicity of infection(MOI) of 5. Simultaneously, human serum albumin (HSA, lOng / ml, Baxalta, West Sacramento, CA) was added to optimize transfection efficiency. The cells were washed to remove the LV particles 24 h after transduction. In contrast, MOCK-T cells in the uninfected group were grown following a procedure like that of CAR-T cells but devoid of any viral infection. T cells were expanded in PRIME-XV T cell chemical defined medium (CDM, Santa Ana, CA) for 4-7 days in the presence of IL-2 (300 lU / ml or 600 lU / ml, Quangang, China) or IL-7 and IL-15 (lOng / ml, SinoBiological, Beijing, China). Cell products were washed twice with PBS for subsequent experiments.

[0191] Analysis of CAR-T cell cytotoxicity

[0192] Effector and target cell cultures were prepared by adjusting the cell density to l*106cells / mL. Mock-T or CAR-T cells were co-cultured with MOLM-13 or HL-60 cells at effector-to-target ratios of 1:1 and 2.5:1. The appropriate numbers of effector and target cells were added to a 24-well plate, with three replicates established for each group. Complete medium was added to each well to achieve a final volume of 800 pL, and the contents were thoroughly mixed. The plate was incubated at 37 °C for 0, 12, 24, and 48 h. At each time point, cells were collected and thoroughly resuspended to obtain a single-cell suspension. A 10 pL aliquot of the suspension was taken for cell counting. The remaining cells were centrifuged at 300g for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in 100 pL of PBS, followed by staining with PC7-CD45, FITC-CD3, and PE-CD33 antibodies. After a 15-minutes incubation at RT, the cells were washed with PBS to remove unbound antibodies. The final cell suspension was resuspended in 500 pL of PBS and prepared for flow cytometry analysis.

[0193] Alternatively, the cytotoxicity of EX-02 CAR-T cells was evaluated using a Cytotoxicity LDH Assay Kit (Dojindo, Shanghai, China; #CK12-05). All procedures were performed following the manufacturer's instructions.

[0194] Cytokine quantitation with ELISA assays

[0195] Commercial ELISA assays (ELK Biotechnology, Wuhan, China) were used according to the provider’s protocol.

[0196] AML xenograft mouse model

[0197] 6-week-old immunodeficient NSG mice, SPF grade, were purchased from Shanghai Model Organisms Pharmachem Biotechnology Co. At day -2, P IO6MOLM-13 cells per mouse were injected into the tail vein. Two days later (d = 2), tumor-bearing mice were randomized into two groups (n=5) by blinded investigators and intravenously administered either MOCK-T (non-transduced T cells, 2*106) or EX-02 CAR-T cells (2*106). Blood, BM or tumor tissues were collected at terminal day when the mice were sacrificed. The animal model has been approved by Institute of Health and Medicine, Hefei Comprehensive National Science Central (Approval No: IHM-AP-2024-084-R1). The period of validity extends from Nov. 4, 2024 to Nov. 4, 2027.

[0198] Statistical analysis

[0199] Statistical analysis was performed using IBM SPSS Statistics 25 and GraphPad Prism 8.0. Continuous data are presented as mean ± standard deviation f,x ± S). For comparison between the two groups, if the data followed a normal distribution, differences were assessed using the t-test, else the data were expressed as median and interquartile ranges and analyzed using a nonparametric rank-sum test. Survival time was defined as the time from the first diagnosis until death (all-cause mortality). A p-value < 0.05 was considered statistically significant.Example 2: The nuclear speckle protein SRRM2 is exposed on the surface of AML cells

[0200] Data from The Cancer Genome Atlas (TCGA) and other public databases indicate that SRRM2 is significantly overexpressed in acute myeloid leukemia (AML) cells compared to normal blood cells (kmplot.com; TCGA at fobinf.com; precog.stanford.edu), but the overall RNA levels of SRRM2 do not correlate with key clinical parameters, such as overall survival. Recent studies have linked SRRM2 phosphorylation to its intracellular translocation and resistance to cytarabine. SRRM2 has also been reported on the surface of multiple myeloma and solid tumor cells. These findings prompted an investigation into the intracellular localization of SRRM2 in AML blasts and normal blood cells, a topic not yet explored in AML.

