Surface SRRM2 as molecular target for prognostic biomarkers and

Abstract

The present invention relates to a method for assessing whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative prognosis or a positive prognosis based on the expression level of SRRM2 on the cell surface of the mother cell of said subject, and the use of the expression level of SRRM2 on the cell surface of the mother cell as a prognostic marker of AML. The invention further relates to the medical use of SRRM2 targeted therapy for the treatment of SRRM2-positive AML patients.

Description

Technical Field

[0001] The present invention relates to a method for evaluating whether a subject diagnosed with acute myeloid leukemia (AML) will have a relatively negative or positive prognosis based on the SRRM2 expression level on the cell surface of the subject's blast cells, and the use of the SRRM2 expression level on the cell surface of blast cells as a prognostic marker for AML. The present invention further relates to the medical use of SRRM2 targeted therapy for treating surface SRRM2-positive AML patients. Background Art

[0002] Acute myeloid leukemia (AML) is a common hematological malignancy characterized by high heterogeneity. Despite recent advances in treatment, the standard treatment regimen remains the "7+3" regimen, i.e., 7 days of cytarabine followed by 3 days of anthracyclines. Unfortunately, the overall clinical outcome of AML patients remains suboptimal, with long-term survival rates hovering around 30%. These findings highlight the urgent need to develop new targeted therapies for relapsed and refractory cases. An important obstacle to the development of new therapies is the lack of a suitable target antigen, which is ideally expressed only in AML blasts and absent or extremely rare in normal hematopoietic stem cells and other healthy tissues. This specificity is critical to reducing the risk of severe on-target / off-tumor side effects.

[0003] Recent studies have shown that post-translational phosphorylation of splicing factors contributes to cytarabine resistance in AML. One such splicing factor that is consistently overexpressed in AML is serine / arginine repeat matrix protein 2 (SRRM2 / SRm300), a serine-arginine (SR) motif-rich RNA-binding protein that plays an important role in the function of nuclear speckles. Although the absolute levels of SRRM2 mRNA did not correlate with clinical parameters of AML, Morales et al. reported that phosphorylation of SRRM2 was directly associated with cytarabine resistance. They also observed increased levels of cytoplasmic SRRM2 in AML blasts from patients with resistance to cytarabine. SRRM2 phosphorylation is essential for its translocation to the cytoplasm, a process also observed in neurons affected by neurodegenerative diseases, resulting in cytoplasmic accumulation of this protein. Although the molecular function of extranuclear SRRM2 is still unclear, these findings highlight its potential role in various diseases, including cancer. Summary of the invention

[0004] The present invention relates to a method for assessing whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative prognosis or a positive prognosis, the method comprising determining in vitro the level of SRRM2 expression on the cell surface of the subject's progenitor cells, wherein (i) subjects having a progenitor cell level of SRRM2 expression of 20% or less are characterized as SRRM2-negative, and (ii) subjects having a progenitor cell level of SRRM2 expression of greater than 20% are characterized as SRRM2-positive, wherein SRRM2-negative subjects have a positive prognosis and SRRM2-positive subjects have a negative prognosis.

[0005] The present invention also relates to an in vitro use of SRRM2 expression levels on the cell surface of blast cells of a subject diagnosed with acute myeloid leukemia (AML) for assessing whether the subject will have a negative prognosis or a positive prognosis, wherein (i) subjects having SRRM2 expression blast cell levels of 20% or less are characterized as SRRM2-negative and have a positive prognosis, and (ii) subjects having SRRM2 expression blast cell levels greater than 20% are characterized as SRRM2-positive and have a negative prognosis.

[0006] The present invention also relates to the use of a binding agent capable of specifically binding to SRRM2 on the surface of a blast cell in the preparation of a composition or a kit for assessing whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative prognosis or a positive prognosis.

[0007] The present invention also relates to a SRRM2-targeted therapy for use in treating AML in a subject, wherein the subject is characterized by having a SRRM2-expressing blast level of greater than 20%.

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

[0009] The present invention also relates to a method of treating AML comprising administering an effective amount of an SRRM2-targeted therapy to a subject in need thereof, wherein the subject is characterized by having a SRRM2-expressing blast level of greater than 20%.

[0010] The present invention also relates to a method of stratifying and treating a subject diagnosed with AML, comprising assessing whether the subject will have a negative prognosis or a positive prognosis according to the method of the present invention, and administering a SRRM2-targeted therapy to the subject if the subject is characterized as SRRM2-positive and / or assessed to have a negative prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] 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 EX-02 antibody to the surface of living cells (top panel) and intracellular SRRM2 (bottom panel). (B) Surface SRRM2 expression in the AML cell line MOLM-13 was confirmed by fluorescence confocal microscopy. SRRM2 (green) was localized on the cell surface of living cells and intracellularly in permeabilized cells, confirming the results of flow cytometry. Cell nuclei were counterstained with DAPI (blue).

[0012] Figure 2 : SRRM2 is only exposed on the surface of AML blasts, but not on normal blood cells. (A) Flow cytometric analysis of blood cell subsets isolated from peripheral blood of AML patients. Upper panel: Staining of live cells with EX-02 antibody demonstrates that SRRM2 is detectable on the surface of blasts, while normal blood cells (lymphocytes, monocytes, granulocytes) show only marginal background staining. Lower panel: Intracellular staining of permeabilized cells of the same type shows that all cell types are positive for intracellular SRRM2 staining. (B) Same as (A), but using peripheral blood derived from healthy donors. After EX-02 staining, no surface SRRM2 was detected. (C) Same as (A), but using bone marrow-derived cells from AML patients. Again only blasts were stained positive for surface SRRM2 by the EX-02 antibody, while all other cell types showed only intracellular SRRM2 staining. (D) Cells isolated from the bone marrow of patients with iron deficiency anemia (IDA). Surface SRRM2 was not detected on lymphocytes, monocytes, granulocytes or hematopoietic stem cells (HSCs). Overall, surface SRRM2 was only detectable on malignant AML blasts but not on normal blood cells.

[0013] Figure 3: Correlation between SRRM2 surface exposure and clinical characteristics in AML patients. (AB) SRRM2 exposure in de novo and secondary AML (A) and various WHO-FAB subtypes (M0, M1, M2, M4) (B) was analyzed by flow cytometry, and no significant differences were observed between these groups. (C) SRRM2 surface exposure in patients with failure of standard induction chemotherapy (incomplete remission, NCR) was significantly higher than that in patients with complete remission (CR). (D) SRRM2 surface exposure in poor and intermediate risk patients stratified according to the 2022 NCCN risk classification was significantly increased compared with good risk patients. (E) Relapsed AML patients showed higher SRRM2 surface exposure compared with newly diagnosed (ND) patients. (F) Longitudinal analysis of 7 AML patients showed that SRRM2 exposure on the surface of AML blasts was significantly increased at relapse. (GH) SRRM2 exposure on the surface of AML blasts from ND patients (G) and relapsed patients (H) was greatly increased compared with other blood cells. (IJ) SRRM2 surface exposure positive (SRRM2 Pos The complete remission rate (CRR) of AML patients with SRRM2 surface exposure was significantly lower than that of AML patients with negative SRRM2 Neg ) patients, and the more SRRM2 surface exposure, the lower the CRR. (KL) Kaplan-Meier analysis showed that SRRM2 Pos Patients have shorter recurrence-free survival (RFS), and high SRRM2 expression is associated with shorter RFS. (MN)SRRM2 Pos The overall survival (OS) of patients was better than that of SRRM2 Neg Patients with shorter. *P<0.05; **P<0.01; ***P<0.001.

[0014] 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) showed minimal surface expression of SRRM2 in lymphocytes, monocytes, neutrophils, and hematopoietic stem cells (HSCs); (B) SRRM2 had strong nuclear expression in all cell types, with almost exclusive positivity. (C) Immunohistochemical analysis showed no significant surface expression of SRRM2 in various normal tissues, including cerebellum, colon, liver, lung, skin, pancreas, heart, kidney, lymph node, muscle, and spleen.

[0015] Figure 5: ROC curve analysis for identifying the optimal expression level cutoff in predicting the response to induction chemotherapy. The ROC curve was used to evaluate SRRM2 expression levels as a predictor of remission after standard induction chemotherapy in ND AML patients. The horizontal axis shows the false positive rate (1-specificity) and the vertical axis shows the true positive rate (sensitivity). The area under the curve (AUC) was 0.711, indicating that SRRM2 levels have moderate predictive performance in distinguishing remission from non-remission. The Youden index was 0.37, reflecting the optimal balance between sensitivity and specificity. The cutoff value of 30.14 was determined to be the point at which SRRM2 levels most accurately predicted remission. DETAILED DESCRIPTION

[0016] Acute myeloid leukemia (AML) is a life-threatening hematological malignancy that presents a major challenge to treatment, characterized by a poor median 5-year survival rate. Although several new therapies have been approved in recent years, the lack of suitable target molecules for the development of specific therapies often hinders the progress of treatment. Serine / arginine repeat matrix 2 (SRRM2) is a known spliceosomal and nuclear speckle protein.

[0017] The inventors of the present application recently developed a novel SRRM2-specific antibody named EX-02 and demonstrated that the protein is expressed on the surface of cancer cells from some solid tumors and malignant blasts from multiple myeloma patients. Notably, in these studies, SRRM2 was mainly localized in the nuclei of normal blood cells and adjacent normal tissues, indicating that SRRM2 translocation is cancer-specific. This makes surface SRRM2 an attractive target antigen for new anticancer therapies.

[0018] Now, the inventors of the present application have surprisingly found that SRRM2 is highly expressed on the surface of AML cell lines and primitive blasts of AML patients. In contrast, SRRM2 is mainly localized in the nuclei of normal hematopoietic stem cells (HSCs), mature blood cells, and various normal tissues examined. Systematic analysis of patient data revealed a direct correlation between surface SRRM2 levels and key clinical parameters, such as gene mutations, relapse rates, treatment resistance, and overall clinical outcomes. In addition, using a human xenograft model of AML, the inventors have elucidated the anti-leukemic efficacy of SRRM2-specific CAR-T cells.

[0019] In 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 the surface of normal blood cells. Notably, the inventors found that elevated levels of surface SRRM2 were associated with advanced and aggressive disease characteristics and resistance to treatment. Surface SRRM2 is also used as an independent prognostic marker for poor outcomes in AML patients. In addition, 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, resulting in significantly prolonged survival of treated animals.

[0020] These surprising findings demonstrate that SRRM2 is significantly exposed on the surface of AML blasts and that surface expression levels are directly correlated with key clinical parameters. This specific translocation expression on AML cells positions surface SRRM2 as a promising target for innovative treatments such as SRRM2-specific CAR-T cell therapy and as a potential biomarker for patient stratification. Furthermore, surface SRRM2 emerges as a promising new biomarker and druggable therapeutic target for the development of novel AML treatments.

[0021] Therefore, the present invention contemplates a method for assessing whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative prognosis or a positive prognosis. The method comprises determining the level of SRRM2 expression on the cell surface of a mother cell of the subject (e.g., obtained from the subject), preferably, the determination is performed in vitro. To this end, subjects having a mother cell level of SRRM2 expression of about 20% or less are characterized as SRRM2-negative, while subjects having a mother cell level of SRRM2 expression of greater than about 20% are characterized as SRRM2-positive. SRRM2-negative subjects may have a positive prognosis, while SRRM2-positive subjects may have a negative prognosis. Therefore, the method may further include the step of indicating that if the subject is assessed to be SRRM2-negative, the subject has a positive prognosis, and / or if the subject is assessed to be SRRM2-positive, the subject has a negative prognosis.

[0022] As used herein, "serine / arginine repeat matrix protein 2" or "SRRM2" is generally not limited by its origin. However, as used herein, "serine / arginine repeat matrix protein 2" or "SRRM2" preferably refers to human SRRM2. Human SRRM may have an 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. The variant may comprise modification of one or more amino acids. Examples of variants 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 with SEQ ID NO:8.

