Bispecific CD123×CD3 diabodies for treatment of hematologic malignancies

Abstract

To provide a method of treating a hematologic malignancy such as acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), including hematologic malignancies that are refractive to chemotherapeutic and / or hypomethylating agents.SOLUTION: The present invention provides a method of treating a chemo-refractory hematologic malignancy in a patient, wherein the method comprises administering to the patient a treatment dosage of a CD123×CD3 bispecific molecule, the dosage being effective to stimulate the killing of cells of the hematologic malignancy in the patient, thereby treating the malignancy.SELECTED DRAWING: None

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application is a joint venture of U.S. Patent Application Serial No. 62 / 878,368 (filed July 25, 2019; pending). No. 62 / 769,078 (filed November 19, 2018; pending), and No. 62 / 7 Claims priority to No. 52,659 (filed October 30, 2018; pending). and each of the above applications is incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference This application contains one or more sequence listings in accordance with 37 CFR §1.821 et seq. These sequence listings are available on a computer readable medium (file name: 1301_0161PCT_S T25.txt, created on September 26, 2019, size: 31,244 bytes) and the above files are incorporated by reference in their entireties into this application.

[0003] The present invention includes hematological malignancies that are refractory to chemotherapy and / or hypomethylating agents. , to treat hematological malignancies such as acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) The present invention is directed to a method of administering a CD123×CD3 bispecific binding molecule to a patient. in an amount effective to stimulate killing of cells of said hematological malignancy in said patient. The present invention further relates to a method for detecting a cell sample derived from the patient, the cell sample being capable of detecting one or more target genes, The baseline level of expression of the gene, e.g., in a plurality of subjects suffering from the hematological malignancy, The expression level of the gene is higher than the baseline level in a reference population of individuals, or the reference gene Of interest is the embodiment of the above method, which shows high expression relative to the level of expression of the gene. [Background technology]

[0004] I.CD123 CD123 (interleukin 3 receptor α, IL-3Ra) is a 40 kDa molecule. , which is part of the interleukin 3 receptor complex (Non-Patent Document 1). (IL-3) promotes early differentiation of pluripotent stem cells into erythroid, myeloid and lymphoid progenitor cells CD123 is expressed on CD34+ committed progenitor cells (Non-Patent Document 2), CD34+ / CD38- is not expressed by normal hematopoietic stem cells. CD123 is expressed by basophils. It is expressed by leukocytes, mast cells, and plasmacytoid dendritic cells, and by monocytes, macrophages, and eosinophils. Somewhat expressed by leukocytes and little or not at all by neutrophils and megakaryocytes Some non-hematopoietic tissues (placenta, Leydig cells of the testes, certain brain cell elements, and some endocrine endothelial cells) express CD123, but expression is mostly cytoplasmic.

[0005] CD123 has been reported to be expressed by leukemic blasts and leukemic stem cells (LSCs). In normal human progenitor populations, CD123 is expressed in hematopoietic progenitors. Although expressed by a subset of hematopoietic stem cells (HPCs), CD123 is also expressed by plasmacytoid dendritic cells (pDCs) and basophils. It is expressed by leukocytes, and to a lesser extent by monocytes and eosinophils (Non-Patent Literature 5~9)

[0006] CD123 is a marker for a wide range of cancers including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). It has been reported that it is overexpressed in malignant cells in a wide variety of hematological malignancies (Non-Patent Literature 1, 2013). Reference 7). Overexpression of CD123 is associated with poor prognosis of AML (Non-Patent Document 10). .

[0007] II.CD3 CD3 is a T cell coreceptor composed of four distinct chains (Non-Patent Document 11). In this species, the complex contains one CD3γ chain, one CD3δ chain, and two CD3ε chains. These chains associate with a molecule known as the T cell receptor (TCR) to bind to the T cell. In the absence of CD3, TCRs do not engage properly and generate activation signals in lymphocytes. CD3 binds to the membrane of all mature T cells and is essentially It has been shown not to bind to the membranes of any other cell types (see Non-Patent Documents 13-15). (see).

[0008] III. AML and MDS Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are generally dormant. (i.e., cells that do not divide rapidly) and therefore are prone to cell death (apoptosis) and conventional chemotherapy This arises in a small population of leukemic stem cells (LSCs) that are resistant to LSCs are characterized by high levels of CD123 expression, which is thought to be a positive They are not present in the corresponding normal hematopoietic stem cells in normal human bone marrow (Non-Patent Documents 4, 3). D123 accounts for 45% to 95% of AML, 85% of hairy cell leukemia (HCL), and acute B It is expressed in 40% of B-ALL. CD123 expression is also seen in It is also associated with multiple malignant / premalignant tumors in the following: chronic myeloid leukemia (CML) precursors (acute transformed CML); Hodgkin / Reed-Sternberg (RS) cells; transformed non-Hodgkin's lymphoma (NHL); some chronic lymphocytic leukemia (CLL) (CD11c +); a subset of acute T-lymphoblastic leukemia (T-ALL) (16%, the most immature, large half adult), plasmacytoid dendritic cell (pDC) DC2 malignancies, and CD34+ / CD38 -Myelodysplastic syndrome (MDS) - Myeloid cell malignancies

[0009] AML is a clonal disease characterized by the proliferation and accumulation in the bone marrow of transformed myeloid progenitor cells. AML is a chronic disease that ultimately leads to hematopoietic failure. The incidence of AML increases with age. The incidence of rheumatoid arthritis is increasing, and older patients typically have poorer outcomes than younger patients (Non-Patent 16) Unfortunately, most adults currently diagnosed with AML die from the disease. He is deceased.

[0010] Treatment of AML initially focuses on inducing remission (induction therapy). Once remission is achieved, Treatment is focused on achieving this remission (post-remission or consolidation therapy) and, in some cases, maintaining remission. The standard induction paradigm for AML has shifted to focus on maintenance therapy. The chemotherapy regimen is anthracycline / cytarabine combination followed by , depending on the patient's ability to tolerate intensive care, and the likelihood of cure with chemotherapy alone (usually consolidation chemotherapy (using higher doses of the same drugs used during the induction period); Alternatively, human stem cell transplantation is performed (see, for example, Non-Patent Document 17).

[0011] Agents frequently used in induction therapy include cytarabine and anthracyclines. Cytarabine (also known as AraC) acts by interfering with DNA synthesis. , which kills cancer cells (and other rapidly dividing normal cells). Side effects include: decreased resistance to infections as a result of decreased white blood cell production; platelets bleeding as a result of decreased production; and anemia due to a potential decrease in red blood cells. Other side effects include nausea and vomiting. bicin, doxorubicin, and idarubicin) inhibit DNA and RNA synthesis, DNA It has multiple mechanisms of action, including disruption of the higher-order structure of β-lactamase and production of cell-damaging free oxygen radicals. The most important side effect of anthracyclines is cardiotoxicity, which can lead to This severely limits the lifetime dose that can be administered and, to some extent, its usefulness.

[0012] Stem cell transplantation is an anti-leukemia therapy for patients with AML in first or subsequent remission. However, it has been established as the most effective form of Despite significant advances in the treatment of newly diagnosed AML, 20% to 40% of patients 0% are the 50% to 60% of patients who did not achieve remission with standard induction chemotherapy and achieved a first complete remission 70% are expected to relapse within 3 years. For patients with relapsed or resistant disease The optimal strategy for this remains uncertain (see non-patent literature 18-23). There is a need for treatment strategies to

[0013] IV. Bispecific molecules Non-monospecific molecules (e.g., bispecific antibodies, bispecific diabodies, BiTEs, etc.) The provision of antibodies, such as those listed in the above patents, allows for the co-ligation and co-localization of cells expressing different epitopes. This offers a significant advantage over monospecific molecules such as natural antibodies: the ability to Bispecific molecules have a wide range of applications including therapy and immunodiagnosis. This provides great flexibility in the design and engineering of diabodies for a wide range of applications, Enhanced avidity for dimeric antigens, cross-linking of different antigens, and dependent on the presence of both target antigens Directed targeting to specific cell types is provided. Of particular importance is the ability to target multiple different cells. Co-ligation, e.g., cross-linking of effector cells, such as cytotoxic T cells, to tumor cells ( Non-patent literature 24, 25).

[0014] A wide range of recombinant bispecific antibodies have been developed to provide molecules with greater potency than natural antibodies. Many of these formats have been developed (e.g., Patent Documents 1 to 7). The peptides can be used to bind additional binding proteins (e.g., scFv, VL, VH, etc.) to the antibody complex. IgA, IgD, IgE, IgG, or IgM. or multiple antibody binding moieties (e.g., two Fab fragments or scFvs) are fused together. Another format uses a linker peptide to bind the binding protein (e.g., sc Fv, VL, VH, etc.) to a dimerization domain, such as a CH2-CH3 domain, or to another polypeptide. In other formats, CL and CH one domain is swapped from its natural position, and / or the VL and VH domains However, they are diversified so that they can bind to two or more antigens (Patent Documents 12 and 13).

[0015] The art further provides diabodies capable of binding to two or more different epitope species. It has been mentioned that it is possible to form stable covalent heterocycles (see, for example, Non-Patent Document 26). Dimeric non-monospecific diabodies have been described (e.g., Patent Documents 14 to 18, Non-Patent Documents 1 to 5, (See References 27-29.) Such diabodies incorporate one or more cysteine ​​residues. For example, a cysteine ​​residue may be incorporated into each of the polypeptide species used. Addition of the carboxy terminus of the carboxyl group allows disulfide bonds between the polypeptide chains, resulting in the formation of a divalent molecule. The resulting heterodimers can be stabilized without interfering with their binding properties. In addition, trivalent molecules containing diabody-like domains have been described. (See, for example, U.S. Patent Nos. 5,333,631 and 5,333,631.) Diabody epitope binding domains also include T CD3, C, expressed on lymphocytes, natural killer (NK) cells, and other mononuclear cells Any immune effector cell surface determinant such as D16, CD32, or CD64 Many studies have targeted effector cell determinants, e.g., Fcγ receptors. Diabodies that bind to FcγRs have also been shown to activate effector cells. (Non-Patent Documents 25, 30, Patent Documents 14 to 18). Usually, effector cell activation is , by binding of antigen-bound antibodies to effector cells via Fc-FcγR interactions. Thus, in this regard, a diabody molecule may or may not contain an Fc domain. Regardless, any of the above (e.g., any of the above known in the art or exemplified herein) may be used. As assayed in an effector function assay (e.g., ADCC assay), ) Ig-like functions. By cross-linking tumor cells and effector cells, The bodies not only deliver effector cells to the vicinity of tumor cells but also induce effective cell killing. (See, for example, non-patent document 31). [Prior art documents] [Patent documents]

[0016] [Patent Document 1] International Publication No. 2008 / 003116 [Patent Document 2] International Publication No. 2009 / 132876 [Patent Document 3] International Publication No. 2008 / 003103 [Patent Document 4] International Publication No. 2007 / 146968 [Patent Document 5] International Publication No. 2009 / 018386 [Patent Document 6] International Publication No. 2012 / 009544 [Patent Document 7] International Publication No. 2013 / 070565 [Patent Document 8] International Publication No. 2005 / 070966 [Patent Document 9] International Publication No. 2006 / 107786 [Patent Document 10] International Publication No. 2006 / 107617 [Patent Document 11] International Publication No. 2007 / 046893 [Patent Document 12] International Publication No. 2008 / 027236 [Patent Document 13] International Publication No. 2010 / 108127 [Patent Document 14] International Publication No. 2006 / 113665 [Patent Document 15] International Publication No. 2008 / 157379 [Patent Document 16] International Publication No. 2010 / 080538 [Patent Document 17] International Publication No. 2012 / 018687 [Patent Document 18] International Publication No. 2012 / 162068

Patent document 19

Patent document 20

Non-licensed literature

[0017]

Non-licensed literature 1

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Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-licensed Document 19

Non-licensed Document 20

Non-licensed Document 21

Non-licensed Document 22

Non-licensed Document 23

Non-licensed Document 24

[0018] CD123 and C-terminal cytoplasmic receptor agonists can mediate T cell targeted killing of CD123-expressing malignant cells. Several bispecific molecules targeting D3 are under development (e.g., Vey, N., et al. (2 017) “Interim Results From A Phase 1 First-In-Human Study Of Flotetuzumab, a CD 123 x CD3 Bispecific DART Molecule In AML / MDS,” Annals of Oncology, 28(S5)5, md x373.001; Godwin, CD, et al. (2017) “Bispecific Anti-CD123 x Anti-CD3 Adaptir (Trademark) Molecules APVO436 and APVO437 Have Broad Activity Against Primary Human AML Cells In Vitro” Blood. 130(S1): 2639; Forslund, A., et al. (2016) “Ex Viv o Activity Profile of the CD123xCD3 Duobody® Antibody JNJ-63709178 Ag ainst Primary Acute Myeloid Leukemia Bone Marrow Samples” Blood 128(22):2875 However, bispecific binding molecules capable of targeting T cells to the location of hematologic malignancies have been Efforts to recruit offspring have not been entirely successful. To develop novel strategies for the treatment of hematological malignancies using D3 bispecific binding molecules The present invention addresses this need and the following: Directly address other demands listed in the [Means for solving the problem]

[0019] The present invention includes hematological malignancies that are refractory to chemotherapy and / or hypomethylating agents. , to treat hematological malignancies such as acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) The present invention is directed to a method of administering a CD123×CD3 bispecific binding molecule to a patient. in an amount effective to stimulate killing of cells of said hematological malignancy in said patient. The present invention further relates to a method for detecting a cell sample derived from the patient, the cell sample being capable of detecting one or more target genes, The baseline level of expression of the gene, e.g., in a plurality of subjects suffering from the hematological malignancy, The expression level of the gene is higher than the baseline level in a reference population of individuals, or the reference gene Of interest is the embodiment of the above method, which shows high expression relative to the level of expression of the gene.