[0201] To begin, the surface expression of SRRM2 across several AML cell lines using FCM with our proprietary antibody EX-02 was assessed. Figure 1A shows flow cytometric analysis of SRRM2 expression in AML cell lines (MOLM-13, MV4-11, SKM-1, and HL-60) which revealed binding of the EX-02 antibody to the surface of vital cells (upper panel) and intracellular SRRM2 (lower panel). In Figure IB surface SRRM2 expression in the AML cell line MOLM-13 was confirmed by fluorescence confocal microscope. SRRM2 (in green; see “Ex-02” and arrows)) was localized both on the cell surface of vital cells and intracellularly in permeabilized cells, corroborating results from flow cytometry. Nuclei were counterstained with DAPI (blue; see “DAPI” and arrows). Cell lines from non-AML, including RAJI , NAML-6, and SU-DHL-4, as well as HEK-293T cells from normal tissue, were analyzed by FCM for surface and intracellular staining. Surface expression levels of SRRM2 were significantly lower in these cell lines compared to AML lines, whereas intracellular expression was consistently high across all samples.Additionally, no significant difference in SRRM2 expression was observed in RAJI cells cultured with conditioned medium. These results indicated that SRRM2 surface expression varies across hematologic malignancies and was not due to non-specific binding, although SRRM2 was primarily an intracellular protein.Example 3: SRRM2 is exposed on blasts from patients with AML

[0202] Having established the presence of SRRM2 on the surface of AML-derived cell lines, we proceeded to conduct FCM analysis on various cell types, including normal lymphocytes, monocytes, granulocytes, and AML blasts freshly isolated from the peripheral blood (PB) and bone marrow (BM) of AML patients. Figure 2 shows that SRRM2 is exposed only on the surface of AML blasts but not on normal blood cells. Figure 2A shows flow cytometric analysis of subgroups of blood cells isolated from the peripheral blood of an AML patients. Upper panel: staining of vital cells with the EX-02 antibody demonstrated that SRRM2 is detectable on the surface of blast cellss, whereas normal blood cells (lymphocytes, monocytes, granulocytes) show only marginal background staining. Lower panel: intracellular staining of the same types of permeabilized cells showed positive intracellular SRRM2 staining of all cell types. Figure 2B is the same as Figure 2A, but with peripheral blood cells derived from a healthy donor. No surface SRRM2 was detectable after EX-02 staining. Figure 2C shows the same staining as in Figure 2A, but of bone marrow-derived cells of an AML patients. Again, only blast cells stained surface SRRM2 positive with EX-02 antibody, while all other types of cells showed only intracellular SRRM2 staining. Figure 2D shows cells isolated from the bone marrow of a patient suffering from iron deficiency anemia (IDA). No surface SRRM2 was detectable on lymphocytes, monocytes, granulocytes or hematopoietic stem cells (HSC). Taken together, surface SRRM2 is exclusively detectable on malignant AML blast cells, but not on normal blood cells.Example 4: Surface SRRM2 is a marker of risk and poor prognosis

[0203] Based on the finding described above, a correlative analysis of SRRM2 surface expression levels and clinical data was conducted in 52 ND AML patients. According to the ‘consensus of Chinese experts on the detection of lineage in acute leukemia by flow cytometry’, surface antigen expression exceeding 20% in the target cell population was defined as positive. Based on this criterion, ND AML patients were stratified into negative SRRM2 surface exposure (SRRM2Neg, <20%) and positive SRRM2 surface exposure (SRRM2Pos, >20%) groups. The basic clinical information including age, gender and disease state of the patients was shown in Table 2.