[0023] "Percent (%) sequence identity" disclosed herein for sequences is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to the amino acid residues or nucleotides in a reference sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percentage of amino acid sequence identity can be achieved in a variety of ways within the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. This also applies to the nucleotide sequences disclosed herein. For the purpose of determining the identity of a sequence, uracil (as in RNA) can be considered to be identical to thymine (as in DNA).

[0024] The group of SRRM2-positive subjects can be further subdivided based on the percentage of blasts expressing SRRM2. Subjects with SRRM2-expressing blast levels greater than 20% and up to 30% can be characterized as low SRRM2-positive. Subjects with SRRM2-expressing blast levels greater than 30% can be characterized as high SRRM2-positive.

[0025] A "negative prognosis" in SRRM2-positive subjects may be associated with a higher failure rate of induction chemotherapy including cytarabine and an anthracycline, such as daunorubicin or idarubicin, a higher relapse rate, a lower complete remission rate (CRR), a shorter relapse-free survival (RFS), or a shorter overall survival (OS) when compared to SRRM2-negative subjects.

[0026] A "positive prognosis" for SRRM2-negative subjects can be associated with lower failure rate of induction chemotherapy including cytarabine and anthracyclines such as daunorubicin or idarubicin, lower relapse rate, higher complete remission rate (CRR), longer relapse-free survival (RFS), and longer overall survival (OS) when compared to SRRM2-positive subjects.

[0027] As used herein, the level of percentage of mother cells expressing SRRM2 on their cell surface relates to the percentage of (AML) mother cells expressing SRRM2 on their surface relative to the total number of (AML) mother cells measured in the subject. Here, SRRM2 expression preferably relates to protein expression. In general, the SRRM2 status of AML mother cells can be determined using any method known to the skilled person. Preferably, the determination of the percentage of AML mother cells expressing SRRM2 is performed in vitro on a sample obtained from a subject. A preferred method for determining the percentage of AML mother cells expressing SRRM2 is to use flow cytometry (FCM), such as the method substantially described in the "Flow Cytometry" section of Example 1. For example, the level of percentage of mother cells expressing SRRM2 on their cell surface can be determined substantially as follows:

[0028] 1. Treat peripheral blood from AML patients with red blood cell lysis buffer and divide it into two tubes for FCM.

[0029] 2. SRRM2 (EX-02) mAb (Eximmium Biotechnologies; Munich, Germany) or isotype control antibody (cat. No. B355601; Abinvivo, Shanghai, China) was added and incubated at room temperature (RT) for 40 min.

[0030] 3. Wash the cells twice with PBS, and then stain 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-conjugated antibody (e.g., cat. no. 150165; Abcam) in the dark at RT for 15 min.

[0031] 4. Perform sample analysis using a flow cytometer such as a Cytoflex flow cytometer (Beckman Coulter), and analyze the results using software such as CytExpert for DxFLEX.

[0032] The percentage of mother cells expressing SRRM2 on their cell surface is preferably measured in a (biological) sample obtained from a subject. The sample can be any sample suitable for determining the percentage of mother cells expressing SRRM2 on their cell surface. Such samples include peripheral blood (PB) samples and bone marrow (BM) samples, wherein PB samples are preferred. In some instances, the sample is a PB sample. In some embodiments, the sample is a BM sample.

[0033] The percentage of blasts expressing SRRM2 on their cell surface is preferably determined by measuring at least 10,000 AML blasts, or in a sample of preferably at least 50 μL, more preferably at least 100 μL of peripheral blood or bone marrow fluid.

[0034] The subject according to the present disclosure is a subject who has been diagnosed with AML. The subject may be a subject newly diagnosed with AML or may be a subject with recurrent AML. The subject may be a therapy intended for treating AML. The subject may also be a subject who has been treated for AML. The subject may be a subject who is receiving or has received AML treatment. In some instances, the method disclosed herein for assessing whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative prognosis or a positive prognosis may be implemented before the subject receives AML treatment.

[0035] The expression of SRRM2 on the cell surface of the mother cell can be determined using a binding agent that can bind to SRRM2. Therefore, according to the method of the present disclosure, the expression of SRRM2 on the cell surface of the mother cell can be determined using a binding agent that binds to SRRM2 on the cell surface of the SRRM2-expressing cell. Such a binding agent may include an antibody or an antigen-binding fragment thereof, such as an antibody that can specifically bind to (human) SRRM2.

[0036] As described above, the present invention includes the discovery that SRRM2 can be detected on the cell surface of AML blasts. Therefore, it is particularly preferred that the binding agent that binds to human SRRM2 binds to human SRRM2 on the surface of blasts. It is also particularly preferred that the binding agent binds to non-permeabilized cells. "Permeabilization of cells" refers to the rupture of the cell membrane by electrical, mechanical or chemical means, that is, the cell membrane becomes permeable. However, in contrast, the cells bound by the binding agent according to the method of the present disclosure preferably have an intact cell membrane on which SRRM2 is expressed, that is, these cells are non-permeabilized. It is also envisioned that the SRRM2-specific binding agent used in the context of the present disclosure binds to human SRRM2 on the surface of living cells. "Surviving cells" are structural and functional units that contain at least an intact cell membrane, a nucleus and a cytoplasm, which control substances that enter and leave the cell and can function independently. Therefore, the blasts disclosed herein are preferably non-permeabilized cells. Therefore, the blasts disclosed herein are preferably living and / or intact blasts.

[0037] As used herein, the term "surface" or specifically "cell surface" refers to the cell membrane. The cell membrane, which may also be referred to as the plasma membrane, is a thin membrane surrounding each living cell, demarcating the cell from its surrounding environment. The cell membrane surrounds the constituents of the cell, which are typically large, water-soluble, highly charged molecules such as proteins, nucleic acids, carbohydrates, and substances involved in cell metabolism. Therefore, the cell membrane has at least two functions: first, a barrier, keeping the constituents of the cell inside and unwanted substances outside, and second, a gate, allowing essential nutrients to be transported into the cell and removing waste from the cell. ER-derived vesicles may also be involved in the construction or formation of the cell membrane.

[0038] Binding agents, such as antibodies that bind to human SRRM2, are preferably those that are able to bind to SRRM2 polypeptides extracellularly. In other words, when SRRM2 is extracellular, such as when SRRM2 is on the surface of a cell, an antibody as described herein is able to bind to such SRRM2. Therefore, antibodies that bind to human SRRM2 are preferably not intracellular antibodies (which are also called intracellular antibodies). "Intracellular antibodies" (from intracellular and antibody) are antibodies that act intracellularly to bind to intracellular proteins. This requires expression of antibodies in target cells, which can be achieved, for example, in transgenic animals or by gene therapy. Therefore, intracellular antibodies are antibodies modified by intracellular localization, and include antibodies produced in prokaryotes or other non-target cells. The term "intracellular antibody" can be applied to several types of protein targeting: the antibody can be retained in the cytoplasm, or it can have a nuclear localization signal, or it can enter the lumen of the endoplasmic reticulum via co-translational translocation across the membrane, provided that it is retained in that compartment by the KDEL sequence.

[0039] The term "antibody" generally refers to a proteinaceous binding molecule with immunoglobulin-like functions. Typical examples of antibodies include, but are not limited to, immunoglobulins, and derivatives or functional fragments thereof, which still retain binding specificity. The techniques for preparing 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 (e.g., IgG1, IgG2, etc.), even if recombinantly produced in an exogenous host using techniques known to those skilled in the art. Exemplary examples of antibodies are full-length immunoglobulins, Fab fragments, F(ab')2, Fv fragments, single-chain Fv fragments (scFv), double antibodies, or domain antibodies. Domain antibodies can be single domain antibodies, single variable domain antibodies, or immunoglobulin single variable domains, which have only one variable domain, which may be VH or VL, which specifically binds to antigens or epitopes independently of other V regions or domains. The definition of the term "antibody" also includes chimeric antibodies, humanized antibodies, monovalent antibodies, multivalent antibodies, low molecular weight antibodies, diabodies or scFv. The term "antibody" may also include antibody fragments, preferably antigen-binding fragments of antibodies.

[0040] 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. Diabodies are dimers comprising two polypeptide chains. Typically, each polypeptide chain constituting the dimer comprises a heavy chain and a light chain variable region connected via a joint on the same chain. The joint in the diabody is typically too short to allow pairing between the heavy chain and light chain variable regions on the same chain. Specifically, the number of amino acid residues constituting the joint is, for example, about 5 residues. Therefore, the heavy chain and light chain variable regions encoded on the same polypeptide chain cannot form a single-chain variable region fragment together. Instead, they form a dimer by pairing with another single-chain variable region fragment. As a result, the diabody has two antigen binding sites.

[0041] As used herein, "low molecular weight antibodies" encompass antibody fragments that lack a portion of a complete antibody (e.g., complete IgG). As long as the resulting antibody fragment is able to bind to its target, such as SRRM2, such partial lack of an antibody molecule is accepted. Preferably, the low molecular weight antibody should contain one or both of the heavy chain variable region (VH) and the light chain variable region (VL). It is also preferred that the low molecular weight antibody should contain CDRs. There is no particular limitation on the number of CDRs contained in the low molecular weight antibody, preferably at least 6 CDRs: heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3.

[0042] As used herein, immunoglobulin preferably refers to a tetrameric glycosylated protein composed of two light chains (L) of about 25 kDa each and two heavy chains (H) of about 50 kDa each.

[0043] The terms "Fab", "Fab region", "Fab portion" or "Fab fragment" are to be understood as defining a region comprising V H , C H 1. V L and C L The polypeptide of immunoglobulin domain. Fab can refer to this region in isolation, or this region in the context of the antibody molecule of the present invention, as well as the full-length immunoglobulin or immunoglobulin fragment. Generally, the Fab region contains the entire light chain of the antibody. The Fab region can be used to define the "arm" of the immunoglobulin molecule. It contains the epitope-binding part of the Ig. The naturally occurring immunoglobulin Fab region can be obtained as a proteolytic fragment by papain digestion. "F(ab')2 part" is a proteolytic fragment of immunoglobulin digested with partial pepsin. "Fab' part" is the product due to the reduction of the disulfide bonds of the F(ab')2 part. The terms "Fab", "Fab region", "Fab part" or "Fab fragment" used herein may further include a hinge region (see above) that defines the C-terminus of the antibody arm. The hinge region corresponds to the C-terminus of the full-length immunoglobulin. H The hinge region is found at the C-terminus of the 1 domain, where the arms of the antibody molecule can be used to define the Y. The art uses the term hinge region because immunoglobulins have some flexibility in this region.

[0044] "Fv" or "Fv fragment" consists only of the VL and VH domains of the "single arm" of an immunoglobulin. Therefore, "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. The "two-chain" Fv fragment consists of a dimer of a heavy chain and a light chain variable region tightly non-covalently linked. The single-chain Fv category (scFv) contains the VH and VL domains of an immunoglobulin, which are present in a single polypeptide chain, in which they are covalently linked to each other by a flexible peptide linker. Generally, in the scFv fragment, the light chain and heavy chain variable domains are connected in a dimer structure, similar to the two-chain Fv category. In the single-chain Fv fragment, the light chain variable domain can be arranged at the N-terminus of the single polypeptide chain, followed by a linker and the heavy chain variable domain arranged at the C-terminus of the polypeptide chain, or vice versa, even if the heavy chain variable domain is arranged at the N-terminus, the light chain variable domain is arranged at the C-terminus, and the peptide linker is arranged between them. The peptide linker can be any flexible linker known in the art, such as a linker formed by glycine and serine residues. The domain connection between the VH and VL domains can also be stabilized by introducing disulfide bonds into the 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 referred to as disulfide-stabilized scFv fragments (ds-scFv).