[0020] In particular, the present invention provides a method for treating chemotherapy-refractory hematological malignancies in a patient, comprising administering to said patient a The method includes administering to the patient a therapeutic dosage of a CD123×CD3 bispecific molecule. and the dosage comprises a dose that stimulates killing of cells of the hematological malignancy in the patient. By so doing, it is effective in treating the above malignant tumors.

[0021] The present invention further relates to a method for treating a patient suffering from atopic dermatitis, comprising administering said CD123×CD3 bispecific molecule to said patient, said method comprising administering said CD123×CD3 bispecific molecule to said patient. Evaluating expression of one or more target and / or reference genes in a cell sample from a patient. The present invention further provides an embodiment of the above method, further comprising: Prior to administration of the 23×CD3 bispecific molecule, one or more of the target genes and / or one or more of the target genes may be The method further comprises the step of evaluating the expression of the above reference genes. The invention also relates to a method comprising administering to said patient a CD123×CD3 bispecific molecule comprising administering to said patient a CD123×CD3 bispecific molecule. evaluating the expression of the target genes and / or the one or more reference genes. , an embodiment of the above method is provided.

[0022] The present invention further provides a method for administering to a patient a CD123xCD3 bispecific antibody for treating a hematological malignancy. The present invention provides a method for determining whether a subject is a favorable responder to the use of a molecule, the method comprising: (a) determining whether the CD123×C and detecting one or more target genes in a cell sample from the patient prior to administration of the D3 bispecific molecule. assessing expression; and (b) determining whether the expression of the one or more target genes is related to the one or more target and / or reference genes; If the expression of CD123×CD 3. Identifying Favorable Responders to Treatment with Bispecific Molecules Includes.

[0023] The present invention further relates to a method comprising: (i) expressing said one or more target genes; and (ii) evaluating one or more reference genes whose expression is not characteristically associated with hematological malignancies, An embodiment of a method is provided.

[0024] The present invention further provides a method for determining the expression of said one or more reference genes in said patient relative to the baseline expression of said one or more reference genes. Embodiments of the above methods are provided that include assessing the expression of one or more target genes. .

[0025] The present invention further relates to a method for the treatment of an individual suffering from a hematological malignancy, or a population of such individuals. For expression of one or more target genes, determining the expression of one or more target genes in a patient The present invention further provides an embodiment of the above method, comprising the step of evaluating The expression of one or more target genes is detected in the individual suffering from a hematological malignancy or in a plurality of individuals. The expression level of the one or more target genes in the population of individuals is higher than the first quartile ( higher than the 2nd quartile (i.e., greater than the bottom 25%), higher than the 2nd quartile (i.e., greater than the bottom 50%), or higher than the 3rd quartile Higher quantiles (ie, greater than the bottom 75%) are provided in accordance with embodiments of the above methods.

[0026] The present invention further provides evidence that hematological malignancies have previously been successfully treated using the methods and compositions of the present invention. For example, for the treatment of hematological malignancies using CD123×CD3 bispecific molecules (i) an individual who has not responded well to the treatment, or (ii) a group of several such individuals; For expression of the above target genes, evaluating the expression of one or more of the above target genes in a patient. The present invention further provides an embodiment of the above method, comprising the step of: The expression of the above target genes is in one of the above individuals or groups of individuals who have not been successfully treated. The expression level of the target gene is higher than the first quartile (i.e., greater than the bottom 25%), Higher than the 2nd quartile (i.e., greater than the bottom 50%) or higher than the 3rd quartile (i.e., lower 75%) % or more of said one or more of said patients. expression of the target gene of said one of said individuals or populations of said individuals who have not been successfully treated At least about 0.4, at least about 0.5, at least about 0.6, at least about 0.8, at least about 0.9, at least about 1.0, at least about 1.2, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.8, at least about 1.9, at least about 1.0, at least about 1.2, at least about 1.4, at least about 1.5 In one embodiment of the present invention, the method further comprises administering to the subject a subject a log2 fold change of at least about 0.6, or more. do.

[0027] The present invention further provides evidence that hematological malignancies have previously been successfully treated using the methods and compositions of the present invention. for the treatment of hematological malignancies using CD123×CD3 bispecific molecules. A population of individuals who have had a favorable response to one or more of the target genes. evaluating expression of the one or more target genes in a patient relative to the expression of the gene. The present invention further provides an embodiment of the method, comprising: Gene expression is associated with one or more target genes in the successfully treated individual or population of individuals. Within the first quartile (i.e., within the bottom 25%) and within the second quartile (i.e., within the bottom 25%) of the expression level of the offspring 25%-50%), or within the third quartile (i.e., the bottom 50-75%). An embodiment is provided.

[0028] The present invention further relates to a method for determining whether the relative expression level of said one or more target genes in said population is Established by averaging gene expression levels in cell samples obtained from a population of individuals In accordance with another embodiment of the present invention,

[0029] The present invention further relates to a method for treating a patient having an abnormality in which the patient has an abnormality in the expression level of at least one of the target genes. Is this: (a) determining the expression level of said target gene in a population of individuals suffering from a hematological malignancy; higher than the first quartile; or (b) Good results for the treatment of hematological malignancies using CD123×CD3 bispecific molecules The first quartile of expression levels of the target gene in the population of non-responsive individuals. higher than the number; or (c) A successful treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression level of the target gene in a population of individuals who were not responsive was low. have a log2 fold change of at least about 0.4; or (d) Use of CD123×CD3 bispecific molecules for the treatment of hematological malignancies At least a first quartile of expression levels of the target gene in a population of responsive individuals. Within a quantile, An embodiment of the above method is provided, which indicates the expression level.

[0030] The present invention further relates to a method for treating a patient having an abnormality in which the patient has an abnormality in the expression level of at least one of the target genes. Is this: (a) determining the expression level of said target gene in a population of individuals suffering from a hematological malignancy; higher than the second quartile; or (b) Good results for the treatment of hematological malignancies using CD123×CD3 bispecific molecules The second quartile of expression levels of the target gene in the population of non-responsive individuals. higher than the number; or (c) A successful treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression level of the target gene in a population of individuals who were not responsive was low. have a log2 fold change of at least about 0.4; or (d) Use of CD123×CD3 bispecific molecules for the treatment of hematological malignancies At least a second quartile of expression levels of the target gene in the population of responsive individuals. Within a quantile, An embodiment of the above method is provided, which indicates the expression level.

[0031] The present invention further relates to a method for treating a patient having an abnormality in which the patient has an abnormality in the expression level of at least one of the target genes. Is this: (a) determining the expression level of said target gene in a population of individuals suffering from a hematological malignancy; higher than the third quartile; or (b) Good results for the treatment of hematological malignancies using CD123×CD3 bispecific molecules The third quartile of expression levels of the target gene in the population of non-responsive individuals. higher than the number; or (c) A successful treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression level of the target gene in a population of individuals who were not responsive was low. have a log2 fold change of at least about 0.6, An embodiment of the above method is provided, which indicates the expression level.

[0032] The present invention further provides a method of treating a hematological malignancy, the method comprising: (a) Employing the method of any one of the above-described embodiments, a patient is diagnosed with a hematological malignancy. -CD123×CD3 BISPECIFIC MOLECULES FOR TREATING SUCCESSFUL BODY OBJECTS determining whether (b) administering to the patient a treatment if the patient is determined to be a favorable responder to such treatment; administering to said patient a therapeutic dosage of said CD123×CD3 bispecific molecule. Including, Administration of the CD123×CD3 bispecific molecule inhibits the progression of hematological malignancies in the patient. Stimulates cell killing.

[0033] The present invention further relates to detecting one or more of said markers in a cell sample obtained from said patient after initiation of said treatment.

[0023] The embodiment of the above method further comprises the step of evaluating the expression of the target gene on one or more occasions. provide.

[0034] The invention further provides an embodiment of the above method, wherein the cell sample is a bone marrow or blood sample. In particular, the present invention provides an embodiment of the above method, wherein the cell sample is a bone marrow sample.

[0035] The present invention further relates to a method for determining the expression level of one or more target genes in a bone marrow sample of the patient. The present invention further provides an embodiment of the above method, further comprising detecting one or more In one embodiment, the method further comprises detecting the expression level of a reference gene on the do.

[0036] The present invention further relates to the use of a method for the treatment of bone marrow necrosis factor (BNC) in said patients, in particular prior to administration of the CD123xCD3 bispecific molecule. The expression levels of the one or more target genes and / or the one or more reference genes in the sample. In accordance with another aspect of the present invention, there is provided an embodiment of the above method, comprising detecting a

[0037] The present invention further relates to the assessment of expression or the administration of CD12 The determination of whether a subject is a favorable responder to the use of the 3×CD3 bispecific molecule is: (a) detecting, using a gene expression platform, each of the target genes in one or more cell samples; determining the gene expression level of (b) comparing the expression level of the target gene with the expression level of one or more reference genes; P The present invention provides an embodiment of the above method, wherein the method is performed by:

[0038] The present invention further relates to the assessment of expression or the administration of CD12 The determination of whether a subject is a favorable responder to the use of the 3×CD3 bispecific molecule is: (a) In a gene expression platform, the raw R determining a level of NA, said gene expression platform being a House K a reference gene set of matching genes; and (b) using the measured RNA level of the internal reference gene to measure the RNA level of the target gene; assigning a relative expression value to each of said determined raw RNA levels. The present invention provides an embodiment of the above method, wherein the method is performed by:

[0039] The present invention further relates to a method for the detection of one or more target genes comprising: (a) one or more of CXCL9, CXCL10, CXCL11, and STAT1; and / or (b)CCL5, CD27, CD274, CD276, CD8A, CMKLR1, CX CL9, CXCR6, HLA‐DQA1, HLA‐DRB1, HLA‐E, IDO1, L AG3, NKG7, PDCD1LG2, PSMB10, STAT1, and TIGIT and / or (c)AREG, CSF3, CXCL1, CXCL2, CXCL3, CCL20, FO One or more of SL1, IER3 (NM_003897.4), IL6, and PTGS2 above; and / or (d) one or more of CCL2, CCL3 / L1, CCL4, CCL7, and CCL8 above; and / or (e) MAGEA3 / A6, MAGEA1, MAGEA12, MAGEA4, MAGE B2, MAGEC1, and MAGEC2; and / or (f) APOL6, DTX3L, GBP1, IFI16, IFI27, IFI35, I FI6, IFIH1, IFIT1, IFIT2, IFIT3, IFITM1, IFITM 2, IRF1, IRF9, ISG15, MX1, OAS1, OAS2, PARP9, PS one or more of MB9, STAT2, TMEM140, and TRIM21; and / or teeth (g) one or more of PSMB8, PSMB9, and PSMB10; and / or (h) IL-10; and / or (i) CD274; and / or (j)PDCD1LG2

[0023] In one embodiment of the method, the method comprises:

[0040] The present invention further relates to a method for the preparation of a method for treating a chronic inflammatory bowel disease, comprising administering to a subject a subject the steps of:

[0023] In one embodiment of the method, the method comprises:

[0041] The present invention further relates to a method for the treatment of a cancer, comprising administering to a patient a therapeutically effective amount of at least one of the following reference genes: ABCF1, G6PD, NRDE2, O AZ1, POLR2A, SDHA, STK11IP, TBC1D10B, TBP, and U BB。 BB.

[0042] The present invention further comprises determining a gene signature score for the one or more target genes. In certain embodiments of the invention, the gene signature The score from the raw RNA level for each of the target genes is: (a) Gene expression platform including a reference gene set of housekeeping genes The raw RNA level for each of the target genes in one or more cell samples is calculated using measuring; (b) comparing each of the measured raw RNA levels with the geometric mean of the housekeeping genes; normalizing to the mean and optionally further normalizing each RNA value to a standard; (c) log-transforming each normalized RNA value; (d) Each log-transformed RNA value was multiplied by the corresponding weighting factor to obtain the weighted RNA value. generating (e) The weighted RNA values ​​are summed together, optionally with an adjustment constant, to produce a single gene Steps for generating child signature scores The amount of the charge is determined by a process including:

[0043] Preferably, the gene signature comprises the target genes provided in Tables 6 and 12A-12G. In a specific embodiment of the present invention, the weighting coefficients are determined using the weighting coefficients in Tables 6 and 12A-1. In certain embodiments of the present invention, an adjustment factor is added to each score. In certain embodiments, the adjustment factors are those provided in Tables 6 and 12B-12G. be.

[0044] The present invention particularly relates to a gene signature score comprising: (a) IFN gamma signaling signature; (b) tumor inflammation signature; (c) bone marrow inflammatory signature; (d) inflammatory chemokine signature; (e) MAGE signature; (f) IFN downstream signaling signature; (g) immune proteasome signature; (h) IL‐10 signature; (i) PD-L1 signature; and / or (j) PD-L2 signature In one embodiment, the method further comprises determining whether the at least one of the following is true:

[0045] The present invention further comprises: (a) detecting one of the target genes in a population of individuals suffering from the hematological malignancy; greater than the first quartile of the score for the gene signature calculated from expression levels of loud; or (b) Good results for the treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression levels of one or more of the target genes in a population of non-responsive individuals greater than the first quartile of the score for the gene signature calculated from the bell; or (c) A successful treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression levels of one or more of the target genes in a population of non-responsive individuals A log 0.4 or greater for the score on the gene signature calculated from the bell have a fold change of 2; or (d) Use of CD123×CD3 bispecific molecules for the treatment of hematological malignancies The expression levels of one or more of the target genes in a population of individuals who were responsive At least within the first quartile of the score for the gene signature calculated from The patient gene signature score was used to predict the efficacy of treatment with the CD123×CD3 bispecific molecule. In one embodiment of the method, a test result is indicative of a relatively favorable patient response to the treatment.