[0204] Tab. 2 Clinical characteristics of AML patients stratified by SRRM2 expressionAll cases SRRM2Ne« SRRM2Pos(n=52) (n=20) (n=32)FAge, years 60 (27-85) 56 (27-85) 65 (36-83) 0.5287 Gender, n(%) 0.8555 male 30 (58) 12 (63) 20 (61)famale 22 (42) 7 (27) 13 (39)Origin of disease 0.0674 De novo AML 42 (82) 14 (33) 28 (67)s-AML 9 (18) 5 (56) 4 (44)FAB, n(%) 0.9410 Ml 4 (8) 1 (25) 3 (75)M2 21 (40) 7 (33) 14 (67)M4 10 (20) 3 (30) 7 (70)u-AML 7 (14) 3 (43) 4 (57) WBC,109 / L 9.90 (1.03-417.3) 9.75 (1.09-417.3) 9.91 (1.03-135.20) 0.9694 HB,g / L 66 (28-147) 65 (28-105) 66.5 (39-147) 0.6156 PLT,109 / L 22 (5-534) 29 (5-345) 21 (8-534) 0.8672 Mono,109 / L 4.5 (0.1-240.2) 4.59 (0.08-240.2) 3.86 (0.21-77.44) 0.8731 Lym,109 / L 3.8 (0.5-171.3) 3.84 (0.54-171.3) 3.47 (0.53-34.43) 0.7533 Neu,109 / L 1.48 (0.1-49.69) 1.74 (0.17-16.26) 1.48 (0.04-49.69) 0.8547 Ret,109 / L 0.025 (0.002-0.498) 0.029 (0.011-0.498) 0.024 (0.002-0.257) 0.3847 Albumin 36.7 (26.9-43.7) 36.55 (28.9-40.7) 36.7 (26.9-43.7) 0.7397 Kalium (mM / L) 3.8 (2.3-7.3) 3.65 (2.49-4.78) 3.8 (2.3-7.3) 0.7333 Calcium (mM / L) 2.07 (1.59-2.3) 2.08 (1.85-2.30) 2.07 (1.59-2.25) 0.6936 Magnesium (mM / L) 0.83 (0.64-1.47) 0.86 (0.74-1.05) 0.83 (0.64-1.47) 0.9604 Cholesterin 2.89 (1.35-4.96) 2.86 (1.89-4.96) 3.23 (1.35-4.82) 0.5631 Triglyceride 1.28 (0.16-7.04) 1.29 (0.48-7.04) 1.16 (0.16-3.95) 0.6109 TCO2 25 (10.7-29.6) 25.1 (18.6-28.9) 25 (10.7-29.6) 0.9157 LDH (U / L) 461 (115-16295) 311.5 (117-722) 594.5 (115-16295) 0.0578 CK (U / L) 41 (3-2363) 39 (3-101) 75 (23-2363) 0.1824 CK-MB,(U / L) 10.5 (4-415) 9 (4-15) 11.5 (5-415) 0.0727 CD34+(%) 66 (0-98) 59 (0-98) 70 (0-96) 0.4868 CD38+(%) 79 (0-99) 79 (35-94) 78 (0-99) 0.8657 CD33+( %) 82 (14-99) 70.5 (21-98) 84.5 (14-99) 0.0928 CD117+(%) 85 (3-117) 86 (70-99) 84.5 (3-117) 0.512 HLA-DR+(%) 53 (0-98) 41 (3-98) 55 (0-95) 0.8539 CyMPO+(%) 48 (1.4-98) 52 (1.4-98) 48 (4-93) 0.6691 PB-Blasts (%) 48 (6.1-96) 47 (6.1-96) 48.5 (9.6-95) 0.8857 BM-Blasts (%) 54 (21-96) 50 (22-96) 55 (21-96) 0.6014

[0205] Baseline characteristics of 52 patients diagnosed with AML were detailed in the table, with stratification based on SRRM2 surface exposure level. The patients were categorized into two groups: SRRM2 surface exposure negative (SRRM2Neg, n=20) and SRRM2 surface exposure positive (SRRM2Pos, n=32). Median values and ranges were reported for continuous variables, including age, white blood cell count (WBC), hemoglobin (HB), platelet count (PLT), and various biochemical markers. Categorical variables, such as gender distribution, disease origin, and French- American-British (FAB) classification types, were presented as percentages.

[0206] Statistical analyses were performed to evaluate differences between the two groups, with p-values indicating significance levels; a p-value of less than 0.05 was considered statistically significant. Near-significant differences were observed in lactate dehydrogenase (LDH) levels (p=0.0578), CD33+ expression (p=0.0928), and creatine kinase-MB (CK-MB) levels (p=0.0727). Although these differences did not achieve statistical significance, they were suggestive of potential correlations between SRRM2 expression and various clinical and laboratory features in AML patients.