[0045] The term "Fc region" or "Fc fragment" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. The Fc portion mediates the effector functions of antibodies, such as the complement system and the activation of the Fc-receptors that bear immune effector cells such as NK cells. In human IgG molecules, the Fc region is generated by papain cleaving the N-terminus of Cys226. Although the boundaries of the Fc region of an immunoglobulin heavy chain may be different, the human IgG heavy chain Fc region is generally defined as a segment from the amino acid residue of site Cys226 or Pro230 to its carboxyl end. The C-terminal lysine (residue 447, according to the EU numbering system) of the Fc region may be removed, for example, during the preparation or purification of antibody molecules, or by recombinant engineering of nucleic acids encoding the heavy chain of antibody molecules. Therefore, the composition of a complete antibody may include antibody groups with all K447 residues removed, antibody groups without K447 residues removed, and antibody groups with or without a mixture of antibodies with K447 residues. Suitable native-sequence Fc regions for antibodies of the present invention include mammals, such as human and mouse IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. Depending on the antibody classification, the Fc region comprises two or three constant domains. In embodiments where the immunoglobulin is IgG, the Fc region has CH2 and CH3 domains.

[0046] The term "variable" refers to the parts of the immunoglobulin domain that exhibit sequence variability and are involved in determining the specificity and binding affinity of a particular antibody (i.e., the "variable domain"). Variability is not evenly distributed throughout the variable domain of an antibody, but is concentrated in subdomains of each heavy and light chain variable region. These subdomains are called "hypervariable regions," "HVRs," or "HVs," or "complementarity determining regions" (CDRs). The more conservative (i.e., non-hypervariable) parts of the variable domain are called "framework" regions (FRs). The variable domains of naturally occurring heavy and light chains each contain four FR regions, primarily in a β-folded configuration, connected by three hypervariable regions to form a connecting loop and, in some cases, form part of the β-folded structure. The hypervariable regions in each chain are clustered together close to the FRs, and together with the hypervariable regions of the other chain, promote the formation of the antigen binding site (see Kabat et al., see below). In general, naturally occurring immunoglobulins contain six CDRs (see below), three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In naturally occurring immunoglobulins, H3 and L3 show the highest variability of the six CDRs, and H3 in particular is considered to play a unique role in conferring good specificity to immunoglobulins. However, immunoglobulins that naturally lack light chains contain three CDRs located in the VHH region. The constant domain is not directly involved in antigen binding, but exhibits a variety of effector functions, such as antibody-dependent, cell-mediated cytotoxicity and complement activation.

[0047] The antibody binding to human SRRM2 according to the present invention may have various forms. It needs to be able to bind to the SRRM2 protein. The antibody is not particularly limited by its source, type, or shape. In some examples, the antibody may have cytotoxic activity. Specifically, the antibody binding to human SRRM2 may be an antibody of non-human animal origin (e.g., mouse, rat, or camel antibody), an antibody of human origin, a chimeric antibody, or a humanized antibody. The antibody binding to human SRRM2 according to the present invention may be a polyclonal or monoclonal antibody, and is preferably a monoclonal antibody.

[0048] Antibodies that bind to human SRRM2 can be obtained as polyclonal or monoclonal antibodies using methods known in the art. For example, such antibodies can be monoclonal antibodies of mammalian origin. Monoclonal antibodies of mammalian origin include, for example, those produced by hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering methods.

[0049] Chimeric antibodies refer to antibodies comprising variable regions and constant regions of different sources connected to each other. For example, mouse-human heterologous chimeric antibodies are antibodies comprising the heavy and light chain variable regions of mouse antibodies and the heavy and light chain constant regions of human antibodies. The DNA encoding the mouse antibody variable regions is connected to the DNA encoding the human antibody constant regions, and the connection product can be integrated into the expression vector to prepare a recombinant vector expressing the chimeric antibody. Cells (recombinant cells) transformed with these vectors can be cultivated to express the DNA insert fragment, thereby obtaining the chimeric antibodies produced during the culture process.

[0050] Typically, chimeric antibodies include antibody variable regions of non-human animal origin and constant regions of human antibody origin. In contrast, humanized antibodies include antibody complementary determining regions (CDRs) of non-human animal origin, framework regions (FRs) of human antibody origin, and constant regions of human antibody origin. Humanized antibodies are sometimes also referred to as reshaped human antibodies. Specifically, for example, humanized antibodies may include non-human animal (e.g., mouse) antibody CDRs transplanted in human antibodies. Because the antigenicity of humanized antibodies in the human body is reduced, they can be used as, for example, therapeutic agents.

[0051] Antibodies according to the present disclosure can be separated antibody molecules. As used herein, the term "separated antibody molecules" refers to antibody molecules identified and separated and / or recovered from the components of its natural environment. The polluting components of its natural environment are substances that will interfere with the use of the antibody, and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In some embodiments, antibody molecules are purified to greater than 95% by weight of antibody, such as greater than 99% by weight, according to the Lowry method. In some embodiments, using a spinning cup sequencer, antibody molecules are purified to a degree sufficient to obtain at least 15 N-terminal residues or internal amino acid sequences. In some embodiments, using Coomassie blue or a preferred silver staining method to judge by SDS-PAGE under reducing or non-reducing conditions, antibodies are purified to homogeneity. Separated antibody molecules may be present in exogenous host cells with components that do not exist in one or more antibody natural environments. Separated antibodies are usually prepared by at least one purification step.

[0052] The binding agent (e.g., antibody) used in the method of the present disclosure may include a detectable label, for example, they may be conjugated to a detectable label. The term "detectable label" generally refers to any suitable chemical substance or enzyme that directly or indirectly produces a detectable compound or signal in a chemical, physical, optical or enzymatic reaction. For example, a fluorescent or radioactive label may be conjugated to an antibody to produce fluorescence or X-rays as a detectable signal. Alkaline phosphatase, horseradish peroxidase, and beta-galactosidase are examples of enzyme labels (and optical labels at the same time), which catalyze the formation of a chromogenic reaction product. Detectable labels refer to detectable entities that can be used to detect a target of interest, for example, in microscopy, immunohistochemistry, flow cytometry, or in vivo applications (e.g., in vivo imaging). Preferably, the labeling does not negatively affect the properties of the binding agent (e.g., antibody) conjugated to the labeling. There are many types of detectable labels, including fluorescent labels, chromophore labels, isotope labels, metal labels, and radioactive labels. Non-exhaustive examples of suitable chromophore labels are alkaline phosphatase or peroxidase exposed to TMB (3,3',5,5'tetramethylbenzidine), DAB (3,3',4,4'diaminobenzidine) and 4CN (4-chloro-1-naphthol). ABTS (2,2'-azino-bis[3-ethyl-benzothiazoline]sulfonate), OPD (o-phenylenediamine) and BCIP / NBT (5-bromo-4-chloro-3-indolyl-phosphate / nitro blue tetrazolium). Non-exhaustive examples of isotopic labels are 13C, 15N, 19F, 27Al, 11B, 127I or different lanthanide isotopes. Non-exhaustive examples of metal labels are Au, Pd, Pb, Pt, Ag, Hg and Os. The label can 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 rhodamine, to name a few. Fluorescently labeled detection means detecting emitted light when the fluorescent label is excited. Non-exhaustive examples of suitable fluorescent labels are "green" emitters (Atto488, Alexa488, Cy2, etc.), "orange" emitters (Atto542, alexa555, Cy3, etc.), "red-far-infrared" emitters (Alexa633, Atto 647N, Cy5, etc.), infrared emitters (Atto700, LiCorIRDye700, LiCor IRDye800, etc.), ultraviolet absorbing fluorescent dyes (Atto390 or Alexa405). Fluorescent labels can also be fluorescent proteins, such as GFP, eGFP, YFP, RFP, CFP, BFP, mCherry or near-infrared fluorescent proteins.

[0053] According to the present disclosure, antibodies that bind to human SRRM2 can be modified (e.g., conjugated) with a variety of molecules (e.g., polyethylene glycol (PEG)). In addition, antibodies that bind to human SRRM2 can also be modified (e.g., conjugated) with cytotoxic substances having cytotoxic activity, such as chemotherapeutic agents, toxic peptides, radioactive chemicals, etc.

[0054] When a binding agent (such as an antibody or antibody molecule / fragment) recognizes its target antigen in a complex mixture of proteins and / or macromolecules, it is said that the binding agent (such as an antibody or antibody molecule / fragment) "specifically" binds to the antigen. In general, a binding agent (such as an antibody) is able to specifically interact and / or bind to its target, but does not substantially bind to another (preferably unrelated) epitope or antigen. If antibody cross-competition is such that only one binding agent is able to bind to the epitope at a given time point, that is, one binding agent prevents the binding or modulation of another binding agent, then the binding agent (such as an antibody) is said to "bind to the same epitope". However, a binding agent (such as an antibody or antibody molecule / fragment) that specifically binds to a particular target can cross-react with similar structures, such as closely related variants of the target to which it specifically binds.

[0055] Typically, binding is considered specific and also has high affinity, for example, when the binding affinity is greater than 10 -6 M(K D In particular, the binding affinity may be about 10 -8 Up to 10 -11 M(K D ), or about 10 -9 Up to 10 -11 M or even higher. If desired, non-specific binding at a binding site can be reduced by changing the binding conditions without substantially affecting specific binding.

[0056] The term "epitope", also referred to as "antigenic determinant", refers to the portion of the antigen to which the antibody specifically binds to form a complex. Therefore, the term "epitope" includes any molecule or protein determinant that can specifically bind to an immunoglobulin or a T-cell receptor. The binding site (antibody binding site) of an antibody molecule described herein can specifically bind / interact with a conformational epitope or a continuous epitope that is unique to the target structure. Epitope determinants are usually composed of chemically active surface components of the molecule such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitope determinants include chemically active surface components of the molecule, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural characteristics, and / or specific charge characteristics. With respect to polypeptide antigens, conformational epitopes or discontinuous epitopes are characterized by the presence of two or more discrete amino acid residues that are separated in the primary sequence, but when the polypeptide is folded into a natural protein / antigen, it is assembled into a continuous structure on the molecular surface (Sela, M., Science (1969) 166, 1365-1374; Laver, WG, et al. Cell (1990) 61, 553-556). The two or more discrete amino acid residues that constitute the epitope may be present in separate segments of one or more polypeptide chains. When the polypeptide chain is folded into a three-dimensional structure, these residues gather on the molecular surface to constitute the epitope. Generally, the epitope may be linear in nature, or it may be a discontinuous epitope. Therefore, as used herein, the term "conformational epitope" refers to a discontinuous epitope formed by the spatial relationship between the antigenic amino acids, rather than a series of unbroken amino acids. The term "epitope" also includes the antigenic determinant of a hapten, which is considered to be a small molecule that can be used as an antigen by displaying one or more immunologically recognized epitopes when binding to a larger substance such as a larger molecule such as a protein.

[0057] The methods of the present disclosure contemplate that any suitable SMMR2 binding agent can be used to determine the level of SRRM2 expression on the surface of a cell. Particularly preferred are SMMR2 binding agents of the present disclosure, such as antibodies that bind to human SRRM2 of the present disclosure.

[0058] In some examples, the antibody binding to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 9, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 10, and a heavy chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 11, and the light chain variable region comprises a light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 12, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 13, and a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 14, or the antibody binding to human SRRM2 according to the present disclosure is an antibody that 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, wherein the amino acid sequence has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or is identical to the amino acid sequence shown in SEQ ID NO: 1 and 2.

[0059] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 15, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 16, and a heavy chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 17, and the light chain variable region comprising a light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 18, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 19, and a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 20, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that 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 has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or is identical to the amino acid sequence shown in SEQ ID NO: 3 and 4.

[0060] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 21, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 22, and a heavy chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 23, the light chain variable region comprising a light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 24, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 25, and a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 26, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that 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 has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or is identical to the amino acid sequence shown in SEQ ID NO: 5 and 6.

[0061] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 21, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 22, and a heavy chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 23, the light chain variable region comprising a light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 27, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 28, and a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 29, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that 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 has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or is identical to the amino acid sequence shown in SEQ ID NO: 5 and 7.

[0062] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region having an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO:2, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that binds to the same epitope.

[0063] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:2, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that binds to the same epitope.

[0064] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region having an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:2, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that binds to the same epitope.

[0065] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region having an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:2, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that binds to the same epitope.

[0066] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region has the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:2.

[0067] In some examples, the 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 and a light chain variable region, wherein the heavy chain variable region has the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region has the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:2.

[0068] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO:3, and the light chain variable region having an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO:4, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that binds to the same epitope.

[0069] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:3, and the light chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:4, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that binds to the same epitope.