[0046] The present invention further comprises: (a) detecting one of the target genes in a population of individuals suffering from the hematological malignancy; greater than the second quartile of the score for the gene signature calculated from expression levels of loud; or (b) Good results for the treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression levels of one or more of the target genes in a population of non-responsive individuals greater than the second quartile of the score for the gene signature calculated from the bell; or (c) A successful treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression levels of one or more of the target genes in a population of non-responsive individuals A log 0.5 or greater for the score on the gene signature calculated from the log have a fold change of 2; or (d) Use of CD123×CD3 bispecific molecules for the treatment of hematological malignancies The expression levels of one or more of the target genes in a population of individuals who were responsive At least within the second quartile of the score for the gene signature calculated from The patient gene signature score was used to predict the efficacy of treatment with the CD123×CD3 bispecific molecule. In one embodiment of the method, a test result is indicative of a relatively favorable patient response to the treatment.

[0047] The present invention further comprises: (a) detecting one or more of the target genes in a population of individuals suffering from a hematological malignancy; Greater than the third quartile of the score for the gene signature calculated from the expression levels above or (b) Good results for the treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression levels of one or more of the target genes in a population of non-responsive individuals greater than the third quartile of the score for the gene signature calculated from the bell; or (c) A successful treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression levels of one or more of the target genes in a population of non-responsive individuals A log 0.6 or greater for the score on the gene signature calculated from the bell With a fold change of 2, The patient gene signature score was used to predict the efficacy of treatment with the CD123×CD3 bispecific molecule. In one embodiment of the method, a test result is indicative of a relatively favorable patient response to the treatment.

[0048] The present invention further comprises: (a) the gene signature is an IFN gamma signaling signature, A patient gene signature score of approximately 2.5 was obtained for the CD123×CD3 bispecific molecule. is indicative of a relatively favorable patient response to the treatment used; and / or (b) the gene signature is a tumor inflammation signature, and is at least about 5.5 The gene signature score is a predictor of response to treatment with the CD123×CD3 bispecific molecule. and / or is indicative of a relatively favorable patient response; (c) the gene signature is an IFN downstream signaling signature, and comprises at least A patient gene signature score of approximately 4.5 was obtained using the CD123×CD3 bispecific molecule. is an indicator of a relatively favorable patient response to a given treatment. An embodiment of the above method is provided.

[0049] The present invention further relates to a method for determining whether the gene signature is an IFN gamma signaling signature, a tumor An inflammatory signature, or an IFN downstream signaling signature, and: (a) detecting one or more of the target genes in a population of individuals suffering from a hematological malignancy; Greater than the first quartile of the score for the gene signature calculated from the expression levels above or (b) Good results for the treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression levels of one or more of the target genes in a population of non-responsive individuals greater than the first quartile of the score for the gene signature calculated from the bell; or (c) A successful treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression levels of one or more of the target genes in a population of non-responsive individuals A log 0.4 or greater for the score on the gene signature calculated from the bell have a fold change of 2; or (d) Use of CD123×CD3 bispecific molecules for the treatment of hematological malignancies The expression levels of one or more of the target genes in a population of individuals who were responsive At least within the first quartile of the score for the gene signature calculated from The patient gene signature score was used to predict the efficacy of treatment with the CD123×CD3 bispecific molecule. In one embodiment of the method, a test result is indicative of a relatively favorable patient response to the treatment.

[0050] The present invention further relates to a method for determining whether the gene signature is an IFN gamma signaling signature, a tumor An inflammatory signature, or an IFN downstream signaling signature, and: (a) detecting one or more of the target genes in a population of individuals suffering from a hematological malignancy; greater than the second quartile of the score of the gene signature calculated from the expression levels above; or (b) Good results for the treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression levels of one or more of the target genes in a population of non-responsive individuals greater than the second quartile of the score for the gene signature calculated from the bell; or (c) A successful treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression levels of one or more of the target genes in a population of non-responsive individuals A log 0.5 or greater for the score on the gene signature calculated from the log have a fold change of 2; or (d) Use of CD123×CD3 bispecific molecules for the treatment of hematological malignancies The expression levels of one or more of the target genes in a population of individuals who were responsive The score of the gene signature calculated from the above is at least within the second quartile. The patient gene signature score was used to predict the efficacy of treatment with the CD123×CD3 bispecific molecule. In one embodiment of the method, a test result is indicative of a relatively favorable patient response to the treatment.

[0051] The present invention also provides gene expression sequences that are characteristic of immune-enhancing and IFN-gamma-dominant tumor microenvironments. Patients exhibiting the syndrome are compared to those treated with the CD123×CD3 bispecific molecule. Provided are embodiments of the above methods that are indicative of a relatively favorable patient response.

[0052] The present invention further relates to a method for producing said CD123×CD3 bispecific molecule comprising administering to said subject a subject the bispecific molecule comprising an scFv. Provided are embodiments of the above methods, which are antibodies or bispecific molecules.

[0053] The present invention further relates to a method for the preparation of said CD123×CD3 bispecific molecule, , XmAb14045, or APVO436.

[0054] The present invention further provides that the CD123×CD3 bispecific molecule comprises two, three or four polypeptides. The embodiment of the above method is a covalent bispecific diabody having a polypeptide chain. provide.

[0055] The present invention further relates to a CD123×CD3 bispecific molecule comprising: (a) a first polypeptide chain having the amino acid sequence of SEQ ID NO:21; and (b) a second polypeptide chain having the amino acid sequence of SEQ ID NO:23. wherein the first polypeptide chain and the second polypeptide chain comprise are covalently bonded to one another by a disulfide bond.

[0056] The present invention further relates to a method for treating a hematological malignancy in a patient, the method comprising the steps of: acute myeloid leukemia (AML); chronic myeloid leukemia (CM); Leukemia (CML); Blast crisis of CML; Abelson oncogene associated with CML (Bc r-ABL translocation; myelodysplastic syndrome (MDS); acute B-lymphoblastic leukemia (B-AL) L); acute T-lymphoblastic leukemia (T-ALL); chronic lymphocytic leukemia (CLL); Richter's syndrome; transformation of Richter's syndrome in CLL; hairy cell leukemia (HCL); blastic Plasmacytoid dendritic cell neoplasm (BPDCN); mantle cell lymphoma (MCL) and small lymphoma Non-Hodgkin's lymphoma (NHL), including splenomegalymphoma (SLL); Hodgkin's lymphoma, all and Burkitt's lymphoma. Provide a form.

[0057] The present invention further relates to a method for treating hematologic malignancies in patients with AML, MDS, BPDCN, or T-AL. L is

[0058] The present invention further relates to a method for treating hematologic malignancies in patients with hematologic malignancies that are refractory to chemotherapy (CTX). , e.g., refractory to cytarabine / anthracycline-based cytotoxic chemotherapy; or refractory to hypomethylating agent (HMA) chemotherapy. do.

[0059] The present invention further relates to a method for determining the expression level of CD123 in blast cells (cancer cells) by comparing the expression level of CD123 in normal peripheral blood mononuclear cells (PBMCs) with that in normal peripheral blood mononuclear cells (MBCs). Compared to corresponding baseline levels of CD123 expressed by cells (PBMCs) An embodiment of the above method is provided, further comprising the step of determining.

[0060] The present invention further provides that the level of expression can be determined by measuring cell surface expression of CD123. The present invention further provides an embodiment of the above method, wherein the cell surface expression of CD123 is determined by The method of claim 1, wherein expression is increased by at least about 20% relative to the baseline level of expression. The present invention further provides a method for the treatment of atopic dermatitis, comprising administering to a patient a therapeutically effective amount of CD123 x CD3 bilayer as described above. and an embodiment of the method, which enhances the patient's response to treatment with a specific molecule. do.

[0061] The present invention further relates to a method for treating a chronic pulmonary disease comprising administering to a patient a therapeutically effective amount of said CD123×CD3 bispecific molecule, said effective amount being 30, 100, 200, 350, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3500, 4000, 5000 , 300, and 500 ng / kg of patient body weight / day. An embodiment is provided.

[0062] The present invention further provides an embodiment of the above method, wherein the therapeutic dosage is administered as a continuous infusion. The present invention further provides a therapeutic dose of 30 ng / kg / day administered by continuous infusion over a period of three days. After administration, a therapeutic dose of 100 ng / kg / day will be administered by continuous infusion for 4 days. The present invention further provides an embodiment of the above method, wherein the therapeutic dosage is administered by continuous infusion. Embodiments of the above methods are provided further comprising administering 500 ng / kg / day.

[0063] The invention further provides embodiments of all of the above methods, wherein the patient is a human patient. [Brief description of the drawings]