[0207] Figure 3 shows the correlation between SRRM2 surface exposure and clinical features in AML patiets. Figure 3 A-B shows that SRRM2 exposure was analyzed by flow cytometry in de novo vs. secondary AML (A) and various WHO-FAB subtypes (M0, Ml, M2, M4) (B) No significant differences were observed between these groups. Figure 3C shows that SRRM2 surface exposure in patients with standard inductive chemotherapy failure (Not complete response, NCR) was higher significantly than in patients with complete response (CR). Figure 3D shows that SRRM2 surface exposure in adverse and intermediate-risk patients stratified by the 2022 NCCN risk classification remarkably increased comparing to in favorable-risk patients. Figure 3E shows that relapsed AML patients showed higher SRRM2 surface exposure compared to newly diagnosed (ND) patients. Figure 3F shows that longitudinal analysis of seven patients with AML revealed that the SRRM2 exposure on surface of AML blast cells at relapse increased considerably.Figure 3G-H show that SRRM2 exposure on surface of AML blast cells from ND patients (Figure 3G) and Relapsed patients (Figure 3H) greatly increased compared to the other blood cells.

[0208] For SRRM2Pospatients, ROC curve analysis determined a cut-off value of 30.14% to distinguish between low and high expression levels, reflecting the differential response to standard induction chemotherapy (Figure 5). Consequently, all newly diagnosed AML patients were categorized into three groups: SRRM2Neg(<20%), SRRM2Low(20%-30.14%), and SRRM2High(>30.14%).

[0209] Figure 3 further shows the following: Figure 3I-J shows that complete remission rates (CRR) in AML patients with SRRM2 surface exposure positive (SRRM2Pos) significantly lower than in patients with SRRM2 surface exposure negative (SRRM2Neg), and more SRRM2 surface exposure along with lower CRR. Figure 3K-L shows that Kaplan-Meier analysis showed shorter relapse-free survival (RFS) in SRRM2Pos patients, with high SRRM2 expression linked to reduced RFS. Figure 3M-N shows that SRRM2Pos patients had shorter overall survival (OS) than SRRM2Neg patients. *P<0.05;**P<0.01;***P<0.001. In summary, SRRM2 surface exposure correlated closely with clinical characteristics in AML patients, with higher SRRM2 exposure associated with poorer prognosis. These findings suggest that SRRM2 could serve as a valuable prognostic biomarker in AML. Moreover, its exposure on the cell surface makes it a promising new target for immunotherapy.Example 5: Surface SRRM2 is not detected on normal hematopoietic stem cells and various normal tissues.

[0210] To evaluate SRRM2 expression in normal hematopoietic cells, including hematopoietic stem cells (HSCs), we conducted flow cytometry (FCM) analysis of bone marrow (BM) cells from patients with iron-deficiency anemia (IDA). As illustrated in Figure 4Aflow cytometric analysis of hematopoietic cells from patients with iron-deficiency anemia (IDA) demonstrated minimal surface expression of SRRM2 in lymphocytes, monocytes, neutrophils, and hematopoietic stem cells (HSCs). In Figure 4B it is apparent that there was strong nuclear expression of SRRM2 across all cell types, with nearly complete positivity noted. Immunohistochemical analysis revealed no significant surface expression of SRRM2 various normal tissues, including cerebellum, colon, liver, lung, skin, pancreas, heart, kidney, lymph node, muscle and spleen (see Figure 4C).