[0070] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:3, and the light chain variable region having an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:4, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that binds to the same epitope.

[0071] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:3, and the light chain variable region having an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:4, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that binds to the same epitope.

[0072] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:3, and the light chain variable region has the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:4.

[0073] In some examples, the 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 and a light chain variable region, wherein the heavy chain variable region has the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:3, and the light chain variable region has the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:4.

[0074] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region having an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO:6, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that binds to the same epitope.

[0075] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:6, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that binds to the same epitope.

[0076] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region having an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:6, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that binds to the same epitope.

[0077] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region having an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:6, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that binds to the same epitope.

[0078] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region has the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:6.

[0079] In some examples, the 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 and a light chain variable region, wherein the heavy chain variable region has the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region has the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:6.

[0080] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region having an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO:7, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that binds to the same epitope.

[0081] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:7, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that binds to the same epitope.

[0082] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region having an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:7, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that binds to the same epitope.

[0083] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region having an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:7, or the antibody that binds to human SRRM2 according to the present disclosure is an antibody that binds to the same epitope.

[0084] In some examples, the antibody that binds to human SRRM2 according to the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region has the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:7.

[0085] In some examples, the 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 and a light chain variable region, wherein the heavy chain variable region has the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region has the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:7.

[0086] According to the present disclosure, the antibody binding to human SRRM2 is preferably an antibody having less than 20% cross-reactivity with SRRM2-related proteins. This means that, preferably, the cross-reactivity of the antibody binding to human SRRM2 with other serine / arginine repeat matrix proteins (such as SRRM1, SRRM3, SRRM4 or SRRM5) is less than 20%. More preferably, the cross-reactivity of the antibody binding to human SRRM2 with SRRM2-related proteins (such as SRRM1, SRRM3, SRRM4 or SRRM5) is less than 15%. Even more preferably, the cross-reactivity of the antibody binding to human SRRM2 with SRRM2-related proteins (such as SRRM1, SRRM3, SRRM4 or SRRM5) is less than 10%. Even more preferably, the cross-reactivity of the antibody binding to human SRRM2 with SRRM2-related proteins (such as SRRM1, SRRM3, SRRM4 or SRRM5) is less than 5%. Even more preferably, the cross-reactivity of the antibody that binds to human SRRM2 with SRRM2-related proteins (such as SRRM1, SRRM3, SRRM4 or SRRM5) is less than 3%. Even more preferably, the cross-reactivity of the antibody that binds to human SRRM2 with SRRM2-related proteins (such as SRRM1, SRRM3, SRRM4 or SRRM5) is less than 2%. Even more preferably, the cross-reactivity of the antibody that binds to human SRRM2 with SRRM2-related proteins (such as SRRM1, SRRM3, SRRM4 or SRRM5) is less than 1%.

[0087] Antibodies that bind to human SRRM2 according to the present disclosure may include antibodies comprising substitutions, deletions, additions and / or insertions of one or more amino acids, and may be prepared or naturally occurring. Examples of methods for introducing mutations in polypeptides 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 known to those skilled in the art for preparing a polypeptide that is functionally equivalent to a certain polypeptide. Those skilled in the art may appropriately introduce mutations into the antibodies of the present invention to prepare antibodies that are functionally equivalent to such antibodies. In addition, amino acid mutations may occur in nature. This antibody having an amino acid sequence containing one or more amino acid mutations derived from an amino acid sequence of an antibody of the present invention is functionally equivalent to the antibody or a variant of the antibody, and is also covered by the antibodies of the present invention. 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 (eg, within 5 amino acids).

[0088] For the amino acid residue to be mutated, it is preferred that the mutation should be conservatively performed between amino acids with the same side chain properties. For example, the following classification based on the side chain properties of amino acids 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 with aliphatic side chains (G, A, V, L, I and P), amino acids with hydroxyl side chains (S, T and Y), amino acids with sulfur atom side chains (C and M), amino acids with carboxylic acid and amide side chains (D, N, E and Q), amino acids with base side chains (R, K and H) and amino acids with aromatic group side chains (H, F, Y and W) (all symbols in brackets represent single letter codes of amino acids).

[0089] It is known that the polypeptide with an amino acid sequence modified from a certain amino acid sequence by deletion and / or addition of one or more amino acid residues and / or substitution with other amino acids maintains the biological activity of the original polypeptide. Specifically, when the amino acids in the amino acid sequence constituting a certain polypeptide are substituted by amino acids classified in the same group as the amino acid sequence, it is generally believed that the polypeptide may maintain its activity.

[0090] In some instances, the disclosed method for assessing whether a subject diagnosed with AML will have a negative or positive prognosis is a method that does not have the purpose of directly diagnosing and / or treating a disease. In some instances, the disclosed method for assessing whether a subject diagnosed with AML will have a negative or positive prognosis is not a diagnostic method performed on humans.

[0091] According to the methods of the present disclosure, SRRM2-positive subjects are suitable for SRRM2 targeted therapy. Such therapy may include therapy using antibodies, antibody constructs, or antibody fragments that bind to SRRM2 on the cell surface of SRRM2-expressing cells. Therefore, the methods of the present disclosure may include assessing whether a subject diagnosed with AML will have a negative prognosis or a positive prognosis, and if the subject has a negative prognosis and / or if the subject is characterized as SRRM2-negative, selecting the subject for SRRM2 targeted therapy. Therefore, the method disclosed herein for assessing whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative prognosis or a positive prognosis is performed before the patient receives SRRM2-targeted therapy.

[0092] The present invention also contemplates a method for stratifying and treating subjects diagnosed with AML. The method comprises implementing the disclosed method for assessing whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative prognosis or a positive prognosis, and administering an SRRM2 targeted therapy to the subject if the subject is assessed as having a negative prognosis and / or SRRM2-positive.

[0093] The present invention also relates to an SRRM2-targeted therapy for use in treating AML in a subject, wherein the subject is characterized by having a level of SRRM2-expressing blasts greater than 20%. The determination of the level of SRRM2-expressing blasts in a subject can be performed by any suitable method, preferably by the methods of the present disclosure, such as the methods of the present invention for assessing whether a subject diagnosed with AML will have a negative prognosis or a positive prognosis. However, it is contemplated that the step of determining the level of SRRM2-expressing blasts may be performed prior to treating the subject. The determining step may not be part of the application of treating AML in a subject.

[0094] The present invention also relates to the use of an SRRM2-targeted therapy in the preparation of a medicament for treating AML in a subject, wherein the subject is characterized by having a level of SRRM2-expressing progenitor cells greater than 20%. The level of SRRM2-expressing progenitor cells in a subject can be determined by any suitable method, preferably by the method disclosed herein, such as the method of the present invention for assessing whether a subject diagnosed with AML will have a negative prognosis or a positive prognosis. However, it is envisioned that the step of determining the level of SRRM2-expressing progenitor cells may be performed before preparing the medicament. The determining step may not be a step in preparing the medicament. However, the (intended) use of the medicament may be limited to subjects in a specific patient group, characterized by having a level of SRRM2-expressing progenitor cells greater than 20%.

[0095] The present invention also relates to a method for treating AML, comprising administering an effective amount of an SRRM2-targeted therapy to a subject in need thereof, wherein the subject is characterized by having a level of SRRM2-expressing progenitor cells greater than 20%. The level of SRRM2-expressing progenitor cells in a subject may be determined by any suitable method, preferably by the methods disclosed herein, such as the methods of the present invention for assessing whether a subject diagnosed with AML will have a negative prognosis or a positive prognosis. The step of determining the level of SRRM2-expressing progenitor cells may be part of the method for treating AML. Alternatively, the step of determining the level of SRRM2-expressing progenitor cells may not be part of the method for treating AML.

[0096] The term "therapeutically effective amount" refers to an amount of an SRRM2 targeted therapy or drug according to the present disclosure that is effective to "treat" a disease, such as AML in a patient. Specifically, in the case of cancer, a therapeutically effective amount of an antibody / drug composition / drug can reduce the number of cancer cells; reduce tumor size; inhibit or stop cancer cell infiltration into peripheral organs; inhibit and stop tumor metastasis; inhibit and stop tumor growth; alleviate one or more symptoms associated with cancer to some extent, or a combination of these effects on cancer cells. To the extent that an SRRM2-targeted therapy prevents the growth of existing cancer cells and / or kills existing cancer cells, it can be referred to as cytostatic and / or cytotoxic.

[0097] Terms such as "treat" or "treatment" refer to both: 1) therapeutic measures that cure, slow, alleviate the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder, and 2) prophylactic or preventative measures that prevent or slow the development of a targeted pathological condition or disorder. Thus, those in need of treatment include those already suffering from the disorder; those predisposed to suffering from the disorder; and those in need of prevention of the disorder. A subject is successfully "treated" according to the methods of the invention or with a pharmaceutical composition or antibody that binds to human SRRM2 according to the invention if the patient exhibits one or more of the following: a decrease in the number of cancer cells or the complete absence of cancer cells; a decrease in tumor size; inhibition or absence of cancer cell infiltration into peripheral organs (including cancer spread to soft tissue and bone); inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; relief of one or more symptoms associated with a particular cancer; reduced morbidity and mortality; and improved quality of life.

[0098] In the context of the present invention, a "subject" or "patient" is a human being. For example, a patient may be a patient suspected of having a disease or clinical condition associated with a blood cancer or a patient diagnosed with such a disease or clinical condition.

[0099] SRRM2-targeted therapy as used herein may include any therapy, such as a therapeutic agent capable of binding to SRRM2 and / or a pharmaceutical composition comprising the therapeutic agent. The pharmaceutical composition may comprise the therapeutic agent, as well as an additional pharmaceutically acceptable carrier, diluent or excipient. The term "pharmaceutically acceptable carrier, diluent or excipient" as used in the context of the present invention may include any pharmaceutically acceptable carrier, diluent or excipient for pharmaceutical compositions known to those skilled in the art. It should be understood that such therapeutic agents or pharmaceutical compositions described herein may be mixed with carriers or diluents that do not interfere with the intended purpose of the present invention. For example, the carrier used in the present invention may be a carrier protein, such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). The carrier may also be selected from, for example, water, saline solution, aqueous buffer, cell culture medium and a combination of the above carriers.

[0100] SRRM2-targeted therapy may include an antibody or an antigen-binding fragment thereof, such as an antibody or an antigen-binding fragment thereof that binds to human SRRM2. Preferably, such SRRM2-targeted therapy comprises an antibody or an antigen-binding fragment thereof that binds to human SRRM2 of the present invention.

[0101] SRRM2-targeted therapy may include an antibody that binds to human SRRM2, preferably an antibody of the present invention, which has antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Therefore, examples of cytotoxic activity according to the present invention may include ADCC and / or CDC activity. In the context of the present disclosure, ADCC activity refers to the activity of destroying target cells by binding to the Fc domain of an antibody that specifically attaches to a cell surface antigen of a target cell through the Fcγ receptor of a cell (immune cell, etc.) carrying an Fcγ receptor. On the other hand, CDC activity refers to cytotoxic activity mediated by the complement system. Whether an antibody has ADCC activity or CDC activity can be determined by methods known in the art.

[0102] Therefore, the antibodies binding to human SRRM2 of the present disclosure may have activities such as ADCC activity, and thus may be used as drugs, preferably as anticancer agents, wherein the cancer is a blood cancer, preferably acute myeloid leukemia (AML).

[0103] SRRM2-targeted therapy may also include an antibody that binds to human SRRM2, preferably an antibody of the present disclosure, which is conjugated to a cytotoxic substance. For example, an antibody that binds to human SRRM2 may be conjugated to a cytotoxic substance, such as a chemotherapeutic 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. Methods for antibody modification have been established in the art.

[0104] Examples of chemotherapeutic agents whose cytotoxic activity is exerted by conjugation to an antibody that binds to SRRM2 may include the following chemotherapeutic agents: azaribine, anastrozole, azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan, camptothecin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin, irinotecan, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin glucuronide, daunorubicin, dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin glucuronide, epirubicin, ethinyl estradiol, estramustine, etoposide, etoposide glucuronide, floxuridine , fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide, leucovorin, lomustine, nitrogen mustard, medroxyprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, methotrexate, mitronquinone, mithramycin, mitomycin, mitotane, phenyl butyrate, prednisolone, procarbazine, paclitaxel, pentostatin, semustine, streptozotocin, tamoxifen, taxanes, taxol, testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uramustine, vinblastine, vinorelbine and / or vincristine.