[0064] [Figure 1A-1C] 1A-1C show the overall structure of an exemplary diabody molecule. FIG. 1A provides the structure of the first and second polypeptide chains of a two-chain CD123×CD3 bispecific diabody (also known as "DART-A" aka Flotetuzumab) with two epitope-binding domains, a Heterodimer-Promoting Domain, and a cysteine-containing linker. FIG. 1B-1C provides the overall structure of a CD123×CD3 bispecific diabody with two epitope-binding domains, composed of three polypeptide chains. Two of the polypeptide chains have CH2 and CH3 domains, such that the associated chains form all or part of an Fc domain. The polypeptide chains comprising the VL and VH domains further comprise a Heterodimer-Promoting Domain and a linker. Cysteine ​​residues may be present in the linker (FIGS. 1A and 1B) and / or in the Heterodimer-Promoting Domain (FIG. 1C). VL and VH domains that recognize the same epitope are shown using the same shading or fill pattern. [Diagram 2]FIG. 2 shows unsupervised hierarchical clustering of 46 IO360 signatures or cell types generated from baseline bone marrow biopsies obtained from patients who had a refractory response to conventional chemotherapy (e.g., patients who had a refractory response to a regimen of treatment with cytarabine administered in combination with daunorubicin (7+3 induction (Ref CTX)), or patients who had a refractory response to a regimen of treatment with the hypomethylating agents decitabine and azacitidine (Ref HMA, including patients with secondary AML)), and all patients who relapsed prior to flotetuzumab treatment (Relapse). Patient response to CD123×CD3 bispecific binding molecule therapy with flotetuzumab is also shown. Such responses were annotated as being an anti-leukemia response (Anti-leuk (A): this included patients showing complete response (CR), complete response with incomplete hematological improvement (CRi), morphological leukemia-free state (MLF), other anti-leukemia effect (OB), or partial response (PR)) or as non-response (NR: this included progressive disease / treatment failure (PD), and stable disease (SD)). To facilitate comparison between signatures, each IO360 signature score was rescaled to a scale of -3 to +3 within the score for this cohort. Immune exhaustion and immune enhancement gene signatures are outlined in bold within the Cluster 2 and Cluster 3 columns, respectively. [Figures 3A-3O]Figures 3A-3O show that chemotherapy-refractory and HMA-refractory patients have differential expression of multiple gene signatures. In particular, gene expression profiles of relapsed patients show features of immune exhaustion, while profiles of HMA-refractory (including HMA-refractory and secondary AML) patients show features of immune exhaustion and adaptive immune resistance, including upregulation of TIGIT, PD-L1 and Treg gene signatures, along with a trend toward increased gene signatures associated with exhausted CD8 T cells, compared to CTX-refractory patients. Figure 3A is a forest plot of the fold change difference between the changes of relapsed patients from all refractory (CTX and HMA). Figure 3B is a forest plot of the fold change difference between the changes of HMA-refractory patients from relapse, and Figure 3C is a forest plot of the fold change difference between the changes of CTX-refractory patients from HMA-refractory patients. Cluster 2 immune exhaustion (C2) and cluster 3 immune enhancement (C3) gene signatures are shown in Figures 3A and 3C. Myeloid (Figure 3D), macrophage (Figure 3E), neutrophil (Figure 3F), B cell (Figure 3G), IFN gamma (IFN-γ, Figure 3H), PD-L1 (Figure 3I), TIGIT (Figure 3J), CTLA-4 (Figure 3K), Th1 (Figure 3L), CTL (Figure 3M), CD8 T cell (Figure 3N), and cytotoxicity (Figure 3O) gene signature scores are plotted for immune-depleted (Depl.), immune-enriched (Enriched), and immune-exhausted (Exh.) profiles. [Figure 4] FIG. 4 shows the percent change (vs. baseline) in bone marrow blasts from 25 patients (relapsed (RL), CTX refractory (CTx), and HMA refractory (HMA) patients) following CD123×CD3 bispecific binding molecule therapy, as well as their response to therapy (CR, complete response; mCR, molecular CR; CRi, complete response with incomplete hematological improvement; MLF, morphological leukemia-free status; PR, partial response; SD, stable disease; PD, progressive disease / treatment failure). [Figure 5A-5C]Figures 5A-5C show that IFN gamma signaling signatures are increased at baseline in responders to flotetuzumab, and therefore predictive of positive response to CD123xCD3 bispecific binding molecule therapy. Figure 5A is a forest plot of baseline fold change differences between OR and NR patients, showing that IFN gamma signaling signatures were increased in baseline samples of OR patients (immune exhaustion (C2) and immune enhancement (C3) gene signatures are shown). Tumor inflammation signatures and IFN downstream signatures were also confirmed to be increased. Figure 5B shows the distribution of IFN gamma signaling signature scores in the NR and OR populations of patients (secondary AML; Ref CTX: refractory to CTX; Ref HMA: refractory to HMA, relapse: primary relapse). FIG. 5C shows the ROC curve (AUC=0.819) illustrating the predictive performance of the baseline IFN gamma signaling signature score. [Figure 6] FIG. 6 shows expression of gene signatures associated with cytotoxic cells or CD8+ T cells examined in RNA from bone marrow samples before treatment ("Base") or after the first cycle of treatment with flotetuzumab ("Cycle 1"). [Figure 7] FIG. 7 shows expression of CD123 in patient populations that are chemotherapy refractory, relapsed, refractory to HMA, or where HMA has failed. [Figure 8] Figure 8 shows the correlation between the level of PD-L1 expression on AML blasts of patients at baseline (BL) and whether they were early progressors or responders to CD123xCD3 bispecific binding molecule therapy. Data are presented as mean + distribution. [Figure 9]Figure 9 shows unsupervised hierarchical clustering of 48 IO360 signatures or cell types generated from baseline bone marrow biopsies obtained from patients who had a refractory response to conventional chemotherapy (P) and from patients who all relapsed prior to flotetuzumab treatment (R). Also shown is the patient response to CD123xCD3 bispecific binding molecule therapy with flotetuzumab. Such responses were annotated as being antileukemic responses (A: this included patients showing complete response (CR), complete response with incomplete hematological improvement (CRi), morphological leukemia-free state (MLF), other antileukemic effects (OB), or partial response (PR)) or as non-response (N: this included progressive disease / treatment failure (PD), and stable disease (SD)). To facilitate comparison between signatures, each IO360 signature score was rescaled to a scale of -3 to +3 within scores for this cohort. Stratification into immune infiltrated and immune depleted clusters is shown. [Figure 10] Figure 10 is a forest plot of baseline fold change differences between relapsed refractory patients with anti-leukemia response (OR) to CD123xCD3 bispecific binding molecule therapy with flotetuzumab and non-responders (NR), with multiple signatures increased in baseline samples from responders, including: IFN gamma signaling signature, IFN downstream signature, and tumor inflammation signature (each outlined in bold). Gene signatures that make up the IFN-dominant module are marked with an asterisk and are also increased in baseline samples from responders. [Figures 11A-11D]Figures 11A-11D show the distribution of scores of multiple gene signatures, with IFN modules of refractory (Refr.) and relapsed (Rel.) patients, OR patients shown as large white circles, and NR patients shown as small black dots. Comparisons were performed using Mann-Whitney U test for paired data. **P<0.01. Figure 11A shows the distribution of IFN gamma signaling signature scores. Figure 11B shows the distribution of IFN downstream signaling signature scores. Figure 11C shows the distribution of tumor inflammation signature (TIS) scores. Figure 11D shows the distribution of IFN dominance module (IFN module) scores. [Fig. 12A-12J] Figures 12A-12J show the distribution of the scores of the nine gene signatures constituting the IFN-dominant module and the tumor inflammation signature (TIS) in non-responders (NR) and responding patients (patients exhibiting antileukemia response) (OR). Figure 12A shows the IFN gamma signaling signature score; Figure 12B shows the IFN downstream signature score; Figure 12C shows the myeloid inflammation signature score; Figure 12D shows the immune proteasome signature score; Figure 12E shows the inflammatory chemokine signature score; Figure 12F shows the MAGE signature score; Figure 12G shows the PD-L1 signature score; Figure 12H shows the PD-L2 signature score; Figure 12I shows the IL10 signature score; Figure 12J shows the tumor inflammation signature (TIS) score. [Figures 13A-13K]Figures 13A-13K show ROC curves showing the predictive performance of the 9 gene signatures constituting the IFN-dominant module, the tumor inflammation signature (TIS), and the baseline scores for the IFN-dominant module for a group of 30 refractory / relapsed patients. Figure 13A shows the ROC curve for the IFN-gamma signaling signature score, with AUC=0.750. Figure 13B shows the ROC curve for the IFN-gamma downstream signaling signature score, with AUC=0.755. Figure 13C shows the ROC curve for the bone marrow inflammation signature score, with AUC=0.69. Figure 13D shows the ROC curve for the immune proteasome signature score, with AUC=0.505. Figure 13E shows the ROC curve for the inflammatory chemokine signature score, with AUC=0.764. Figure 13F shows the ROC curve for the MAGE signature score, AUC=0.736. Figure 13G shows the ROC curve for the PD-L1 signature score, AUC=0.699. Figure 13H shows the ROC curve for the PD-L2 signature score, AUC=0.727. Figure 13I shows the ROC curve for the IL10 signature score, AUC=0.745. Figure 13J shows the ROC curve for the TIS score, AUC=0.852. Figure 13K shows the ROC curve for the IFN dominant module score, AUC=0.806. [Figure 14A-14D] Figures 14A-14D show the distribution of scores for tumor inflammation (TIS, Figure 14A), IFN gamma signaling (Figure 14B), antigen processing machinery (APM, Figure 14C), and PD-L1 (Figure 14D) gene signatures examined in RNA from bone marrow samples before treatment ("Pre") or after the first cycle of treatment with flotetuzumab ("Post-C1"), with OR patients indicated by large white circles and NR patients indicated by small black dots. Comparisons were made using the Mann-Whitney U test for paired data. Pre=baseline. C1=cycle 1. **P<0.01. ***P<0.001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] The present invention includes hematological malignancies that are refractory to chemotherapy and / or hypomethylating agents. , to treat hematological malignancies such as acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) The present invention is directed to a method of administering a CD123×CD3 bispecific binding molecule to a patient. in an amount effective to stimulate killing of cells of said hematological malignancy in said patient. The present invention further relates to a method for detecting a cell sample derived from the patient, the cell sample being capable of detecting one or more target genes, The baseline level of expression of the gene, e.g., in a plurality of subjects suffering from the hematological malignancy, The expression level of the gene is higher than the baseline level in a reference population of individuals, or the reference gene Of interest is the embodiment of the above method, which shows high expression relative to the level of expression of the gene.

[0066] As mentioned above, chemotherapy resistance and relapse are common in children with acute myeloid leukemia (AML). It remains a significant cause of death in adults and children. Only 26.9% of patients are expected to survive beyond five years.

[0067] Treatment approaches for patients with acute myeloid leukemia (AML) have changed over the past 30 years. Standard frontline therapy is daunorubicin in combination with The two-drug regimen (the so-called 7+3 induction regimen, referred to herein as cytarabine) Hypomethylating Agents (abbreviated herein as "HMA") Decitabine and azacitidine are generally used in older patients or those who are not eligible for CTX regimens. However, literature estimates suggest that 10% of patients Up to 45% of cases have been shown to be refractory to standard frontline chemotherapy. Further intensification of conventional cytotoxic chemotherapy is needed to prevent the normal cell deaths commonly induced by these agents. This has been deemed unfeasible due to the severity of the acute and long-term side effects on cells. (Tasian, SK (2018 “Acute Myeloid Leukemia Chimeric Antigen Receptor T-Cell I mmunotherapy: How Far Up The Road Have We Traveled?,” Ther. Adv. Hematol. 9(6): 135-148; Przespolewski, A. et al. (2018) “Advances In Immunotherapy For Acute M 14(10):963-978; Shimabukuro-Vornhagen, A. et al. (2018) “Cytokine Release Syndrome,” J. Immunother. Cancer. 6(1):56 pp. 1-14; Milone, MC et al. (2018) “The Pharmacology of T Cell Therapies,” Mol. Ther. Methods Clin. Dev. 8:210-221; Dhodapkar, MV et al. (2017) “Hematologic Malign ancies: Plasma Cell Disorders,” Am. Soc. Clin. Oncol. Educ. Book. 37:561-568; K roschinsky, F. et al. (2017) “New Drugs, New Toxicities: Severe Side Effects Of Modern Targeted And Immunotherapy Of Cancer And Their Management,” Crit. Care 14;21(1):89).

[0068] Bispecific antibodies that bind to T cells stimulate the release of proinflammatory cytokines. Such cytokines act both by direct cytotoxicity and by activating immune cells to fight tumors. By recruiting them to the tumor site, their anti-leukemic efficacy can be increased (Hoseini, SS et al. (2107) “Acute Myeloid Leukemia Targets For Bispecific Antibodies,” Bl Food Cancer Journal 7:e522, doi:10.1038 / bcj.2017.2; pp. 1-12). Treatment with CD123×CD3 bispecific binding molecules, known as mabs, has been shown to be effective in treating relapsed / refractory (R It is being tested in a Phase 1 / 2 trial for AML. Despite the impressive ability of immunotherapy to selectively target cells (e.g. Koch, J. et al. (2017) “Recombinant Antibodies to Arm Cytotoxic Lymphocytes in Cancer Immunot herapy,” Transfus. Med. Hemother. 44:337-350; Lichtenegger, FS et al. (2017) “Recent Developments In Immunotherapy Of Acute Myeloid Leukemia,” J. Hematol. Oncol. 10:142, pp. 1-20), a dual-specific antibody that can target T cells to the location of hematologic malignancies. Efforts to employ heteromeric binding molecules have not been entirely successful.

[0069] Therefore, the discovery of new therapeutic strategies, including immunotherapy, remains a priority. AML patients with IFN-gamma-dominant tumor microenvironment ("TME") have relapse-free survival This is a significant advantage over standard induction chemotherapy. (Vadakekolathu, J. et al. (2017) “Immune Gene Expression Profile” filing in Children and Adults with Acute Myeloid Leukemia Identifies Distinct Ph enotypic patterns,” Blood 130:3942A).

[0070] As used herein, the term "gene expression signature" refers to a set of "Signature" refers to a specific cell type and / or biological process. It is intended to refer to the pattern of gene expression of a group of distinct genes (e.g. For example, Stenner, F. et al. (2018) “Cancer Immunotherapy and the Immune Response in Follicular Lymphoma,” Front. Oncol. 8:219 doi: 10.3389 / fonc.2018.00219, pages 1 -7; Cesano, A. et al. (2018) “Bringing The Next Generation Of Immuno-Oncology Biomarkers To The Clinic,” Biomedicines 6(14) doi: 10.3390 / biomedicines6010014, pages 1-11; Shrestha, G. et al. (2016) “The Value Of Genomics In Dissecting Th e RAS-Network And In Guiding Therapeutics For RAS-Driven Cancers,” Semin. Cell Dev. Biol. 58:108-117; Gingras, I. et al. (2015) “CCR 20th Anniversary Commenta ry: Gene-Expression Signature in Breast Cancer--Where Did It Start and Where Are We Now?,” Clin. Cancer Res. 21(21):4743-4746; Eberhart, C.G. (2011) “Molecula r Diagnostics In Embryonal Brain Tumors,” Brain Pathol. 21(1):96-104; Baylin, S .B. (2009) “Stem Cells, Cancer, And Epigenetics,” StemBook, ed. The Stem Cell Research Community, StemBook, doi / 10.3824 / stembook.1.50.1, pages 1-14; Asakura, M. et al. (2009) “Global Gene Expression Profiling In The Failing Myocardium,” Circ. J. 73(9):1568-1576; Shaffer, A.L. et al. (2001) “Signatures Of The Immun e Response,” Immunity 15(3):375-385; Staudt, L.M. et al. (2005) “The Biology O f Human Lymphoid Malignancies Revealed By Gene Expression Profiling,” Adv. Immu Nol. 87:163-208). The observed gene expression signatures and / or altered ( or unchanged) in one or more biological processes. can be used to assess the presence, nature, and / or severity of a pathogenic medical condition.

[0071] A central aspect of the present invention is the presence of an IFN-gamma dominant AML tumor microenvironment ("TME") Recognition of CD123×CD3 doublets, in contrast to predicting resistance to standard chemotherapy, Therapy using bispecific binding molecules (CD123×CD3 bispecific binding molecule flotetizumab) The present invention relates to the recognition that the use of ribozymes to predict favorable responses to therapeutic approaches, including those employing ribozymes. This is due, in part, to the specific subtype of patients with refractory hematologic malignancies (e.g., acute myeloid leukemia). Treatment of the population with a CD123×CD3 bispecific binding molecule (e.g., flotetuzumab). The members of this subpopulation are particularly suited to the immune system of the above members. These results demonstrate a gene expression signature characteristic of immune-enhanced and the presence of an IFN-gamma-dominant tumor microenvironment. They can be easily identified by their ability to

[0072] I. Patient populations particularly suitable for treatment with the CD123×CD3 bispecific binding molecules of the invention Identification of the group A. How to determine a "gene expression signature" A patient presented with a genetic mutation characteristic of immune enhancement and the presence of an IFN-gamma dominant tumor microenvironment. By determining whether the present signature is exhibited, the method and composition of the present invention are used Cells obtained from patients to identify those specifically suited for the treatment of hematological malignancies An RNA sample derived from the sample is evaluated to identify an RNA sequence that correlates with the above signature. We will determine whether the above is evidence of increased expression of the "target" genes. Such evaluation may utilize existing detection and / or measurement of gene expression, or incorporating one or more steps of detecting and / or measuring such gene expression. As used herein, the term "cell sample" refers to a sample that is "Cell sample" refers to a sample containing cells or an extract of cells.