[0211] Our findings lead us to postulate that SRRM2, typically a nuclear protein, is specifically exposed on the surface of AML blasts. In contrast, SRRM2 is either minimally expressed or completely absent on the surface of normal blood cells, including hematopoietic stem cells. Furthermore, various normal tissues exhibited surface SRRM2 levels below detection limits. Notably, we observed a direct correlation between SRRM2 surface levels on AML blasts and high-risk stratification, as well as adverse prognosis. This association underscores the potential clinical relevance of SRRM2 as a biomarker in AML. Targeting EX-02 CAR-T cells displayed effect against AML in vitro and in vivo.Example 6: SRRM2-specific CAR-T cells exhibit anticancer activity against AML cells in vitro and in vivo

[0212] CAR-T cell therapy has demonstrated remarkable efficacy in treating various hematological malignancies. However, its application in acute myeloid leukemia (AML) has been limited due to the absence of specific and suitable target molecules on AML blasts, resulting in no commercially available CAR-T products for this condition. Our research identified surface SRRM2 as a promising target molecule uniquely expressed on AML blasts while absent on normal blood cells. Consequently, we aimed to evaluate the anti-tumor efficacy of SRRM2-specific CAR-T cells both in vitro and using a human xenograft model of AML. To achieve this, we engineered a CAR incorporating the single-chain variable fragment (scFV) derived from the EX-02 antibody and created lentiviral vectors as previously described. The EX-02 CAR-T cells exhibited potent cytolytic activity against all tested AML cell lines, including MOLM-13, MV4-11, SKM-1, and HL-60, at effector-to-target (E : T) ratios of 1:1 and 2.5:1. This cytotoxicity was accompanied by significantly elevated levels of cytokines IFN-y and TNF-a in the supernatants of EX-02 CAR-T cells co-cultured with target cells compared to those incubated with MOCK-T cells. Furthermore, a lactate dehydrogenase (LDH) release assay confirmed a dose-dependent increase in tumor cell lysis after 24 hours of co-culture, with higher E:T ratios correlating with enhanced cytolytic activity.

[0213] Having confirmed that EX-02 CAR-T cells have anti-tumor activity in vitro, we next set up a human xenograft model in immunocompromised NSG mice to assess the therapeutic potential of EX-02. For this, 1 * 106MOLM-13 cells per mouse were injected into the tail vain. 2 days later, mice were randomized into three groups and treated with MOCK-T (non-transduced T cells, n=5) or EX-02 CAR-T cells (2* 106cells per mouse, n=5) and PBS (Blank group, n=3). Bioluminescence images (BLI) of the tumor-bearing mice taken at Days 0, 6, 12, 18, 21, and 24. At Day 12, the CAR-T-treated group exhibited significantly reduced tumor burden compared to both the MOCK-T and Blank groups. Bioluminescence quantification at Day 24 confirmed significantly reduced tumor activity in the CAR-T group (p < 0.001) compared to both the MOCK-T and Blank groups. Survival analysis showed a significant extension of survival in the EX02 CAR-T treated group. With the CAR-T group exhibiting significantly prolonged survival compared to both the MOCK-T (p = 0.0026) and Blank groups (p = 0.0016). No significant survival difference was observed between the MOCK-T and Blank groups (p = 0.6554), indicating that CAR-T therapy significantly prolonged survival in this AML xenograft model. Addtional, mice in the MOCK-T group experienced significant weight loss (p = 0.02) starting from Day 12. The strong in vivo efficacy ofEX-02 CAR-T cells and underscoring the potential of SRRM2-specific CAR-T cells as a viable and promising therapeutic strategy for AML.Example 7: Discussion

[0214] While antibody-based immunotherapies have revolutionized the treatment of various cancers, progress in addressing AML has been limited. This challenge primarily arises from the scarcity of suitable target molecules that are present on malignant cells but not on normal blood cells, including hematopoietic stem cells (HSCs). Our research employing EX-02, a proprietary antibody specifically designed to target the surface-exposed variant of SRRM2 has produced significant findings in this respect. These studies reveal that SRRM2, typically recognized as a nuclear protein, is notably expressed on the surface of acute myeloid leukemia (AML) cell lines and in primary blasts from AML patients. While data from The Cancer Genome Atlas (TCGA) indicate that SRRM2 is overexpressed at the total RNA level in AML, this overexpression does not correlate with clinical parameters such as progression- free and overall survival. In stark contrast, our findings demonstrate that elevated levels of surface SRRM2 are not only directly and significantly associated with poorer clinical outcomes but also with treatment resistance and lower complete remission rates. This finding shows that extranuclear SRRM2, whether located in the cytoplasm or at the cell surface, may play a crucial role in the malignancy of AML and potentially other cancer types.