[0105] Chemotherapeutic agents are preferably low molecular weight chemotherapeutic agents. Low molecular weight chemotherapeutic agents are unlikely to interfere with antibodies, even after they are conjugated to antibodies. Low molecular weight chemotherapeutic agents generally have a molecular weight of 100 to 2000, preferably 200 to 1000. All chemotherapeutic agents exemplified above are low molecular weight chemotherapeutic agents. These chemotherapeutic agents encompass prodrugs that are converted into active chemotherapeutic agents in vivo. The activation of the prodrug can be an enzymatic conversion or a non-enzymatic conversion.

[0106] Examples of toxic peptides are snake venom peptides, including three-finger toxins (3FTxs), disintegrins, Kunitz-type inhibitors, natriuretic peptides or sarafotoxins. Further examples are trypsin inhibitors, penicillins, pallotoxins or amatoxins.

[0107] Examples of radioactive chemicals are chemicals with cytotoxic radionuclides, such radionuclides may be, for example, iodine-131, indium-111, yttrium-90, lutetium-177, actinium-225, gallium-68 or bismuth-213. Antibodies conjugated to cytotoxic substances can be used to specifically deliver cytotoxic substances to target cells, which can reduce undesirable side effects.

[0108] SRRM2-targeted therapy may also include bispecific antibodies, wherein the bispecific antibody preferably comprises a portion that binds to human SRRM2, such as an antibody or an antigen-binding fragment thereof. Preferably, such SRRM2-targeted therapy comprises an antibody or an antigen-binding fragment thereof that binds to human SRRM2 of the present disclosure.

[0109] As used herein, the term "bispecific construct" refers to a bispecific antibody, ie, the antibody 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.

[0110] The bispecific antibody constructs disclosed herein can be bispecific single-chain antibodies. Typically, single-chain antibodies also include a polypeptide linker between the VH and VL domains that enables it to form a desired structure that allows antigen binding. Plugkthun discussed single-chain antibodies in detail in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994). Various methods for producing 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 may also be human and / or humanized and / or synthetic. As used herein, the term "di-scFv" or "ta-scFv" (tandem scFv) refers to two scFvs fused together. Such a di-scFv or ta-scFv may include a linker between the two scFv portions. Generally, the arrangement of the VH and VL domains on the polypeptide chain in each scFv can be in any order. This means that a "di-scFv" or "ta-scFv" can be arranged in the order of 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), wherein (1) and (2) represent the first and second scFv, respectively. The bispecific single-chain antibody is preferably or comprises a "di-scFv" or "ta-scFv".

[0111] In some examples, the bispecific antibody can be a BiTE. As used herein, "bispecific T cell engager" or "BiTE" refers to a bispecific antibody construct that can recruit T cells to target cells (such as cancer cells, such as AML cells). Typically, a BiTE molecule is a fusion protein comprising two single-chain variable fragments (scFv) of different antibodies. Typically, one binding domain can bind to a target on the surface of a T cell, while the other binding domain can bind to a tumor antigen or a tumor-associated antigen (such as SRRM2). BiTE is preferably a bispecific single-chain antibody.

[0112] In some examples, the bispecific antibody constructs of the present disclosure may comprise a first binding domain capable of specifically binding to a first target that is an immunomodulatory antigen on the surface of an immune effector cell and a second binding domain capable of specifically binding to SRRM2.

[0113] The term "binding domain" as used herein characterizes a domain capable of specifically binding to / interacting with / recognizing a given target epitope or a given target site on a target molecule (antigen), such as SRRM2.

[0114] As used herein, the term "immune effector cell" may refer to any leukocyte or precursor involved in, for example, protecting the body against cancer, diseases induced by infectious agents, foreign substances, or autoimmune reactions. For example, the immune effector cell may be a T cell or a NK cell.

[0115] 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 subpopulations of T cells, each with a different function.

[0116] Cytotoxic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells, and are also involved in transplant rejection. These cells are also referred to as CD8+T cells because they express CD8 glycoproteins on their surfaces. These cells recognize their targets by binding to antigens associated with MHC class I molecules, which are present on all nucleated cell surfaces. Through IL-10, adenosine and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated to an anergic state, which prevents autoimmune diseases.

[0117] Natural killer (NK) cells are CD56+CD3-large granular lymphocytes that can kill virally infected and transformed cells and constitute a key cell subset of the innate immune system (Godfrey J et al., Leuk Lymphoma 2012 53:1666-1676). Unlike cytotoxic CD8+T lymphocytes, NK cells produce cytotoxicity to tumor cells without the need for prior sensitization, and can also eradicate MHC-I-negative cells (Narni-Mancinelli E et al. Int Immunol 2011 23:427-431). NK cells are safer effector cells because they can avoid cytokine storms (Morgan RA et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter DL et al. N Engl J Med 2011 365:725-733) and potential lethal complications of targeted, non-tumor effects.

[0118] As used herein, the term "immunomodulatory antigen" refers to an antigen, which is preferably a receptor. The antigen or preferably the receptor is capable of receiving and / or transducing signals, and it is believed that its engagement affects the quality and intensity of the innate immune cell response. These antigens include inhibitory receptors, activating receptors, adhesion molecules and co-stimulatory molecules. Such "immunomodulatory antigens" include, but are not limited to: CD3, CD16, NKG2A, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 (CD226), SLAMF7 (CD319), CD244 (2B4), OX40, CD47, SIRPα, 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. Preferred immunomodulatory antigens are CD3.

[0119] SRRM2-targeted therapy may also include cell therapy, such as immune cells comprising antigen receptors that can specifically bind to SRRM2 on the cell surface of SRRM2-expressing cells. Preferably, the immune cells are immune effector cells, preferably T cells or NK cells, such as cytotoxic T cells and / or CD8+T cells.

[0120] In some instances, such immune effector cells (such as T cells or NK cells) can be engineered. Such engineered cells can express one or more recombinant receptors. Among these receptors, there are antigen receptors and receptors containing one or more of its components. Recombinant receptors may include chimeric receptors (e.g., those containing ligand binding domains or their binding fragments and intracellular signaling domains or regions), functional non-TCR antigen receptors, chimeric antigen receptors (CAR), T cell receptors (TCR) (e.g., recombinant or transgenic TCR), chimeric autoantibody receptors (CAARs) and any of the aforementioned receptor components. Recombinant receptors, such as CAR, generally include extracellular antigen (or ligand) binding domains, which are connected to one or more intracellular signaling components by joints and / or transmembrane domains in some cases. In some instances, engineered cells express two or more receptors containing different components, domains or regions. In some instances, two or more receptors allow the specificity, activity, antigen (or ligand) binding, function and / or expression of recombinant receptors to be regulated or controlled spatially or temporally.

[0121] In some examples, such as chimeric antigen receptors containing one or more domains, the one or more domains will provide a ligand binding domain (e.g., an antibody or antibody fragment) for the desired antigen (e.g., a tumor antigen) and a combination of an intracellular signaling domain. Preferably, the intracellular signaling domain is an activation intracellular domain portion, such as a T cell activation domain, thereby providing a primary activation signal. In some examples, the intracellular signaling domain contains or additionally contains a co-stimulatory signaling domain to promote effector function. When the chimeric receptor is genetically engineered into an immune cell, it may be preferred to regulate T cell activity, and in some cases may regulate T cell differentiation or homeostasis, thereby producing genetically engineered cells with improved lifespan, survival and / or persistence in vivo, for example, for use in adoptive cell therapy methods.

[0122] Chimeric receptors, such as CARs, can generally include an extracellular antigen binding domain, such as a portion of an antibody molecule, typically a variable heavy (VH) chain region and / or a variable light (VL) chain region of an antibody, for example, a scFv antibody fragment.

[0123] Antigen receptors include intracellular domains directly or indirectly connected to the extracellular domain. In some instances, chimeric antigen receptors include transmembrane domains connecting extracellular domains and intracellular signaling domains. For example, the intracellular signaling domain may include ITAM. For example, an antigen recognition domain (e.g., an extracellular domain) is generally connected to one or more intracellular signaling components (such as a signaling component that simulates activation and / or conduction of signals by another cell surface receptor in the case of CAR by an antigen receptor complex (e.g., a TCR complex). In some instances, CAR is included in a transmembrane domain connected or fused between an extracellular domain (e.g., scFv) and an intracellular signaling domain. Therefore, in some instances, an antigen binding component (e.g., an antibody or a fragment or variant thereof) is connected to one or more transmembrane domains and an intracellular signaling domain.

[0124] Antigen receptors (such as CAR) can generally include at least one or more intracellular signaling components. In some instances, CAR includes a primary cytoplasmic signaling sequence for the primary activation of the TCR complex. The primary cytoplasmic signaling sequence that works in a stimulating manner can contain a signaling motif, which is referred to as an activation motif or ITAM based on immunoreceptor tyrosine. Examples of primary cytoplasmic signaling sequences containing ITAM include those derived from CD3ζ chains, FcRγ, CD3γ, CD3δ, and CD3ε. In some instances, one or more cytoplasmic signaling molecules in CAR contain cytoplasmic signaling domains, portions thereof, or sequences derived from CD3ζ. In some instances, receptors include intracellular components of TCR complexes, such as TCR CD3 chains, such as CD3ζ chains, that mediate T cell activation and cytotoxicity.

[0125] Chimeric antigen receptors also contain intracellular domains of T cell costimulatory molecules.For example, CAR includes signaling domains and / or transmembrane portions of costimulatory receptors (such as CD28, 4-1BB, OX40, DAP10, and ICOS).In some instances, the same CAR includes both activation and costimulatory components.Chimeric antigen receptors contain intracellular domains derived from T cell costimulatory molecules or their functional variants, such as between transmembrane domains and intracellular signaling domains.T cell costimulatory molecules are selected from CD28 or 41BB, preferably CD28.

[0126] Transmembrane domains can generally be derived from natural or synthetic sources. In the case where the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein. Transmembrane regions include those derived from (i.e., comprising at least one or more transmembrane regions thereof): α, β or ζ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. For example, a transmembrane domain can contain the transmembrane portion of CD28.

[0127] In some examples, antibodies that bind to human SRRM2 (preferably antibodies that bind to human SRRM2 of the present disclosure) may be modified to form a chimeric antigen receptor (CAR) expressed by an immune cell, preferably a T cell, NK cell, NK-T cell or macrophage, most preferably a T cell (CAR T cell). Therefore, the extracellular region of the CAR may include light chain and heavy chain variable regions of an antibody that recognizes human SRRM2 (preferably an antibody that binds to human SRRM2 of the present disclosure). The light chain and heavy chain variable regions may form a single chain variable fragment (scFv). The CAR described in this way may cause T cell activation after binding to SRRM2.

[0128] In some examples, the antibodies that bind to human SRRM2 of the present disclosure may be modified to form a chimeric antigen receptor (CAR) expressed by immune cells, including but not limited to T cells, NK cells, NK-T cells, or macrophages, wherein the immune cells may be derived from induced pluripotent stem cells (iPSCs).

[0129] In some examples, the antibodies that bind to human SRRM2 of the present disclosure may be modified to form a chimeric antigen receptor (CAR) expressed by immune cells, including but not limited to T cells, NK cells, NK-T cells, or macrophages, wherein the immune cells are immune cells in which β2-microglobulin has been inactivated.

[0130] Other SRRM2-specific CARs and CAR-T cells are described in PCT / EP2024 / 075361, which is incorporated herein by reference in its entirety.

[0131] The present invention also relates to the in vitro use of SRRM2 expression levels on the cell surface of blast cells of a subject diagnosed with acute myeloid leukemia (AML) for assessing whether the subject will have a negative prognosis or a positive prognosis, wherein (i) subjects having SRRM2 expression blast cell levels of 20% or less are characterized as SRRM2-negative and have a positive prognosis, and (ii) subjects having SRRM2 expression blast cell levels greater than 20% are characterized as SRRM2-positive and have a negative prognosis.