[0073] Gene expression characteristic of immune-enhanced and IFN-gamma-dominant tumor microenvironments in some patients Sources of RNA or protein for use in determining whether a signature is present Any cell sample may be used as the target gene. The comparison of expression may be performed from bone marrow (BM) samples, or from blood samples, or from a population of patients or donors. This is performed using RNA obtained from blast cells (cancer cells). The expression levels employed were determined when RNA was obtained from such cells to provide a baseline expression level. A mean of the mean values ​​may be used (e.g., a geometric mean may be employed). In certain embodiments, the patient exhibits at least The expression level of one or more target genes in a population of individuals suffering from a hematological malignancy; and determining a level of expression of the compound in the blood of a patient suffering from said hematological malignancy, and The population of individuals who were not good responders (i.e., those receiving CD123×CD3 bispecific molecules) a population of individuals who have not responded well to the previously used hematological malignancy treatments); and and / or the patient is afflicted with the hematological malignancy at the time of determining the reference expression level and subsequently suffers from the hematological malignancy. A population of individuals whose tumors were successfully treated using the methods and compositions of the present invention (i.e., CD12 3×CD3 bispecific molecules have shown to be effective in treating hematological malignancies. The expression level of the target gene is compared to that of a population of individuals. When the group is a population suffering from a hematological malignancy, such a population is preferably Patients include individuals suffering from hematological malignancies. Such populations include individuals who have received chemotherapy relapsed after previous treatment with chemotherapy and / or is refractory to treatment with chemotherapy agents The comparison population may include individuals who have previously undergone CD123×CD3 doublet immunization (i.e., primary refractory). have had or have had a good response to treatment of hematological malignancies with specific molecules In the case of a population of individuals who have not been affected by the same hematological malignancy, said population is preferably The term includes as patients individuals who are

[0074] As used herein, expression of a gene may be measured relative to a baseline or other comparator (e.g., For example, the expression of such a gene in a population is at least about 10% More, at least about 20% more, at least about 30% more, at least about 40% more, less At least about 50% more, at least about 60% more, at least about 70% more, at least About 80% more, at least about 90% more, at least about 1.5 times more, at least about 2 times more, at least about 2.5 times more, at least about 3 times more, at least about 3.5 times more, At least about 4 times more, at least about 4.5 times more, at least about 5 times more, at least About 5.5 times more, at least about 6 times more, at least about 6.5 times more, at least about 7 times more more, at least about 7.5 times more, at least about 8 times more, at least about 8.5 times more, "Increased" means that the number of cases is at least about 9 times higher or at least about 10 times higher. Such an increase is expressed as the log2 fold change (log2-f sed). Regarding the increase in expression, for example, a l of 0.4 A log2 fold change of 0.5 corresponds to approximately 30% more expression; a log2 fold change of 0.5 corresponds to approximately 40% more expression. A log2 fold change of 0.6 corresponds to approximately 50% more expression; a log2 fold change of 0.7 corresponds to approximately 50% more expression. A log2 fold change of 0.8 corresponds to approximately 70% more expression. A log2 fold change of 0.9 corresponds to approximately 90% more expression; a log2 fold change of 1 corresponds to approximately 2 a log2 fold change of 1.5 corresponds to a 2.8-fold increase; a log2 fold change of 2 corresponds to a A log2 fold change of 2.5 corresponds to a 5.7-fold increase; A log2 fold change of 1.5 corresponds to an 8-fold increase; a log2 fold change of 3.5 corresponds to an 11.3-fold increase a log2 fold change of 4 corresponds to a 16-fold increase. Log2 fold changes are generally It is used to compare counts with array data and is also suitable for t-tests.

[0075] Alternatively, the increase may be referred to as a "gene signature score." In a paper published in Nature Communications, the authors describe a cluster of target genes that are expressed in a single cluster of 100 genes. Normalized to one or more housekeeping genes and / or internal standards, log-transformed, and overlaid The scores are then summed to generate a single gene signature score. Methods for calculating α are known in the art, and specific methods are described herein. (see Example 1 below).

[0076] As used herein, a gene signature (e.g., an IFN gamma signaling signature) is a gene signature that is a The expression of the gene signature score is at least about 2, or at least about 2 0.5, or at least about 3.0, or at least about 3.5, or at least about 4, or less At least about 4.5, or at least about 5, or at least about 5.5, or at least about 6 or greater than about 6.5.

[0077] Additionally, the patient's gene signature score is consistent with the overall population of individuals suffering from hematological malignancies. The first step of calculating the gene signature score from the expression levels of such target genes in the The second quartile of the gene signature score above the quartile (i.e., above the bottom 25%) higher than the third quartile of the gene signature score (i.e., greater than the bottom 50%) High (i.e., above the bottom 75%), above 85% of the gene signature scores, 90% It is said to be "enhanced" when the level exceeds 100% or exceeds 95%.

[0078] Additionally, patients' gene signature scores are associated with better response to treatment for hematological malignancies. A group of individuals who did not have hematological malignancies (e.g., using CD123×CD3 bispecific molecules) Such a population of individuals who have not responded well to treatment for chronic tumors The gene signature score calculated from the expression level of the target gene is higher than the first quartile. (i.e., greater than the bottom 25%), and higher than the second quartile of the gene signature score (i.e., above the third quartile of the gene signature score (i.e., the bottom 70%) 5%), more than 85%, more than 90% of the gene signature score above, or If it exceeds 95%, it is said to be "increasing."

[0079] Additionally, patients' gene signature scores are associated with better response to treatment for hematological malignancies. A group of individuals who did not have hematological malignancies (e.g., using CD123×CD3 bispecific molecules) Such a population of individuals who have not responded well to treatment for chronic tumors For a gene signature score calculated from the expression levels of the target genes, A log2 factor of 0.4, or at least about 0.5, or at least about 0.6, or even higher When there is a change, it is said to be "increasing."

[0080] The patient's gene signature score can also be determined by analyzing blood samples taken in the past using the methods and compositions of the present invention. A population of individuals whose malignancies have been successfully treated (e.g., using CD123×CD3 bispecific molecules) This is a population of individuals who had a good response to treatment for hematological malignancies. At least a first gene signature score calculated from the expression levels of such target genes More preferably, the gene signature score is within a quartile (i.e., within the bottom 25%). At least within the second quartile (i.e., the bottom 25%–50%) of Within a few percent (i.e., the bottom 50% to 75%), more than 85%, more than 90%, or 95% When it exceeds this, it is said to be "increasing."

[0081] The discovery of an increased gene signature score indicates that the CD123×CD3 bispecific molecule of the invention It is an indicator of a relatively favorable patient response to treatment of hematological malignancies with

[0082] In one embodiment, the patient is evaluated for the expression of the target gene, which is a predictor of the patient's health. The expression profile of the above genes during the patient's overall health or before the patient was diagnosed with a hematological malignancy. to baseline levels, or during or after the patient's chemotherapy treatment regimen. At some point during the course of the patient's treatment regimen with the CD123×CD3 bispecific binding molecule. The expression of the gene in the immune system is then "increased" or not. These results demonstrate a gene expression signature characteristic of immune-enhanced and the presence of an IFN-gamma-dominant tumor microenvironment. and thus are useful for the treatment of hematological malignancies using the methods and compositions of the present invention. These have been identified as being particularly suitable for

[0083] In a second embodiment, the patient is diagnosed with a hematological malignancy by assessing the level of expression of one or more target genes. The mean or weighted expression of one or more such target genes in a population of individuals suffering from By comparing with baseline levels, immune enhancement and IFN-gamma dominant tumor microenvironment These have been identified as exhibiting gene expression signatures characteristic of the presence of small environments, and thus Identified as particularly suitable for the treatment of hematological malignancies using the disclosed methods and compositions. Target genes that are expressed above the average or weighted baseline level are The methods and compositions of the present invention are described as exhibiting "increased" levels of expression in such patients. For example, the methods and compositions of the invention are particularly suitable for use in treating hematological malignancies. of expression of one or more target genes in a population of individuals suffering from a hematological malignancy. higher than the first quartile of expression levels of one or more target genes (i.e., greater than the bottom 25%); It is particularly suitable for use in the treatment of hematological malignancies in patients who exhibit "elevated" levels. The methods and compositions of the invention are directed to determining whether expression of one or more target genes is increased in patients suffering from hematological malignancies. higher than the second quartile of expression levels of one or more of the target genes in a population of individuals (i.e. Use in the treatment of hematological malignancies in patients with "elevated" levels of The methods and compositions of the present invention are particularly suitable for use in the detection of the expression of one or more target genes in blood. A method for determining the expression level of one or more target genes in a population of individuals suffering from a malignant tumor. Hematological abnormalities in patients with "elevated" levels higher than the third quartile (i.e., greater than the bottom 75%) The methods and compositions of the present invention are particularly suitable for use in the treatment of cancer. and determining whether or not expression of a target gene is associated with one or more of the above targets in a population of individuals suffering from a hematological malignancy. "Increased" refers to more than 85%, more than 90%, or more than 95% of the expression level of a gene. The subject invention is particularly suitable for use in the treatment of hematological malignancies in patients exhibiting elevated levels of IL-1.

[0084] In a third embodiment, the patient is provided with a method for determining the level of expression of one or more target genes in a patient previously treated with the present invention. A population of individuals with hematological malignancies not successfully treated using the methods and compositions of Good response to treatment of hematological malignancies with D123×CD3 bispecific molecules The mean or weighted mean of expression of one or more such target genes in a population of individuals who had not By comparing with baseline levels of immune enhancement and IFN-gamma dominant tumors, These have been identified as exhibiting gene expression signatures characteristic of the presence of a microenvironment, and thus The same are particularly suited for the treatment of hematological malignancies using the methods and compositions of the invention. Target genes expressed at or above the mean or weighted baseline level are designated as “increased The methods and compositions of the present invention are described as exhibiting "significant" levels of expression in such patients. The methods and compositions of the invention are particularly suitable for use in the treatment of hematological malignancies. For example, The expression of one or more target genes in a population of poorly treated individuals as described above The expression level of one or more of the target genes is higher than the first quartile (i.e., lower than the bottom 25%). and are particularly suitable for use in the treatment of hematological malignancies in patients exhibiting "elevated" levels. The methods and compositions of the present invention provide for successful treatment of the expression of one or more target genes, as described above. the second quartile of the expression level of said one or more target genes in a population of untreated individuals of hematological malignancies in patients with "elevated" levels higher than the baseline (i.e., greater than the bottom 50%) The methods and compositions of the present invention are particularly suitable for use in therapy. expression of one or more of the above targets in a population of poorly treated individuals as described above An "increased" level is one that is higher than the third quartile of expression levels of a gene (i.e., greater than the bottom 75%). The methods and methods of the present invention are particularly suitable for use in the treatment of hematological malignancies in patients with The composition includes a method for inhibiting the expression of one or more target genes in an undertreated individual as described above. More than 85% or more than 90% of the expression levels of one or more of the target genes in the population or greater than 95% of patients exhibiting "enhanced" levels of It is particularly suitable for use.

[0085] In a fourth embodiment, the patient is provided with a method for determining the level of expression of one or more target genes in a patient previously treated with the method of the present invention. A population of individuals successfully treated for hematological malignancies using the methods and compositions of the present invention (e.g., CD12 3×CD3 bispecific molecules have shown to be effective in treating hematological malignancies. The average or weighted basis of expression of one or more such target genes in a population of individuals By comparing with line levels, the presence of immune enhancement and an IFN-gamma dominant tumor microenvironment was The present invention provides a method and apparatus for identifying a gene expression signature that is characteristic of the present invention. and compositions have been identified as being particularly suitable for the treatment of hematological malignancies. Target genes that are expressed above the mean or weighted baseline level are considered to have "increased" expression. and the methods and compositions of the invention are described as exhibiting levels of hematological malignancies in such patients. For example, the methods and compositions of the invention are particularly suitable for use in the treatment of one or more targets. and detecting expression of one or more of the target genes in a population of successfully treated individuals as described above. The expression level of the target gene is at least within the first quartile (i.e., within the bottom 25%). The present invention is particularly suitable for use in treating hematological malignancies in patients exhibiting elevated levels of erythropoietin. The methods and compositions include a method for reducing the expression of one or more target genes in a successfully treated individual as described above. At least within the second quartile of the expression level of one or more of the target genes in the population (i.e. Use in the treatment of hematological malignancies in patients with "elevated" levels (<25-50%) The methods and compositions of the present invention are particularly suitable for use in the detection of expression of one or more target genes, as described above. and determining the expression level of said one or more target genes in a population of successfully treated individuals such as Patients with "elevated" levels, at least within the third quartile (i.e., the bottom 50%-75%) The methods and compositions of the present invention are particularly suitable for use in treating hematological malignancies in subjects. The expression of one or more target genes in a population of successfully treated individuals as described above. At least within the fourth quartile (i.e., higher than the bottom 75%) of the expression levels of the target genes and further suitable for use in treating hematological malignancies in patients exhibiting "elevated" levels. be.

[0086] However, whether expression of a target gene is "increased" depends on the expression of the target gene. The levels are compared to one or more markers that are not disease-associated or that do not show increased expression as a result of a disease state. By comparing the expression levels of a gene (a "reference" gene) It is preferable to determine whether a reference gene is expressed at multiple different levels. The geometric mean of the expression of the reference genes can be used to calculate the scaling coefficients. Multiply each gene by each sample value in the dataset, and take the n-th root of the product (n is the number of samples in the dataset). The geometric mean is the center of a set of numbers. It is similar to the arithmetic mean in that it shows a trend. However, unlike the arithmetic mean, it shows a trend. The mean is less sensitive to the magnitude of variation in count levels between probes. When comparing biological signatures across a set of one or more "reference genes," The geometric mean was used to normalize individual samples across the data set to obtain a biological Comparisons between genes are independent of differences due to technical variables such as sample mass input and sample quality. It is possible to do so.