[0215] Traditionally, SRRM2 is recognized as an RNA-binding nuclear protein that plays a critical role in splicing processes. Previous studies have established its involvement in the formation and function of nuclear speckles and maintaining cellular homeostasis.

[0216] Surface expression of SRRM2 on cancer cells including AML blasts was unexpected, yet not without precedent, as other splicing proteins have also been identified on the surfaces of cancer cells. For example, Christian et al. (The Journal of cell biology. 2003;163(4):871-8) discovered nucleolin on the surface of endothelial cells within the tumor vasculature. Similarly, Gillissen et al. (Blood. 2018; 131(1): 131-43) reported U5 snRNP200's presence on AML blasts, while Tonapi et al. (Cell chemical biology. 2019;26(5):756-64.e6) identified a splicing complex comprising at least 13 core components on the surface of non-Hodgkin’s lymphoma.

[0217]

[0218] Our findings reveal that SRRM2 is present on the surface of AML blasts while being nearly absent from normal blood cells, including hematopoietic stem cells (HSCs). Notably, SRRM2-specific CAR-T cells exhibit significant anti-cancer activity both in vitro and in vivo. This suggeststhat SRRM2-targeted immunotherapeutic approaches hold great promise for the treatment of AML. By focusing on more selective targets like SRRM2, we may enhance the efficacy of CAR-T-based immunotherapy while minimizing adverse effects associated with current antigen targets.Example 8: Conclusions and Future Directions

[0219] Our study identifies SRRM2 as a novel and highly specific target for acute myeloid leukemia (AML) therapy. The unique surface expression of SRRM2 in AML cells, coupled with its nuclear confinement in normal tissues, creates a compelling opportunity for the development of targeted immunotherapies, such as EX-02 CAR-T cells. This approach has the potential not only to enhance outcomes for high-risk and relapsed AML patients but also to pave the way for personalized therapeutic strategies in AML.

[0220] Embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present embodiments have been specifically disclosed by preferred embodiments and optional features, modification and variations thereof may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. Each of the narrower species and subgeneric groupings falling within the generic disclosure also forms part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0221] Equivalents: Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0222] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. Theterminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0223] All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer’s specifications, instructions, etc.) are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0224] Further embodiments will become apparent from the following claims.

Claims

CLAIMS1. A method for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative or positive prognosis, comprisingdetermining in vitro the level of SRRM2 expression on the cell surface of blast cells of said subject, wherein(i) a subject having a level of 20% or less SRRM2 expressing blast cells is characterized as being SRRM2-negative, and(ii) a subject having a level of more than 20% SRRM2 expressing blast cells is characterized as being SRRM2-positive,wherein an SRRM2-negative subject has a positive prognosis, whereas an SRRM2-positive subject has a negative prognosis.

2. The method of claim 1, wherein an SRRM2-positive subject has either a level of more than 20% and up to 30% SRRM2 expressing blast cells and is characterized as being SRRM2- positive low or has a level of more than 30% SRRM2 expressing blast cells and is characterized as being SRRM2-positive high.

3. The method of claim 1 or 2, wherein a negative prognosis of an SRRM2-positive subject is associated with a higher failure to induction chemotherapy including cytarabine and an anthracycline drug, such as daunorubicin or idarubicin, higher relapse rate, lower complete remission rate (CRR), shorter relapse-free survival (RFS), or shorter overall survival (OS) when compared to SRRM2-negative subjects.

4. The method of claim 1 or 2, wherein a positive prognosis of an SRRM2-negative subject is associated with a lower failure to induction chemotherapy including cytarabine and an anthracycline drug, such as daunorubicin or idarubicin, lower relapse rate, higher complete remission rate (CRR), longer relapse-free survival (RFS), longer overall survival (OS) when compared to SRRM2-positive subjects.

5. The method of any one of the preceding claims, wherein the level of SRRM2 expression on the cell surface of blast cells is determined by flow cytometry.

6. The method of any one of the preceding claims, wherein the level of SRRM2-expressing blast cells is in relation to the total number of blast cells of said subject which are measured.

7. The method of any one of the preceding claims, wherein said subject is intended for or is subject to a therapy for treating AML.