[0132] The use of SRRM2 expression levels on the cell surface of blasts of subjects diagnosed with acute myeloid leukemia (AML) may further include reclassifying subjects with SRRM2 expression blast levels greater than 20%. Thus, SRRM2-positive subjects with SRRM2 expression blast levels greater than 20% and up to 30% may be characterized as low SRRM2-positive. SRRM2-positive subjects with SRRM2 expression blast levels greater than 30% may be characterized as high SRRM2-positive.

[0133] In some examples, the in vitro use of SRRM2 expression levels on the cell surface of blast cells of a subject diagnosed with acute myeloid leukemia (AML) is not directly intended for diagnosis and / or treatment of the disease.

[0134] The use of SRRM2 expression levels on the cell surface of blast cells of a subject diagnosed with acute myeloid leukemia (AML) can be used in any method, including any method for assessing whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative prognosis or a positive prognosis. Therefore, any features disclosed herein in the context of a method for assessing whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative prognosis or a positive prognosis are also applicable to the in vitro use of SRRM2 expression levels on the cell surface of blast cells of a subject diagnosed with acute myeloid leukemia (AML) of the present disclosure.

[0135] The present invention also relates to the use of a binding agent in the preparation of a composition or kit for assessing whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative prognosis or a positive prognosis, wherein the binding agent is capable of specifically binding to SRRM2 on the surface of a mother cell. Generally, the binding agent can be any binding agent disclosed herein. Such a binding agent can be or include an antibody and / or an antigen-binding fragment thereof that is capable of specifically binding to SRRM2, such as an antibody or an antigen-binding fragment thereof that binds to human SRRM2 disclosed herein.

[0136] The use of a binding agent capable of specifically binding to SRRM2 on the surface of a mother cell in the preparation of a composition or kit for assessing whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative prognosis or a positive prognosis can be used in any method disclosed herein for assessing whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative prognosis or a positive prognosis. Therefore, any features disclosed herein in the context of a method for assessing whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative prognosis or a positive prognosis are also applicable to the use of a binding agent capable of specifically binding to SRRM2 on the surface of a mother cell disclosed herein in the preparation of a composition or kit for assessing whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative prognosis or a positive prognosis.

[0137] The description of the sequences shown in the sequence listing and used in the context of the present invention is as follows:

[0138] SEQ ID NO: 1 shows the amino acid sequence of the VH-region of antibody 13F11.

[0139] SEQ ID NO: 2 shows the amino acid sequence of the VL region of antibody 13F11.

[0140] SEQ ID NO: 3 shows the amino acid sequence of the VH-region of antibody 23A7.

[0141] SEQ ID NO: 4 shows the amino acid sequence of the VL-region of antibody 23A7.

[0142] SEQ ID NO: 5 shows the amino acid sequence of the VH-region of antibody 18A4 and antibody 18A4-2.

[0143] SEQ ID NO: 6 shows the amino acid sequence of the VL-region of antibody 18A4.

[0144] SEQ ID NO: 7 shows the amino acid sequence of the VL-region of antibody 18A4-2.

[0145] SEQ ID NO: 8 shows the amino acid sequence of SRRM2 as shown in the UniProt database entry Q9UQ35 (version 2, March 6, 2007).

[0146] SEQ ID NO: 9 shows the amino acid sequence of VH-CDR1 of antibody 13F11.

[0147] SEQ ID NO: 10 shows the amino acid sequence of VH-CDR2 of antibody 13F11.

[0148] SEQ ID NO: 11 shows the amino acid sequence of VH-CDR3 of antibody 13F11.

[0149] SEQ ID NO: 12 shows the amino acid sequence of VL-CDR1 of antibody 13F11.

[0150] SEQ ID NO: 13 shows the amino acid sequence of VL-CDR2 of antibody 13F11.

[0151] SEQ ID NO: 14 shows the amino acid sequence of VL-CDR3 of antibody 13F11.

[0152] SEQ ID NO: 15 shows the amino acid sequence of VH-CDR1 of antibody 23A7.

[0153] SEQ ID NO: 16 shows the amino acid sequence of VH-CDR2 of antibody 23A7.

[0154] SEQ ID NO: 17 shows the amino acid sequence of VH-CDR3 of antibody 23A7.

[0155] SEQ ID NO: 18 shows the amino acid sequence of VL-CDR1 of antibody 23A7.

[0156] SEQ ID NO: 19 shows the amino acid sequence of VL-CDR2 of antibody 23A7.

[0157] SEQ ID NO: 20 shows the amino acid sequence of VH-CDR3 of antibody 23A7.

[0158] SEQ ID NO: 21 shows the amino acid sequence of VH-CDR1 of antibody 18A4 and antibody 18A4-2.

[0159] SEQ ID NO: 22 shows the amino acid sequence of VH-CDR2 of antibody 18A4 and antibody 18A4-2.

[0160] SEQ ID NO: 23 shows the amino acid sequence of VH-CDR3 of antibody 18A4 and antibody 18A4-2.

[0161] SEQ ID NO: 24 shows the amino acid sequence of VL-CDR1 of antibody 18A4.

[0162] SEQ ID NO: 25 shows the amino acid sequence of VL-CDR2 of antibody 18A4.

[0163] SEQ ID NO: 26 shows the amino acid sequence of VL-CDR3 of antibody 18A4.

[0164] SEQ ID NO: 27 shows the amino acid sequence of the VL-region of antibody 18A4-2.

[0165] SEQ ID NO: 28 shows the amino acid sequence of VL-CDR1 of antibody 18A4-2.

[0166] SEQ ID NO: 29 shows the amino acid sequence of VL-CDR2 of antibody 18A4-2.

[0167] 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 submitted for formal reasons, the sequences of Table 1 replace the sequences of the sequence listing):

[0168] Table 1:

[0169]

[0170]

[0171]

[0172]

[0173]

[0174] In the context of the present invention the following abbreviations are used: VH = variable heavy chain; VH = variable heavy chain; VL = variable light chain; CDR = complementarity determining region; VH-CDR = CDR of the variable heavy region; VL-CDR = CDR of the variable light region; CDRs can be determined by using the Kabat algorithm, for example http: / / abysis.org / abysis / .

[0175] Note that as used herein, the singular forms "a," "an," and "the" include plural referents and vice versa unless the context clearly dictates otherwise.

[0176] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series.

[0177] 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 also intended to be encompassed by the present invention.

[0178] Wherever used herein, the term "and / or" includes the meanings of "and," "or," and "all or any other combination of elements connected by the term."

[0179] As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. However, it also includes specific numbers, for example, about 20 includes 20.

[0180] Throughout the specification and claims, unless the context requires otherwise, the word "comprise" and variations such as "include" and "comprising" should 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 integers or steps. When used herein, the term "comprise" may be replaced with the term "contains" or "includes", or sometimes when used herein, with the term "having".

[0181] When used in this article, "consisting of" excludes any elements, steps or ingredients not specified in the claim elements. When used in this article, "consisting essentially of" does not exclude materials or steps that do not substantially affect the basic and novel characteristics of the claim.

[0182] In each case herein, any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with any of the other two terms. For example, when the present invention uses the term "comprising", the present invention also encompasses the replacement of the term "comprising" with the terms "consisting essentially of" and "consisting of", and vice versa. For example, the term "comprising" is intended to provide clear support for replacing it with "consisting essentially of" and / or "consisting of", the term "consisting essentially of" is intended to provide clear support for replacing it with "comprising" and / or "consisting of", and the term "consisting of" is intended to provide clear support for replacing it with "consisting essentially of" and "comprising". The possibility of replacing the terms with each other should not be understood as meaning that the terms are synonymous. To avoid ambiguity, the terms "comprising", "consisting essentially of" or "consisting of" as clearly defined in the respective context are preferred terms, while replacing them with any of the other two terms is less preferred.

[0183] Example

[0184] Example 1: Materials and Methods

[0185] Cell lines

[0186] MOLM-13 (AML associated with myeloproliferation) and HL-60 (promyelocytic leukemia) are purchased from Nanjing Saihongrui Biotechnology Co., Ltd. MV4-11 (biphenotype B-myelomonocytic leukemia, FLT3-ITD positive) is purchased from Wuhan Hua Erna Biotechnology Co., Ltd. and SKM-1 (a kind of AML-MDS cell line, myelodysplastic syndrome is to the transformation of acute myeloid leukemia) is from Hefei Zhong Tuoda Biotechnology Co., Ltd. HL-60 and MV4-11 cells are cultured in the IMDM culture medium (Cytiva, SH30228.01) supplemented with 10% fetal bovine serum (FBS, Lonsera, S711-001S). Cells are maintained in RPMI 1640 (Cytiva, SH30255.01) containing 10% FBS, and at 37 ℃, contain 5% CO2 in a humid atmosphere and cultivate.

[0187] patient

[0188] In this study, 76 patients with acute myeloid leukemia (AML) admitted to the Second Affiliated Hospital of Anhui Medical University from January 2022 to March 2024 were included. The group included 52 newly diagnosed (ND) and 24 relapsed AML cases. Normal controls were selected from patients who underwent bone marrow puncture diagnosis and were subsequently confirmed to be free of hematological malignancies, including iron deficiency anemia (IDA). The use of human materials for this study was approved by the Ethics Committee of the Second Affiliated Hospital of Anhui Medical University (approval number YJ-YX2019-015). The diagnosis of AML followed the 5th edition of the World Health Organization Classification of Hematolymphoid Neoplasms: Myeloid and Histiocyte / Dendritic Neoplasms, and combined molecular, cytogenetic, and clinical parameters to ensure accurate 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 surface SRRM2 expression analysis using flow cytometry (FCM). Comprehensive patient information is available in the supplementary materials.

[0189] Flow cytometry

[0190] Peripheral blood from AML patients was treated with red blood cell lysis buffer and divided into two tubes for FCM. SRRM2 (EX-02) mAb (Eximmium Biotechnologies; Munich, Germany) or isotype control antibody (cat. No. B355601; Abinvivo, Shanghai, China) was added and incubated for 40 minutes at room temperature (RT). 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-conjugated antibody (cat. no. 150165; Abcam) in the dark at RT for 15 minutes. Samples were analyzed using a Cytoflex flow cytometer (Beckman Coulter), and the results were analyzed using CytExpert for DxFLEX software.

[0191] For intracellular staining, 6 × 10 6 PBMCs were fixed in 100 μL of fixative (cat. no. A07803; Beckman Coulter) at RT for 15 minutes. Then, 4 mL of PBS was added, the cells were centrifuged and the supernatant was discarded. Permeabilization was performed by adding 100 μL of Reagent 2 for 5 minutes and then gently stirring. The cells were incubated with EX-02 or isotype antibodies for 30 minutes, washed twice in PBS, and then incubated with Alexa488-coupled secondary antibodies (cat. no. 150165; Abcam) in the dark for 15 minutes. After the last PBS wash, the cells were analyzed by flow cytometry.

[0192] Preparation of conditioned medium and culture of RAJI cells for SRRM2 expression analysis

[0193] To investigate the effect on SRRM2 expression on RAJI cells, conditioned medium was prepared using MOLM-13 cells with high SRRM2 surface expression. MOLM-13 cells were cultured in RPMI-1640 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 min to remove cell debris, and filtered through a 0.22 μm filter to obtain conditioned medium. RAJI cells were cultured at 1 × 106 The density of cells / well was seeded in 6-well plates and cultured in conditioned medium derived from MOLM-13 cells for 24, 48 and 72 hours. RAJI cells cultured in standard RPMI-1640 medium containing 10% FBS were used as a control group. After the specified incubation period, RAJI cells were harvested, washed twice with PBS, and analyzed by FCM.