[0087] A preferred "reference gene" is one that is constitutively expressed at the same level in normal and malignant cells. Housekeeping genes, such as genes required for maintaining basic cell functions (Eise nberg, E. et al. (2003) “Human Housekeeping Genes Are Compact,” Trends in Gene tics. 19(7):362-365; kon Butte, AJ et al. (2001) “Further Defining Housekeeping ng, Or "Maintenance," Genes Focus On 'A Compendium Of Gene Expression In Normal Human Tissues',” Physiol. Genomics. 7(2):95-96; Zhu, J. et al. (2008) “On The Nature Of Human Housekeeping Genes,” Trends in Genetics 24(10):481-484; erg, E. et al. (2013) “Human Housekeeping Genes, Revisited,” Trends in Genetic s. 29(10):569-574) are a preferred class of reference genes.

[0088] In a further embodiment, a patient is particularly susceptible to treatment with CD123×CD3 binding molecule therapy. The suitability determination further includes: (a) Steps to assess the level of CD123 expression in the patient's blast cells (cancer cells). The levels are measured before or at some point during the patient's chemotherapy treatment regimen. , associated with expression of CD123 in blast cells of the above patients, or in relapsed individuals or Associated with CD123 expression on blast cells in a population of individuals refractory to HMA therapy and compared to corresponding baseline levels of CD123. The findings suggest that CD123×CD3 combination therapy may be beneficial for patients with hematological malignancies. Determination of CD123 expression is a further indication of patient suitability. by assessing the presence of RNA or expression of CD123 in cell lysates or extracts. Alternatively, determining CD123 expression can be accomplished by assessing the presence of CD123 By assessing the presence of CD123 molecules arranged on the cell surface of expressing cells (e.g., blast cells), This can be achieved by increasing the expression of CD123 (e.g. At least a 10% increase in expression, at least a 15% increase in expression, at least a 20% increase in expression % increase in expression, at least a 25% increase in expression, at least a 30% increase in expression, or (at least 40% increase) is a promising candidate for CD123×CD3 combination therapy for hematological malignancies. It is an indicator of the patient's suitability to receive; and / or (b) assessing the level of PD-L1 expression in the patient's blast cells (cancer cells); In certain embodiments, the level of PD-L1 expression is measured across a sample of patient blast cells. The percentage of blast cells expressing PD-L1 will be assessed by evaluating In the present study, the level of PD-L1 expression in the blast cells of a patient was compared with that of the chemotherapy treatment of the patient. Compared with PD-L1 expression in blast cells of these patients at time points over the course of the regimen Thus, the level of PD-L1 expression on patient cells was determined by CD123 × CD The low expression of PD-L1 can be assessed prior to and / or following administration of any of the three binding molecules. The current findings provide a useful guide to the benefits of receiving CD123×CD3 combined molecular therapy for hematological malignancies. The finding of high PD-L1 expression is an indication of patient suitability for hematological malignancies. Combination of CD123×CD3 binding molecule therapy with antagonists of the PD-1 / PD-L1 axis Determination of PD-L1 expression is an indicator of a patient's suitability to receive CD4+ chemotherapy. By assessing the presence of mRNA encoding PD-L1 or in cell lysates or This can be achieved by assessing the presence of PD-L1 in the extracts. Expression was determined by measuring PD-L1 expression on the cell surface of PD-L1-expressing cells (e.g., PBMCs). This may be achieved by assessing the presence of PD-L1 molecules. Increased expression of 1 (e.g., at least 10% of blast cells express, at least at least 15% of blast cells express; at least 20% of blast cells express; at least 20% of blast cells express 5% of blast cells express, at least 30% of blast cells express, or at least 40% of blast cells express 0% of patients) for hematological malignancies, and Patient suitability for receiving a combination with an antagonist of the PD-1 / PD-L1 axis is an indicator (see, for example, WO 2017 / 214092).

[0089] In a further embodiment, CD8+ T lymphocytes are treated with a CD123xCD3 bispecific molecule. Monitor for an increase in the proportion of CD8+ T lymphocytes in the tumor microenvironment during and / or after treatment. do.

[0090] In a further embodiment, the CD123×CD3 binding molecule therapy of the invention further comprises an anti-human PD- anti-human PD-L1 binding molecules, such as anti-human PD-L1 antibodies, or dyes having a human PD-L1 binding domain. The anti-human PD-L1 binding abody that may be used in accordance with this embodiment. Molecules include atezolizumab, avelumab, and durvalumab (e.g. (See U.S. Patent No. 9,873,740; U.S. Patent No. 8,779,108). Zumab (WHO Drug Information, 2015, Recommended INN: List 74, 29(3):387), Dupa Ruvalumab (WHO Drug Information, 2015, Recommended INN: List 74, 29(3):393-394 ), and avelumab (WHO Drug Information, 2015, Recommended INN: List 74, 30(1): The amino acid sequences of the complete heavy and light chains of 100-101) are known in the art.

[0091] In a further alternative embodiment, the CD123×CD3 binding molecule therapy of the invention further comprises an anti-human P Anti-human PD-1 binding molecules such as D-1 antibodies or diazonium complexes having a human PD-1 binding domain Anti-human PD-1 binding molecules that may be used according to this embodiment. Nivolumab (5C4, BMS-936558, ONO-4538, MDX-1 Also known as OPDIVO 106, a Bristol-Myers Squibb company. ), pembrolizumab (formerly known as lambrolizumab, MK-3475, SC Also known as H-900475, marketed by Merck as KEYTRUDA® ), EH12.2H7 (commercially available from BioLegend), and pidilizumab (CAS Registry Number 1 036730-42-3, also known as CT-011, CureTech), hPD-1 mAb 7(1.2)IgG4(P), and DART-I (International Publication No. 2017 / 01 No. 9846) (e.g., U.S. Pat. No. 5,952,136; U.S. Pat. No. No. 7,488,802; U.S. Patent No. 7,521,051; U.S. Patent No. 8,008,44 No. 9; U.S. Patent No. 8,088,905; U.S. Patent No. 8,354,509; U.S. Patent No. 8,552,154; U.S. Patent No. 8,779,105; U.S. Patent No. 8,900,58 No. 7; U.S. Patent No. 9,084,776; U.S. Patent Publication No. WO 2004 / 056875 No.; International Publication No. 2006 / 121168; International Publication No. 2008 / 156712; International Publication No. 2012 / 135408; International Publication No. 2012 / 145493; International Publication No. 013 / 014668;WO 2014 / 179664;WO 2014 / 194302; International Publication No. 2015 / 112800; International Publication No. 2017 / 0198 46; and WO 2017 / 214092).

[0092] 1. Exemplary "Target" Genes IFN-gamma activates primary response genes such as IRF, Fc gamma receptor (FCGR), and GBP (guanylate-binding protein), major histocompatibility complex (MHC) class I and class II II molecules, proteins involved in antigen presentation, antiviral proteins such as PKR, and It stimulates gene expression of over 200 genes, including OAS proteins (Boehm, U. et al. (1997) “Cellular Responses To Interferon-γ,” Annu. Rev. Immunol. 15:74 9-795; Schroder, K. et al. (2003) “Interferon-Gamma: An Overview Of Signals, Me chanisms And Functions,” J. Leukoc. Biol. 75(2):163-189).

[0093] Table 1 shows exemplary target genes and representative, non-limiting GenBan sequences for each gene. (Der, SD et al. (1988) “Identification of Genes Differentially Regulated By Interferon α, β, or γ Using Oligonucleotide Arrays,” Proc. Natl. Acad. Sci. (USA) 95:15623-15628; Schneider, WM et al (2014) “Interferon-Stimulated Genes: A Complex Web of Host Defenses,” Annu. Rev. Immunol. 32:513-545), and those disclosed in Schroder, K. et al. (2003) (“ Interferon-Gamma: An Overview Of Signals, Mechanisms And Functions,” J. Leukoc. Biol. 75(2):163-189; these references are incorporated herein by reference).

[0094] [Table 1] JPEG2024178163000002.jpg205156

[0095] As provided herein, the present invention relates to a method for treating a patient having an immune-enhanced and IFN-gamma dominant tumor microenvironment. A highly favorable gene expression signature for determining whether a patient had This is called the "IFN gamma signaling signature." IFN gamma signaling The signature genes were: CXCL9, CXCL10, CXCL11, and STAT1. (Table 6). The IFN gamma signaling signature may further include IFNG (e.g., For example, see NCBI sequence accession number NM_000619.2. Increased expression of the nulling signature indicates response to CD123×CD3 bispecific binding molecule therapy This is particularly relevant to the suitability of the patient for the procedure.

[0096] Additional suitable target genes can be added. Such additional target genes can be Using the EROME database, genes downstream of IFN-gamma could be easily identified. (Samarajiwa1, SA et al. (2009) “INTERFEROME: The Database Of Interferon Re ("Nucleic Acids Research 37: D852-D857"). The genes include PDCD1 (also referred to herein by the generic name PDL1), PDCD1LG 2 (also referred to herein by its generic name PDL2), IL10, CTLA4 (Table 13) and / or of genes present in the following gene signatures provided in the Examples below: One or more of the following: "Interferon (IFN) downstream signature" (the gene is "bone marrow inflammation signature" (the genes of which are listed in Table 12C); "inflammatory chemokine signature" (the genes of which are listed in Table 12D) ); "MAGES signature" (the genes of which are listed in Table 12E); and / or "Immunoproteasome signature" (the genes of which are listed in Table 12F).

[0097] In particular, the expression of multiple genes and signatures is classified as a "module." By assessing the population, we were able to determine the response of patients to CD123×CD3 bispecific binding molecule therapy. Patients with immune-infiltrated (immune-boosting) IFN-dominant tumor microenvironments can be assessed for suitability. One particularly preferred module that can be used to determine whether It is called the "IFN Dominant Module." Target genes associated with the FN dominant module include: PDL1, PDL2, IL10, C TLA4, as well as genes present in each of the following gene expression signatures: IF N-gamma signaling signature, interferon downstream signature, bone marrow inflammation signature , inflammatory chemokine signature, MAGES signature, and immune proteasome signature Necha (Table 10).

[0098] An IFN-dominant module has a module score of at least about 24, at least about 25, At least about 26, at least about 27, at least about 28, at least about 29, at least at least about 30, at least about 31, at least about 32, at least about 33, or at least about 35 is described as "increasing."

[0099] Additionally, the IFN-dominant module scores of patients are consistent with the population of individuals suffering from hematological malignancies. The IFN-dominant module score calculated from the expression levels of such target genes in IFN-dominant module score above the first quartile (i.e., above the bottom 25%) of IFN dominance module score above the second quartile (i.e., above the bottom 50%) 8 of the above IFN dominance module scores higher than the third quartile (i.e., greater than the bottom 75%) "Increased" is expressed when it is greater than 5%, greater than 90%, or greater than 95%. can be.

[0100] Additionally, patients' IFN-dominant module scores indicate a favorable response to treatment for hematological malignancies. A population of individuals who have not had a sexual relationship (e.g., blood samples using CD123×CD3 bispecific molecules) Such a population of individuals who had not responded well to treatment for hematologic malignancies ( The first quartile of the IFN-dominant module score calculated from the expression levels of such target genes the second quartile of the IFN dominance module score above the number (i.e., above the bottom 25%) Higher (i.e., greater than the bottom 50%) than the third quartile of the IFN dominance module score higher than 85% of the above IFN-dominant module scores (i.e., greater than the lower 75%); More than 90% or more than 95% is said to be "enhanced."

[0101] In addition, the IFN-dominant module score of a patient may be determined by the method and composition of the present invention. A population of individuals with successfully treated hematological malignancies (e.g., CD123×CD3 bispecific molecules) A population of individuals who had a good response to treatment of hematological malignancies with The IFN dominance module score calculated from the expression level of the target gene such as More preferably, both of the above IFN-dominant modules are within the first quartile (i.e., within the bottom 25%). at least within the second quartile of the HR score (i.e., the bottom 25%–50%), is within the third quartile (i.e., the bottom 50% to 75%), above 85%, or above 90% , or more than 95%.

[0102] Gene signatures associated with the IFN-dominant module (CD274, PDCD1LG2, IL10, CTLA4, IFN-gamma signaling signature, interferon downstream signature signature, bone marrow inflammation signature, inflammatory chemokine signature, MAGES signature, and immune proteasome signatures) separately, 3. Patient suitability for bispecific binding molecule therapy can be assessed.

[0103] As further provided herein, the patient is diagnosed with a disorder associated with a suppressed adaptive immune response in the tumor. The gene expression signature (herein referred to as the “Tumor Inflammation Signature”) that "Transfer Identification Signature (TIS)" or simply "Transfer Identification Signature (TIS)" As another set of highly favorable target genes, These include the following genes: CCL5, CD27, CD274, CD276, CD8 A, CMKLR1, CXCL9, CXCR6, HLA‐DQA1, HLA‐DRB1, H LA‐E, IDO1, LAG3, NKG7, PDCD1LG2, PSMB10, STAT 1, and / or TIGIT (Table 12A). Increased expression of tumor inflammatory signatures was associated with CD1 This is particularly relevant for patient suitability for 23xCD3 bispecific binding molecule therapy.