8. The method of any one of the preceding claims, wherein an SRRM2-positive subject is amenable for the therapy with an antibody binding to SRRM2 on the cell surface of SRRM2 expressing cells.

9. The method of any one of the preceding claims, wherein SRRM2 expression on the cell surface of blast cells is determined with an antibody binding to SRRM2 on the cell surface of SRRM2 expressing cells.

10. The method of claim 9, wherein said antibody binds to non-permeabilized SRRM2 expressing cells.

11. The method of claim 9 or 10, wherein said SRRM2 expressing cells are living and intact blast cells.

12. The method of any one of claims 8 to 11, wherein said antibody is an antibody comprising (a) a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 9, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 10, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 11, anda light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 12, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 13, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 14;(b) a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 15, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 16, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 17, and53a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 18, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 19, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 20;(c) a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 21, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 22, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 23, anda light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 24, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 25, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 26; or(d) a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 21, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 22, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 23, anda light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 27, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 28, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 29.

13. The method of any one of claims 8 to 12, wherein said antibody is an antibody comprising (a) a heavy chain variable region having the amino acid sequence as set forth in SEQ ID NO: 1 and a light chain variable region having the amino acid sequence as set forth in SEQ ID NO: 2,(b) a heavy chain variable region having the amino acid sequence as set forth in SEQ ID NO: 3, and a light chain variable region having the amino acid sequence as set forth in SEQ ID NO: 4,(c) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:5, and light chain variable region having the amino acid sequence as set forth in SEQ ID NO: 6, or(d) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:5, and light chain variable region having the amino acid sequence as set forth in SEQ ID NO: 7.5414. The method of any one of claims 1 to 15, wherein the immediate purpose of the method is not to diagnose and / or treat diseases.

15. In vitro use of the level of SRRM2 expression on the cell surface of blast cells of a subject diagnosed with acute myeloid leukemia (AML) for the evaluation whether said subject will have a negative or positive prognosis, wherein(i) a subject having a level of 20% or less SRRM2 expressing blast cells is characterized as SRRM2-negative and has a positive prognosis, whereas(ii) a subject having a level of more than 20% SRRM2 expressing blast cells is characterized as SRRM2-positive and has a negative prognosis.

16. The use of claim 15, wherein an SRRM2-positive subject has either a level of more than 20% and up to 30% SRRM2 expressing blast cells and is characterized as being SRRM2- positive low or has a level of more than 30% SRRM2 expressing blast cells and is characterized as being SRRM2-positive high.

17. The use of claim 15 or 16, wherein the immediate purpose of the use is not to diagnose and / or treat diseases.

18. Use of a binding agent which is capable of specifically binding SRRM2 on the surface of a blast cell for the manufacture of a composition or a kit for the evaluation whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative or positive prognosis.

19. The use of claim 18, wherein the binding agent is or comprises an antibody and / or an antigen-binding fragment thereof, which is capable of specifically binding to SRRM2.

20. The use of claim 19, wherein said antibody and / or antigen-binding fragment thereof is an antibody as defined in any one of claims 12 or 13 or an antigen-binding fragment thereof.

21. An SRRM2 -targeting therapy for use in treating AML in a subject, wherein the subject is characterized by having a level of more than 20% SRRM2 expressing blast cells.5522. Use of an SRRM2 -targeting therapy for the manufacture of a medicament for treating AML in a subject, wherein the subject is characterized by having a level of more than 20% SRRM2 expressing blast cells.

23. A method of treating AML comprising administering to a subj ect in need thereof an effective amount of an SRRM2 -targeting therapy, wherein the subject is characterized by having a level of more than 20% SRRM2 expressing blast cells.

24. A method of stratifying and treating subject diagnosed with AML, comprising evaluatiing whether said subject will have a negative or positive prognosis according to the method of any one of claims 1 to 14, and administering to the subject an SRRM2 -targeting therapy, if the subject is evaluated with a negative prognosis.

25. The SRRM2 -targeting therapy for the use, the use, or the method of any one of claims 21 to 24, wherein the SRRM2-targeting therapy is or comprises an anti-SRRM2 antibody or antigen-binding fragment thereof and / or an immune effector cell comprising an antigen receptor that specifically binds to SRRM2.

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