[0194] Immunofluorescence

[0195] The cells were grown on glass coverslips and fixed with 4% paraformaldehyde (Biosharp, BL539A). The cells were blocked in 2% normal donkey serum (Solarbio, SL050) at RT for 30 minutes. The cells were incubated overnight at 4 ° C with anti-SRRM2 antibody (EX02) dissolved in PBST (Servicebio, G2157-1L). After washing three times with PBST, the cells were incubated with goat anti-rat IgG H&L / AF488 (Abcam, ab150165) antibodies at RT for 2 hours. The nuclei were stained with Antifade Mounting medium with DAPI (P0130; Beyotime, Shanghai, China). Images were captured using a fluorescence microscope (Axio Scope A1, Zeiss, Oberkochen, Germany).

[0196] Immunohistochemistry

[0197] Bone marrow smears of AML (n = 2) and IDA (n = 2) patients used in this study (which were obtained from our research institution) were prepared according to standard protocols. The normal tissue samples used in this study were obtained from Shanghai Outdo Biotech Company (Shanghai, China; Cat no.HOrgC120PG04; Lot no.XT19-008). The project named Development and Application of Multi-Organ Tissue Chip Products was evaluated and approved by the Ethics Committee of Shanghai Outdo Biotech to ensure compliance with ethical standards. The smears were dewaxed in xylene, rehydrated by gradient ethanol solutions (100% twice, 5 minutes each, 95%, 80% and 70% each gradient 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 minutes. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 5 minutes. Antigen retrieval was achieved by heating the smears at 95°C for 20 minutes in citrate buffer (1.8 mM citric acid, 8.2 mM sodium citrate). Nonspecific binding was blocked with PBS / 5% DSA for 1 hour. The smears were then incubated with EX-02 (1:200) at 4°C overnight, followed by incubation with HRP-labeled anti-rat IgGH&L antibody (Abcam, Shanghai, China; ab7097; 1:200) at RT for 1 hour, and detected using a DAB substrate kit (No.SD3102; CELNOVTE, Suzhou, China) until optimal staining was achieved. Counterstaining was performed with hematoxylin (Biosharp, Hefei, China; #71025010). The smears were mounted with neutral balsam and examined under an optical microscope, and images were captured with a microscopic imaging system.

[0198] Construction of EX-02CAR and CAR-T cells

[0199] The 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 second-generation CAR backbone with 4-1BB / CD137 and CD3-ζ transactivation domains and checked for integrity by sequencing. The lentiviral vector was produced by co-transfecting HEK293 cells with VSV-G envelope; GAG-pol packaging factor and Rev plasmid. The supernatant was collected after 72 hours, filtered, concentrated by centrifugation, tested on HEK293 cells to calculate the titer of infectious virus, and finally stored at -80°C until use.

[0200] Peripheral blood mononuclear cells (PBMCs) were isolated from PB of healthy volunteers by Ficoll separation. Cells were plated at 2 × 10 6The cells were inoculated at a density of 1 cell / ml and stimulated for 24 hours using CD3 / CD28 monoclonal antibodies (mAbs) (100 IU / ml or 300 IU / ml, Miltenyi, Bergisch-Gladbach, Germany). T cells were transduced with LV at a multiplicity of infection (MOI) of 5, and human serum albumin (HSA, 10 ng / ml, Baxalta, West Sacramento, CA) was added to optimize transfection efficiency. Cells were washed 24 hours after transduction to remove LV particles. In contrast, the simulation (MOCK)-T cells in the uninfected group were grown in a similar manner to CAR-T cells, but without any viral infection. In the presence of IL-2 (300 IU / ml or 600 IU / ml, Quangang, China) or IL-7 and IL-15 (10 ng / ml, SinoBiological, Beijing, China), T cells were expanded for 4-7 days in PRIME-XV T cell chemical composition defined medium (CDM, Santa Ana, CA). The cell culture products were washed twice with PBS and used in subsequent experiments.

[0201] Cytotoxicity analysis of CAR-T cells

[0202] By adjusting the cell density to 1 × 10 6 Effector and target cell cultures were prepared at 1:1 and 2.5:1 ratios of effector and target cells per mL. Simulated-T or CAR-T cells were co-cultured with MOLM-13 or HL-60 cells at effector to target cell ratios of 1:1 and 2.5:1. An appropriate number of effector and target cells were added to a 24-well plate, and three replicates were established for each group. Complete medium was added to each well to reach a final volume of 800 μL, and the contents were thoroughly mixed. The plate was incubated at 37 ° C for 0, 12, 24, and 48 hours. At each time point, the cells were collected and thoroughly resuspended to obtain a single cell suspension. An aliquot of 10 μL of the suspension was taken for cell counting. The remaining cells were centrifuged at 300 g for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in 100 μL PBS and then stained with PC7-CD45, FITC-CD3, and PE-CD33 antibodies. After incubation at room temperature for 15 minutes, the cells were washed with PBS to remove unbound antibodies. The final cell suspension was resuspended in 500 μL PBS and prepared for flow cytometry analysis.

[0203] Alternatively, the cytotoxicity of EX-02CAR-T cells was assessed using a cytotoxicity LDH assay kit (Dojindo, Shanghai, China, #CK12-05). All procedures were performed according to the manufacturer's instructions.

[0204] Quantification of cytokines by ELISA analysis

[0205] A commercial ELISA assay (ELK Biotechnology, Wuhan, China) was used according to the supplier's protocol.

[0206] AML xenograft mouse model

[0207] Six-week-old immunodeficient NSG mice, SPF grade, were purchased from Shanghai Model Organisms Pharmachem Biotechnology Co. On day -2, 1×10 6 MOLM-13 cells / mouse were injected into the tail vein. Two days later (d=2), blind researchers randomly divided the tumor-bearing mice into two groups (n=5) and intravenously administered simulated-T (untransduced T cells, 2×10 6 ) or EX-02CAR-T cells (2×10 6 ). Blood, BM or tumor tissues were collected on the last day of killing mice. This animal model was approved by the Hefei Comprehensive National Science Center Institute of Big Health (approval number: IHM-AP-2024-084-R1). The validity period is extended from November 4, 2024 to November 4, 2027.

[0208] Statistical analysis

[0209] Statistical analysis was performed using IBM SPSS Statistics 25 and GraphPad Prism 8.0. Continuous data are expressed as mean ± standard deviation For comparison between the two groups, if the data followed a normal distribution, differences were assessed using a t-test, otherwise the data were expressed as median and interquartile range and analyzed using a nonparametric rank sum test. Survival was defined as the time from the first diagnosis until death (all-cause death). A p value < 0.05 was considered statistically significant.

[0210] Example 2: Nuclear plaque protein SRRM2 is exposed on the surface of AML cells

[0211] 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 total 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 have prompted studies of the intracellular localization of SRRM2 in AML blasts and normal blood cells, a topic that has not been explored in AML.

[0212] First, the surface expression of SRRM2 in several AML cell lines was assessed using FCM and our proprietary antibody EX-02. Figure 1 A 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 both surface (upper panel) and intracellular SRRM2 (lower panel) of live cells. Figure 1 In B, surface SRRM2 expression in AML cell line MOLM-13 was confirmed by fluorescence confocal microscopy.SRRM2 (green; see "Ex-02" and arrows) is localized to the cell surface of living cells and intracellularly in permeabilized cells, which confirms the results from flow cytometry.Counterstained nuclei with DAPI (blue; see "DAPI" and arrows). Cell lines from non-AML, including RAJI, NAML-6 and SU-DHL-4, and surface and intracellular staining of HEK-293T cells from normal tissues were analyzed by FCM.Compared with AML lines, the surface expression level of SRRM2 is significantly lower in these cell lines, while intracellular expression is always high in all samples.In addition, no significant difference in SRRM2 expression was observed in RAJI cells cultured with conditioned medium.These results show that although SRRM2 is mainly an intracellular protein, SRRM2 surface expression changes in hematological malignancies, and this is not due to nonspecific binding.

[0213] Example 3: Exposure of SRRM2 to blasts from AML patients

[0214] After confirming the presence of SRRM2 on the surface of AML-derived cell lines, we performed FCM analysis of various cell types including normal lymphocytes, monocytes, granulocytes and AML blasts freshly isolated from peripheral blood (PB) and bone marrow (BM) of AML patients. Figure 2 It was shown that SRRM2 is only exposed on the surface of AML blasts, but not on normal blood cells. Figure 2 A shows flow cytometric analysis of blood cell subsets isolated from peripheral blood of AML patients. Upper panel: Staining of live cells with the EX-02 antibody demonstrates that SRRM2 is detectable on the surface of blast cells, while normal blood cells (lymphocytes, monocytes, granulocytes) show only marginal background staining. Lower panel: Intracellular staining of permeabilized cells of the same type shows that all cell types stain positive for intracellular SRRM2. Figure 2 B and Figure 2 Same as A, but using peripheral blood derived from healthy donors. No surface SRRM2 was detected after EX-02 staining. Figure 2C shows the Figure 2 A Same staining, but using bone marrow-derived cells from AML patients. Again only blasts were stained surface SRRM2 positive by the EX-02 antibody, while all other cell types showed only intracellular SRRM2 staining. Figure 2 D shows cells isolated from the bone marrow of a patient with iron deficiency anemia (IDA). No surface SRRM2 was detected on lymphocytes, monocytes, granulocytes, or hematopoietic stem cells (HSC). In general, surface SRRM2 was only detectable on malignant AML blasts, but not on normal blood cells.

[0215] Example 4: Surface SRRM2 is a marker of risk and poor prognosis

[0216] Based on the above findings, a correlation analysis between SRRM2 surface expression levels and clinical data was performed in 52 ND AML patients. According to the "Domestic Expert Consensus on Detection of Acute Leukemia Lineages by Flow Cytometry", surface antigen expression of more than 20% in the target cell population was defined as positive. Based on this criterion, ND AML patients were stratified into negative SRRM2 surface exposure (SRRM2 Neg ,<20%) and positive SRRM2 surface exposure (SRRM2 Pos Table 2 shows the basic clinical information including patient age, gender and disease status.

[0217] Table 2: Clinical characteristics of AML patients stratified by SRRM2 expression

[0218]

[0219]

[0220] The baseline characteristics of the 52 patients diagnosed with AML are described in detail in the Table, with stratification based on the level of SRRM2 surface exposure. Patients were divided into two groups: SRRM2 surface exposure negative (SRRM2 Neg , n = 20), SRRM2 surface exposure positive (SRRM2 Pos , 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 sex distribution, disease origin, and French-American-British (FAB) typing, were expressed as percentages.

[0221] Statistical analysis was performed to evaluate the differences between the two groups, where the p-value indicated the level of significance; p-values ​​less than 0.05 were considered statistically significant. Nearly 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 reach statistical significance, they suggest a potential correlation between SRRM2 expression and various clinical and laboratory features in AML patients.

[0222] Figure 3 Correlation between SRRM2 surface exposure and clinical characteristics in AML patients is shown. Figure 3 AB show SRRM2 exposure in de novo and secondary AML (A) and various WHO-FAB subtypes (M0, M1, M2, M4) (B) analyzed by flow cytometry, with no significant differences observed between these groups. Figure 3 C shows that the SRRM2 surface exposure in patients who failed standard induction chemotherapy (incomplete remission, NCR) was significantly higher than that in patients with complete remission (CR). Figure 3 D shows that SRRM2 surface exposure was significantly increased in poor and intermediate risk patients stratified according to the 2022 NCCN risk classification compared with good risk patients. Figure 3 E shows that relapsed AML patients showed higher SRRM2 surface exposure compared with newly diagnosed (ND) patients. Figure 3 F shows a longitudinal analysis of seven AML patients demonstrating a significant increase in SRRM2 exposure on the surface of AML blasts at relapse. Figure 3 GH showed that compared with other blood cells, those from ND patients ( Figure 3 G) and relapsed patients ( Figure 3 H) SRRM2 exposure on the surface of AML blasts is greatly increased.

[0223] For SRRM2 Pos In patients, ROC curve analysis determined a cutoff value of 30.14% to distinguish between low and high expression levels, reflecting different responses to standard induction chemotherapy ( Figure 5 ). Therefore, all newly diagnosed AML patients were divided into three groups: SRRM2 Neg (<20%), SRRM2 低 (20%-30.14%) and SRRM2 高 (>30.14%).