[0104] 2. Exemplary "reference" genes Housekeeping genes constitutively expressed at the same levels in normal and malignant cells Genes constitute one preferred class of reference genes. As housekeeping genes Genes involved in general gene expression (e.g., transcription factors, repressors, RNA splicers, etc.) isolating factor, translation factor, tRNA synthetase, RNA-binding protein, ribosomal protein Proteins, mitochondrial ribosomal proteins, RNA polymerase, protein processing binding factors, heat shock proteins, histones, cell cycle regulators, apoptosis, cancer genetics genes involved in metabolism (e.g., genes encoding metabolism, citric acid cycle, lipid metabolism, amino acid metabolism, NADH dehydrogenase, cytochrome P450 Chromium C oxidase, ATPases, lysosomal enzymes, proteasome proteins, genes encoding enzymes such as ribonuclease and thioreductase), and the integrity of the cell structure Genes involved in (e.g., cytoskeletal proteins, proteins involved in organelle synthesis, genes encoding mitochondrial proteins, etc.), as well as cell surface proteins (e.g. Cell adhesion proteins, ion channels and transporters, receptors, HLA / immunoglobulins (genes encoding phospho / cell recognition proteins, etc.) and kinase / signaling proteins These include genes involved in protein synthesis (e.g., growth factors, tissue necrosis factor, casein kinase, etc.). Suitable reference genes for this purpose include those encoding: Sterol regulatory element binding proteins (e.g. ATF1, ATF2, ATF4, ATF 6, ATF7, ATF7, BTF3, E2F4, ERH, HMGB1, ILF2, IER 2, JUND, TCEB2, etc.); Repressors (e.g. PUF60); RNA splicing proteins (e.g. BAT1, HNRPD, HNRPK, PA BPN1, SRSF3, etc.); Translation factors (e.g. EIF1, EIF1AD, EIF1B, EIF2A, EIF2AK 1, EIF2AK3, EIF2AK4, EIF2AK1, EIF2B2, EIF2B3, EIF2B4, EIF2S2, EIF3A, EIF3B, EIF3D, EIF3G, EI F3I, EIF3H, EIF3J, EIF3K, EIF3L, EIF3M, EIF3S5 , EIF3S8, EIF4A1, EIF4A2, EIF4A3, EIF4E2, EIF4 G1, EIF4G2, EIF4G3, EIF4H, EIF5, EIF5, EIF5A, E IF5AL1, EIF5B, EIF6, TUFM etc.); tRNA synthetases (e.g. AARS, AARS2, AARSD1434, CARS, CARS2, DARS, DARS2, EARS2614, FARS2, FARSA, FA RSB, GARS, HARS, HARS2, IARS, IARS2, KARS, LARS 2, MARS, MARS2, NARS, NARS2, QARS, RARS, RARS2, SARS, TARS, VARS2, WARS2, YARS, YARS2436, etc.); ·RNA-binding proteins (e.g. ELAVL1); Ribosomal proteins (e.g. RPL5, RPL8, RPL9, RPL10A, RP L11, RPL14, RPL25, RPL26L1, RPL27, RPL30, RPL3 2, RPL34, RPL35, RPL35A, RPL36AL, RPS5, RPS6, R PS6KA3, RPS6KB1, RPS6KB2, RPS13, RPS19BP1, RP S20, RPS23, RPS24, RPS27, RPN1, etc.); Mitochondrial ribosomal proteins (e.g. MRPL9, MRPL1, MRPL1 0, MRPL11, MRPL12, MRPL13, MRPL14, MRPL15, MRP L16, MRPL17, MRPL18, MRPL19, MRPL2, MRPL20, MR PL21, MRPL22, MRPL23, MRPL24, MRPL27, MRPL28, MRPL3, MRPL30, MRPL32, MRPL33, MRPL35, MRPL36 , MRPL37, MRPL38, MRPL4, MRPL40, MRPL41, MRPL4 2, MRPL43, MRPL44, MRPL45, MRPL46, MRPL47, MRP L48, MRPL49, MRPL50, MRPL51, MRPL52, MRPL53, M RPL54, MRPL55, MRPL9, MRPS10, MRPS11, MRPS12, MRPS14, MRPS15, MRPS16, MRPS17, MRPS18A, MRPS 18B, MRPS18C, MRPS2, MRPS21, MRPS22, MRPS23, M RPS24, MRPS25, MRPS26, MRPS27, MRPS28, MRPS30 , MRPS31, MRPS33, MRPS34, MRPS35, MRPS5, MRPS6 , MRPS7, MRPS9 etc.); RNA polymerases (e.g. POLR1C, POLR1D, POLR1E, POLR 2A, POLR2B, POLR2C, POLR2D, POLR2E, POLR2F, PO LR2G, POLR2H, POLR2I, POLR2J, POLR2K, POLR2L, POLR3C, POLR3E, POLR3GL, POLR3K etc.); Protein processing proteins (e.g. PPID, PPIE, PPIF, PPI G, PPIH, CANX, CAPN1, CAPN7, CAPNS1, NACA, NACA 2, PFDN2, PFDN4, PFDN5, PFDN6, SNX2, SNX3, SNX4 , SNX5, SNX6, SNX9, SNX12, SNX13, SNX17, SNX18, SNX19, SNX25, SSR1, SSR2, SSR3, SUMO1, SUMO3, etc.) ; Heat shock proteins (e.g. HSPA4, HSPA5, HSPA8, HSPA9, HSPA14, HSBP1, etc.); Histones (e.g. HIST1H2BC, H1FX, H2AFV, H2AFX, H2A FY, H2AFZ, etc.); Cell cycle proteins (e.g. ARHGAP35, ARHGAP5, ARHGDIA, ARHGEF10L, ARHGEF11, ARHGEF40, ARHGEF7, RAB1 0, RAB11A, RAB11B, RAB14, RAB18, RAB1A, RAB1B, RAB21, RAB22A, RAB2A, RAB2B380, RAB3GAP1, RAB 3GAP2, RAB40C, RAB4A, RAB5A, RAB5B, RAB5C, RAB 6A, RAB7A, RAB9A, RABEP1, RABEPK, RABGEF1, RAB GGTA, RABGGTB, CENPB, CTBP1, CCNB1IP1, CCNDBP 1, CCNG1, CCNH, CCNK402, CCNL1, CCNL2, CCNY, PP P1CA, PPP1CC, PPP1R10, PPP1R11, PPP1R15B, PPP 1R37, PPP1R7, PPP1R8, PPP2CA, PPP2CB552, PPP2 R1A, PPP2R2A, PPP2R2D, PPP2R3C, PPP2R4, PPP2R 5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP4C , PPP4R1, PPP4R2, PPP5C, PPP6C, PPP6R2, PPP6R3 , RAD1, RAD17, RAD23B, RAD50, RAD51C, IST1 etc.); · Apoptotic proteins (e.g. DAD1, DAP3, DAXX, etc.); Oncogene proteins (e.g. ARAF, MAZ, MYC, etc.); ·DNA repair / replication proteins (e.g. MCM3AP, XRCC5, XRCC6, etc.); Metabolic proteins (e.g. PRKAG1, PRKAA1, PRKAB1, PRKACA , PRKAG1, PRKAR1A, PRKRIP1, etc.); Carbohydrate metabolism proteins (e.g. ALDOA, B3GALT6, B4GALT3, B 4GALT5, B4GALT7, GSK3A, GSK3B, TPI1, PGK1, PGA M5, ENOPH1, LDHA, TALDO1, TSTA3); Citric acid cycle proteins (e.g. SDHA, SDHAF2, SDHB, SDHC , SDHD, etc.); Lipid metabolism proteins (e.g. HADHA, etc.); · Amino acid metabolism proteins (e.g. COMT, etc.); NADH dehydrogenases (e.g. NDUFA2, NDUFA3, NDUFA4, N DUFA5, NDUFA6, NDUFA7, NDUFA8, NDUFA9, NDUFA1 0, NDUFA11, NDUFA12, NDUFA13, NDUFAF2, NDUFAF 3, NDUFAF4, NDUFB2, NDUFB3, NDUFB4, NDUFB5, ND UFB6, NDUFB7, NDUFB10, NDUFB11, NDUFB8, NDUFB 9, NDUFC1, NDUFC2, NDUFC2, NDUFS5, NDUFV2, NDU FS2, NDUFS3, NDUFS4, NDUFS5, NDUFS6, NDUFS7, N DUFS8, NDUFV1, NDUFV2 etc.); Cytochrome C oxidases (e.g. COX4I1, COX5B, COX6B1, CO X6C, COX7A2, COX7A2L, COX7C, COX8, COX8A, COX1 1, COX14, COX15, COX16, COX19617, COX20, CYC1, UQCC, UQCR10, UQCR11, UQCRB, UQCRC1, UQCRC2, U QCRHL591, UQCRQ, ATPase, ATP2C1, ATP5A1, ATP5 B, ATP5C1, ATP5D, ATP5F1, ATP5G2, ATP5G3, ATP5 H, ATP5J, ATP5J2, ATP5J2, ATP5L, ATP5O, ATP5S, ATP5SL, ATP6AP1, ATP6V0A2, ATP6V0B, ATP6V0C, ATP6V0D1, ATP6V0E1, ATP6V1C1, ATP6V1D, ATP6V 1E1, ATP6V1F, ATP6V1G1, ATP6V1H, ATPAF2, ATPI F1 etc.); Lysosomal proteins (e.g. CTSD, CSTB, LAMP1, LAMP2, M6 PR etc.); Proteasome proteins (e.g. PSMA1, PSMA2, PSMA3, PSMA 4, PSMA5, PSMA6, PSMA7, PSMB1, PSMB2, PSMB3, PS MB4, PSMB5, ​​PSMB6, PSMB7, PSMC2, PSMC3, PSMC4, PSMC5, PSMC6, PSMD1, PSMD10, PSMD11, PSMD12, P SMD13, PSMD14, PSMD2, PSMD3, PSMD4, PSMD5, PSM D6, PSMD7, PSMD8, PSMD9, PSME2, ​​PSME3, PSMF1, P SMG2, PSMG3, PSMG4591, UBA1, UBA2, UBA3, UBA5, UBA52, UBAC2, UBALD1, UBAP1, UBAP2L, UBB, UBC, UBE2A, UBE2B, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2F, UBE2G2, UBE2H, UBE2I, U BE2J1, UBE2J2, UBE2K, UBE2L3, UBE2M, UBE2N, UB E2NL989, UBE2Q1, UBE2R2, UBE2V1, UBE2V2, UBE2 W, UBE2Z, UBE3A, UBE3B, UBE3C, UBE4A, UBE4B, US P10, USP14, USP16, USP19, USP22, USP25, USP27X 073, USP33, USP38, USP39, USP4, USP47, USP5, US P7, USP8, USP9X590 etc.); Ribonucleases (e.g. RNH, etc.); Thioreductases (e.g. TXN2, TXNDC11, TXNDC12, TXNDC 15, TXNDC17, TXNDC9, TXNL1, TXNL4A, TXNL4B, TX NRD1, cytoskeleton, ANXA6, ANXA7, ARPC1A, ARPC2, ARPC5 L, CAPZA2, CAPZB, RHOB, RHOT1, RHOT2, TUBB, WDR 1st prize); Proteins involved in organelle synthesis (e.g., BLOC1S1, BLOC1S2, B LOC1S3, BLOC1S4, BLOC1S6, AP1G1, AP1M1, AP2A1 , AP2A2, AP2M1, AP2S1, AP3B1, AP3D1, AP3M1, AP3 S1, AP3S2, AP4B1, AP5M1, ANXA6, ANXA7, AP1B1, C LTA, CLTB, CLTC, etc.); · Mitochondrial proteins (e.g. MTX2, etc.); Cell surface proteins (e.g. AP2S1, CD81, GPAA1, LGALS9, M GAT2, MGAT4B, VAMP3, etc.); Cell adhesion proteins (e.g., CTNNA1, CTNNB1, CTNNBIP1, CT NNBL1, CTNND1458, etc.); Ion channel and transporter proteins (e.g., ABCB10, ABCB7, ABCD3, ABCE1, ABCF1, ABCF2, ABCF3, CALM1, MFSD 11, MFSD12, MFSD3, MFSD5, SLC15A4, SLC20A1, SL C25A11, SLC25A26, SLC25A28, SLC25A3, SLC25A3 2, SLC25A38, SLC25A39, SLC25A44, SLC25A46, SL C25A5, SLC27A4, SLC30A1, SLC30A5, SLC30A9, SL C35A2, SLC35A4, SLC35B1, SLC35B2, SLC35C2, SL C35E1, SLC35E3, SLC35F5, SLC38A2, SLC39A1, SL C39A3, SLC39A7, SLC41A3, SLC46A3, SLC48A1, receptor body, ACVR1, ACVR1B, CD23, etc.); HLA / immunoglobulin / cell recognition proteins (e.g. BAT1, BSG, MIF, TAPBP etc.); Kinase / signaling proteins (e.g. ADRBK1, AGPAT1, ARF1 , ARF3, ARF4, ARF5, ARL2, CSF1, CSK, DCT, EFNA3, FKBP1A, GDI1, GNAS1, GNAI2, HAX1, ILK, MAPKAPK 2, MAP2K2, MAP3K11, PITPNM, RAC1, RAP1B, RAGA, STK19, STK24, STK25, YWHAB, YWHAH, YWHAQ, YWHA Z, etc.); Growth factors (e.g. AIF1, HDGF, HGS, LTBP4, VEGFB, ZFP3 6L1, tissue necrosis factor, CD40, casein kinase, CSNK1E, CSNK2B, etc.) and Miscellaneous proteins (e.g. ALAS1, ARHGEF2, ARMET, AES, BE CN1, BUD31, CKB, CPNE1, ENSA, FTH1, GDI2, GUK1, HPRT, IFITM1, JTB, MMPL2, NME2, NONO, P4HB, PRD X1, PTMA, RPA2, SULT1A3, SYNGR2, TTC1, C11Orf1 3, C14orf2, C21orf33, SPAG7, SRM, TEGT, DAZAP2 , MEA1, etc.).