[0224] Figure 3 The following is further shown: Figure 3IJ shows SRRM2 surface exposure positive (SRRM2 Pos The complete remission rate (CRR) of AML patients with SRRM2 surface exposure was significantly lower than that of AML patients with negative SRRM2 Neg ) patients, and the more SRRM2 surface exposure, the lower the CRR. Figure 3 KL shows Kaplan-Meier analysis showing SRRM2 Pos Patients had shorter recurrence-free survival (RFS), and high SRRM2 expression was associated with shorter RFS. Figure 3 MN shows SRRM2 Pos The overall survival (OS) of patients was better than that of SRRM2 Neg Patients with short. *P<0.05; **P<0.01; ***P<0.001. In conclusion, SRRM2 surface exposure was closely associated with clinical features in AML patients, and higher SRRM2 exposure was associated with worse prognosis. These findings suggest that SRRM2 may serve as a valuable prognostic biomarker in AML. In addition, its exposure on the cell surface makes it a promising new target for immunotherapy.

[0225] Example 5: Surface SRRM2 is not detected in normal hematopoietic stem cells and various normal tissues.

[0226] To evaluate SRRM2 expression in normal hematopoietic cells, including hematopoietic stem cells (HSCs), we performed flow cytometric (FCM) analysis of bone marrow (BM) cells from patients with iron deficiency anemia (IDA). Figure 4 A, flow cytometric analysis of hematopoietic cells from patients with iron deficiency anemia (IDA) shows that SRRM2 has minimal surface expression in lymphocytes, monocytes, neutrophils, and hematopoietic stem cells (HSCs). Figure 4 In B, it is evident that SRRM2 has strong nuclear expression in all cell types, with almost exclusive positivity. Immunohistochemical analysis showed no significant surface expression of SRRM2 in various normal tissues, including cerebellum, colon, liver, lung, skin, pancreas, heart, kidney, lymph node, muscle, and spleen (see Figure 4 C).

[0227] Our findings led us to postulate that SRRM2, normally a nuclear protein, is specifically exposed on the surface of AML blasts. In contrast, SRRM2 is minimally expressed or completely absent on the surface of normal blood cells, including hematopoietic stem cells. In addition, various normal tissues exhibited surface SRRM2 levels below the limit of detection. Notably, we observed a direct correlation between SRRM2 surface levels on AML blasts and high-risk stratification as well as poor prognosis. This association highlights the potential clinical relevance of SRRM2 as an AML biomarker. Targeted EX-02 CAR-T cells showed anti-AML effects both in vitro and in vivo.

[0228] Example 6: SRRM2-specific CAR-T cells exhibit anticancer activity against AML cells both in vitro and in vivo

[0229] CAR-T cell therapy has been shown to have significant efficacy in the treatment of various hematological malignancies. However, the application of this therapy in acute myeloid leukemia (AML) is limited due to the lack of specific and suitable target molecules on AML blasts, resulting in no commercially available CAR-T products for this disease. Our study found that surface SRRM2 is a promising target molecule that is uniquely expressed on AML blasts and not present on normal blood cells. Therefore, we aimed to evaluate the anti-tumor efficacy of SRRM2-specific CAR-T cells in vitro and using a human xenograft model of AML. To achieve this goal, we engineered a CAR with a single-chain variable fragment (scFV) derived from the EX-02 antibody as described above, and produced a lentiviral vector. EX-02CAR-T cells exhibited effective cytolytic activity against all tested AML cell lines (including MOLM-13, MV4-11, SKM-1, and HL-60) at an effector-target (E:T) ratio of 1:1 and 2.5:1. This cytotoxicity was accompanied by significantly elevated levels of cytokines IFN-γ and TNF-α in the supernatants of EX-02CAR-T cells co-cultured with target cells compared to those incubated with mock (MOCK)-T cells. In addition, lactate dehydrogenase (LDH) release assays confirmed a dose-dependent increase in tumor cell lysis after 24 hours of co-culture, with higher E:T ratios associated with enhanced cytolytic activity.

[0230] After confirming the antitumor activity of EX-02CAR-T cells in vitro, we then established a human xenograft model in immunodeficient NSG mice to evaluate the efficacy of EX-02. 6 MOLM-13 cells / mouse were injected into the tail vein. Two days later, the mice were randomly divided into three groups and treated with mock-T (untransduced T cells, n=5) or EX-02CAR-T cells (2×10 6Cells / mouse, n=5) and PBS (blank group, n=3) were treated. Bioluminescent images (BLI) of tumor-bearing mice were taken on days 0, 6, 12, 18, 21, and 24. On day 12, the CAR-T treated group showed a significant reduction in tumor burden compared with the mock-T and blank groups. Bioluminescence quantification on day 24 confirmed that tumor activity in the CAR-T group was significantly reduced compared with the mock-T and blank groups (p<0.001). Survival analysis showed a significant prolongation of survival in the EX02 CAR-T treatment group. The CAR-T group showed a significantly prolonged survival compared with 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. In addition, mice in the mock-T group experienced significant weight loss starting from day 12 (p=0.02.) The strong in vivo efficacy of EX-02 CAR-T cells emphasizes the potential of SRRM2-specific CAR-T cells as a feasible and promising therapeutic strategy for AML.

[0231] Example 7: Discussion

[0232] While antibody-based immunotherapy has revolutionized the treatment of various cancers, progress in addressing AML has been limited. This challenge is primarily due to the lack of suitable target molecules present on malignant cells but not on normal blood cells, including hematopoietic stem cells (HSCs). Our studies using EX-02, a proprietary antibody specifically designed to target surface-exposed variants of SRRM2, have produced significant findings in this regard. These studies revealed that SRRM2, which is generally considered a nuclear protein, is significantly expressed on the surface of acute myeloid leukemia (AML) cell lines and on the surface of blasts from AML patients. Although data from The Cancer Genome Atlas (TCGA) indicate that SRRM2 is overexpressed at the total RNA level in AML, this overexpression is not associated with clinical parameters such as progression-free survival and overall survival. In stark contrast, our findings demonstrate that elevated levels of surface SRRM2 are not only directly and significantly associated with poor clinical outcomes, but also with treatment resistance and lower complete remission rates. This finding suggests that extranuclear SRRM2, whether located in the cytoplasm or on the cell surface, may play a key role in the malignancy of AML and potentially other cancer types.

[0233] Traditionally, SRRM2 has been considered an RNA-binding nuclear protein with a key role in the splicing process. Previous studies have identified its involvement in the formation and function of nuclear speckles and in maintaining cellular homeostasis.

[0234] The surface expression of SRRM2 on cancer cells, including AML blasts, was unexpected, but not without precedent, as other splicing proteins have also been found on the surface of cancer cells. For example, Christian et al. (The Journal of cellbiology.2003; 163(4):871-8) found nucleolar proteins on the surface of endothelial cells in tumor blood vessels. Similarly, Gillissen et al. (Blood.2018; 131(1):131-43) reported the presence of U5 snRNP200 on AML blasts, and Tonapi et al. (Cell chemical biology.2019; 26(5):756-64.e6) identified a splicing complex containing at least 13 core components on the surface of non-Hodgkin lymphoma.

[0235] Our findings revealed that SRRM2 is present on the surface of AML blasts and is almost absent in normal blood cells, including hematopoietic stem cells (HSCs). Notably, SRRM2-specific CAR-T cells exhibited significant anticancer activity both in vitro and in vivo. This suggests that immunotherapy approaches targeting SRRM2 hold great promise in the treatment of AML. By focusing on more selective targets such as SRRM2, we can enhance the efficacy of CAR-T-based immunotherapy while reducing adverse reactions associated with existing antigen targets.

[0236] Example 8: Conclusion and future directions

[0237] Our study identifies SRRM2 as a novel and highly specific target in the treatment of acute myeloid leukemia (AML). The unique surface expression of SRRM2 in AML cells, combined with its nuclear restriction in normal tissues, creates a compelling opportunity for the development of targeted immunotherapies such as EX-02CAR-T cells. This approach not only has the potential to improve treatment outcomes for high-risk and relapsed AML patients, but also paves the way for personalized treatment strategies for AML.

[0238] The embodiments described exemplarily herein can be appropriately implemented in the absence of any one or more elements, one or more limitations not specifically disclosed herein. Therefore, the terms and expressions adopted herein are used as terms of description rather than terms of limitation, and there is no intention to exclude any equivalents or parts of the features shown and described when using these terms and expressions, but to recognize that various modifications can be within the scope of the present invention. Therefore, it should be understood that although these embodiments have been specifically disclosed by preferred embodiments and optional features, those skilled in the art can seek modifications and changes thereof, and such modifications and changes are considered to be within the scope of the present invention. Each narrower category and sub-category grouping falls within the disclosure scope of the genus and also forms a part of the present invention. This includes a general description of the present invention, and has conditions or negative limitations to take out any subject matter from the category, regardless of whether the removed material is specifically stated in this article. In addition, when the feature is described in the form of a Markush group, those skilled in the art will recognize that the present invention is also described in the form of any individual member or member subgroup in the Markush group.

[0239] 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. The following claims are intended to cover such equivalents.

[0240] It should be understood that the present invention is not limited to the specific methodology, protocols, materials, reagents and substances, etc. described herein, and thus may vary. The terminology used herein is only for the purpose of describing specific embodiments and is not intended to limit the scope of the present invention, which is limited only by the claims.

[0241] All publications cited throughout this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions for use, etc.) are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosures by virtue of prior invention. To the extent that material incorporated by reference is inconsistent or inconsistent with this specification, this specification will supersede any such material.

[0242] Further embodiments will become apparent from the following claims.

Claims

1. A method for assessing whether a subject diagnosed with acute myeloid leukemia (AML) will have a negative prognosis or a positive prognosis, the method comprising The level of SRRM2 expression on the cell surface of the subject's progenitor cells is determined in vitro, wherein (i) characterizing a subject having SRRM2-expressing blast levels of 20% or less as SRRM2-negative, and (ii) characterizing a subject having a SRRM2-expressing blast level of greater than 20% as SRRM2-positive, Among them, SRRM2-negative subjects had a positive prognosis, while SRRM2-positive subjects had a negative prognosis.

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

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

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

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

6. The method of any one of the preceding claims, wherein the level of SRRM2 expressing blasts is correlated to the total number of blasts measured in the subject.

7. The method according to any one of the preceding claims, wherein the subject is intended for or undergoing therapy to treat AML.

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

9. The method according to any one of the preceding claims, wherein the expression of SRRM2 on the cell surface of the mother cell is determined using an antibody that binds to SRRM2 on the cell surface of SRRM2-expressing cells.

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

11. The method according to claim 9 or 10, wherein the SRRM2-expressing cells are viable and intact mother cells.

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

29.

13. The method according to any one of claims 8 to 12, wherein the antibody is an antibody comprising: (a) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 2, (b) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 4, (c) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:5 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:6, or (d) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:5 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:

7.

14. The method according to any one of claims 1 to 13, wherein the direct purpose of the method is not to diagnose and / or treat a disease.

15. An in vitro use of the expression level of SRRM2 on the cell surface of blast cells of a subject diagnosed with acute myeloid leukemia (AML) for assessing whether the subject will have a negative prognosis or a positive prognosis, wherein (i) Subjects with SRRM2 expressing blast levels of 20% or less are characterized as SRRM2-negative and have a positive prognosis, while (ii) Subjects with SRRM2 expressing blast levels greater than 20% are characterized as SRRM2-positive and have a negative prognosis.

16. The use according to claim 15, wherein a SRRM2-positive subject has a SRRM2-expressing blast cell level greater than 20% and up to 30% and is characterized as low SRRM2-positivity, or a SRRM2-positive subject has a SRRM2-expressing blast cell level greater than 30% and is characterized as high SRRM2-positivity.

17. The use according to claim 15 or 16, wherein the direct purpose of the use is not the diagnosis and / or treatment of a disease.

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

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

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

21. An SRRM2-targeted therapy for use in treating AML in a subject, wherein the subject is characterized by having SRRM2 expressing blast levels greater than 20%.

22. Use of a SRRM2-targeted therapy in the preparation of a medicament for treating AML in a subject, wherein the subject is characterized by having a SRRM2-expressing blast level greater than 20%.

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

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

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

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