[0105] Preferred housekeeping genes include those listed in Table 2. Table 2 also provides representative and non-restrictive NCBI accession numbers for each gene. Any combination or subcombination of these genes (and / or their splice variants) A combination of these may also be used.

[0106] [Table 2] JPEG2024178163000004.jpg99155

[0107] The following reference genes are particularly preferred: ABCF1, G6PD, NRDE2, OAZ1, P OLR2A, SDHA, STK11IP, TBC1D10B, TBP, and UBB.

[0108] 3. Exemplary Methods for Assessing Expression of Target and Reference Genes The level of expression of one or more target genes relative to a baseline or one or more reference genes To determine the amount of mRNA in the cell sample corresponding to each target gene being evaluated, Determine and normalize against baseline or mRNA expression corresponding to one or more reference genes Any suitable method may be employed to accomplish such analysis. The method used was the nCOUNTER® analysis system (NanoString Technology). The company will use the nCOUNTER (registered trademark) analysis system. In this system, the RNA from a sample is subjected to detection with a gene-specific reporter probe and a capture probe. To allow the sample RNA to hybridize to the probe in the presence of Each reporter probe carries a fluorescent barcode and each The capture probe captures the hybridized complex onto a rigid support for data collection. After hybridization, the excess probe is removed and the top The support is scanned under an automated fluorescent microscope. The barcodes for each target molecule are counted. Data Analysis The analysis is preferably carried out using nSolver® 4.0 analysis software (NanoStri The method is carried out using a fusion technology provided by NG Technologies, Inc. The data presented are based on the PanCancer IO360™ Gene Expression Panel Kit ( NanoString Technologies, Inc.) The kit detects 770 different genes (tumor, tumor microenvironment, and immune response boundaries) 750 genes covering major pathways in cerebrospinal fluid (CSF) and 20 genes that can be used to normalize data It contains a set of probes for internal reference genes (Table 5). A is calculated as follows: The raw data counts for each gene were compared against the housekeeping data selected for each sample ( HK) genes (e.g., ABCF1, NRDE2, G6PD, OAZ1, POLR2A, S DHA, STK11IP, TBC1D10B, TBP, UBB) To become. Next, the HK normalized data is compared with the IO360 panel standard, preferably with the same color as the test sample. Normalize against a standard run on the cartridge. Next, each normalized gene count is log-transformed. After normalization and logarithmic transformation, each gene is multiplied by a weight value. These weighted numbers are then summed to produce a single score. The final calculated score is A constant adjustment factor is added to the score (for example, +7). The adjustment factor is used to adjust the score range from 0 to 10. Derived from the lowest observed score (from TCGA and / or cell line analysis) to exceed can.

[0109] In Table 3, genes are classified as follows: column 1: gene name; column 2: internal reference gene; Column 3: Cell type (B: B cells; CD8: CD8T cells; Cyto: cytotoxic cells; C D45: CD45 expressing cells; CD56d: NK CD56 dim cells;DC: dendritic cells; eCD8: exhausted CD8 cells; M: macrophages; MC: mast cells; N: neutrophils; N K: NK cells; T: T cells; Th1: Th1 cells; Treg: Treg cells); Column 4: Cancer Antigen release; Column 5: Cancer antigen presentation; Column 6: T cell priming and activation; Column 7: Tumor Localization of immune cells against cancer; Column 8: stromal factors; Column 9: recognition of cancer cells by T cells; Column 10: Cancer cell killing; Column 11: Myeloid cell activity; Column 12: NK cell activity; Column 13: Cell cycle and proliferation growth; column 14: tumor intrinsic factors; column 15: immunometabolism; column 16: common signaling pathways. Genes associated with additional pathways and cell types that constitute specific gene expression signatures, as well as Methods for calculating the gene expression signature score are provided in the Examples below.

[0110] [Table 3] JPEG2024178163000006.jpg240164JPEG2024178163000007.jpg235164JPEG2024178163000008.jpg23816 2JPEG2024178163000009.jpg238167JPEG2024178163000010.jpg240170JPEG2024178163000011.jpg23916 4JPEG2024178163000012.jpg248167JPEG2024178163000013.jpg245167JPEG2024178163000014.jpg24716 5JPEG2024178163000015.jpg241168JPEG2024178163000016.jpg240166JPEG2024178163000017.jpg24016 5JPEG2024178163000018.jpg238164JPEG2024178163000019.jpg244165JPEG2024178163000020.jpg24016 2JPEG2024178163000021.jpg239164JPEG2024178163000022.jpg239165JPEG2024178163000023.jpg24316 6JPEG2024178163000024.jpg240164JPEG2024178163000025.jpg242164JPEG2024178163000026.jpg23816 3JPEG2024178163000027.jpg239164JPEG2024178163000028.jpg246169JPEG2024178163000029.jpg61168

[0111] B. Analysis of "Gene Expression Signatures" Gene expression analysis of baseline bone marrow (BM) cell samples identified chemotherapy-refractory We divided patients into three classes within the immunological continuum: AML, HMA-refractory (including secondary AML), and relapsed patients. Group: Patients with immune exhaustion gene expression signature, The present invention stratifies patients into those who exhibit an immune enhancing gene expression signature, and those who exhibit an immune enhancing gene expression signature.

[0112] As described in more detail below, patients with primary refractory disease (those who have completed two or more induction trials) Refractory to chemotherapy, first CR <6 months, or ≥4 cycles of hypomethylating agent HMA failure after relapse) exhibits a gene expression signature of the immune-infiltrated tumor microenvironment, which Approximately 33% higher inflammatory chemokine levels (3.27 vs. levels observed in patients) ±0.22 vs 2.46±0.07, p=0.026).

[0113] Among this group, chemotherapy-refractory and HMA-refractory patients were classified as having immune-exhausted tumor microenvironment. The first subpopulation exhibiting environmental gene signatures (Figure 2, shown for cluster 2, bold line (see the boxed signature) and interferon gamma (herein referred to as “IFN-gamma”). Gene signatures of immune-enhanced tumor microenvironments, including the immune-enhancing tumor microenvironment signaling signature (also called “immunoglobulin”) The second subpopulation, which shows a necha (Figure 2, signatures shown for cluster 3, outlined in bold). HMA-refractory patients are further stratified into those with immune exhaustion and adaptive immune resistance. This is characterized by an approximately 44% increase in TIGIT expression in chemotherapy-refractory patients. large (5.55±0.34 vs 3.85±0.24, p=0.006), approximately 48% Increased PD-L1 expression in patients with PD-L1 (3.55 ± 0.18 vs. 2.4 ± 0.29, p =0.009), and an increase in Treg cell-specific expression of approximately 32% (4.87±0.2 3 vs 3.69±0.19, p=0.0009). HMA-refractory patients also Compared with chemotherapy-refractory patients, (CD244, EOMES, LAG3, and PTGER 4) tend to become increasingly exhausted.

[0114] Chemotherapy-refractory (i.e., refractory to ≥2 induction trials, first CR <6 months) and looking only at relapsed AML patients (i.e., not including HMA-refractory patients), (By aggregating the scores of the three signature modules as explained below Further analysis of a broader set of genes (to be performed) may provide insight into the role of these genes in relapsed and refractory AML patients. BM samples from subjects were collected at baseline to determine immune infiltration, as defined herein. "immune-exhausted" and "immune-depleted" subtypes The researchers stratified the disease into two immune subtypes, called

[0115] II. Exemplary CD123×CD3 Bispecific Binding Molecules A.JNJ‐63709178 JNJ-63709178 is a humanized IgG4 with silenced Fc function This antibody is a bispecific antibody. The antibody was generated using a 10-kDa antibody that binds to both CD123 on tumor cells and CD3 on T cells. JNJ-63709178 can recruit T cells to CD123-expressing tumor cells. can induce the killing of these tumor cells in vitro (MOLM-13, OCI-AM L5, and KG-1; EC50 = 0.51-0.91 nM). JNJ-63709178 (2016) “Development of fa CD123 x CD3 Bispecific Antibody (JNJ-63709178) for the Treatment of Acute My eloid Leukemia (AML),” Blood 128:2824; and Forslund, A. et al. (2016) “Ex Vivo Activity Profile of the CD123 x CD3 Duobody® Antibody JNJ-63709178 A gainst Primary Acute Myeloid Leukemia Bone Marrow Samples,” Blood 128:2875 JNJ-6370, which are incorporated herein by reference. 9178 and / or related antibodies: 13RB179, 13RB180, 13RB181, 1 3RB182, 13RB183, 13RB186, 13RB187, 13RB188, 1 3RB189, CD3B19, 7959, 3978, 7955, 9958, 8747, 8 The amino acid sequences of the heavy and light chains of 876, 4435 and 5466 are described in WO 2016 This is disclosed in US Pat. No. 5,993,367.

[0116] B.XmAb14045 XmAb14045 (also known as bibecotamab) is a mAb that binds the CD123 binding domain and XmAb is a tumor-directed antibody that contains both the cytotoxic T cell binding domain (CD3) and the CD3 domain. The bispecific Fc domain acts as a scaffold for these two antigen-binding domains, forming XmAb 14045, providing long circulating half-life, stability, and ease of manufacture. Binding of CD3 by 14045 resulted in highly potent and targeted binding of CD123-expressing tumor cells. Activating T cells for enhanced killing (U.S. Patent Publication No. 2017 / 0349660; Chu, SY et al. (2014) “Immunotherapy with Long-Lived Anti-CD123 x CD3 Bispeci fic Antibodies Stimulates Potent T Cell-Mediated Killing of Human AML Cell Lines and of CD123+ Cells in Monkeys: A Potential Therapy for Acute Myelogenous Leuke mia," Blood 124(21):2316, which are incorporated herein by reference. The heavy and light chain amines of B14045 and similar CD123×CD3 bispecific binding molecules are The amino acid sequence is described in U.S. Patent Publication No. 2017 / 0349660 and WHO Drug Information, Disclosed in Proposed INN: List 120, 2018, 32(4):658-660.

[0117] C.APVO436 APVO436 has an anti-CD123 scFv portion and an anti-CD3 scFv portion. ADAPTIR™ is a CD123×CD3 bispecific binding molecule. Each molecule contains an Fc domain modified to abolish ADCC / CDC effector function. APVO436 has an EC 50 Human CD123 and CD 3 expressing cells and at low effector:target ratios, It has been disclosed to exhibit potent target-specific activity against tumor cell lines. In experiments with primary AML subject samples and normal donor samples, It has been disclosed that it can potently induce endogenous T cell activation and proliferation accompanied by cell depletion. . APVO436(Comeau, MR et al. (2018) “APVO436, a Bispecific anti-CD123 x anti-CD3 ADAPTIR(TM) Molecule for Redirected T-cell Cytotoxicity, Induces P otent T-cell Activation, Proliferation and Cytotoxicity with Limited Cytokine Re lease,” AACR Annual Meeting April 2018, Abstract 1786; Godwin, CD et al. (201 7) “Bispecific Anti-CD123 x Anti-CD3 ADAPTIR(TM) Molecules APVO436 and APVO 437 Have Broad Activity Against Primary Human AML Cells In Vitro,” American Soc iety of Hematology Annual Meeting, December 2017, Blood 130:2639; Comeau, MR e t al. (2017) “Bispecific anti-CD123 x anti-CD3 ADAPTIR(TM) Molecules for Re directed T-cell Cytotoxicity in Hematological Malignancies,” AACR Annual Meeting g April 2017, Abstract 597). APVO436 CD123×CD3 bispecific binding The amino acid sequences of the heavy and light chains of the synthetic molecule are described in WO 2018 / 057802A1. This is disclosed in.

[0118] D.DART‐A DART-A (also known as Flotetuzumab, CAS number: 1664355-28-5 ) is a preferred CD123×CD3 bispecific binding molecule of the present invention. The sequence of the nucleotide ... Optimized bispecific diabody CD3 ("CD123 x CD3" bispecific diabody) (U.S. Patent Publication No. 2016-0200827, International Publication No. 2015 / 02689 No. 2, Al-Hussaini, M. et al. (2016) “Targeting CD123 In Acute Myeloid Leukemia Using A T-Cell-Directed Dual-Affinity Retargeting Platform,” Blood 127:122-131, in Vey, N. et al. (2017) “A Phase 1, First-in-Human Study of MGD006 / S80880 (CD 123 x CD3) in AML / MDS,” 2017 ASCO Annual Meeting, June 2-6, 2017, Chicago, IL: Abstract TPS7070, each of which is incorporated herein by reference in its entirety. RT-A is a novel, sequence-optimized CD123×CD3 bispecific diabody with similar composition. Since it has been shown to show enhanced functional activity compared to This is called the "CD123×CD3 bispecific diabody" (ence-optimized). International Application No. PCT / US2017 / 050471 describes a method for administering DART-A to a patient. No. 6,399,423, which is incorporated by reference in its entirety into this application. do.

[0119] DART-A comprises a first polypeptide chain and a second polypeptide chain (Figure 1). The first polypeptide chain of the bispecific diabody is, in the N-terminal to C-terminal direction, At the end, a light chain variable domain (VL domain) of a monoclonal antibody capable of binding to CD3 (V L CD3 ), an intervening linker peptide (Linker 1), and a monoclonal antibody capable of binding to CD123 The heavy chain variable domain (VH domain) of a human antibody CD123 ), and the C-terminus. do.

[0120] Such a VL CD3 A preferred sequence for the domain is SEQ ID NO:1: QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG It is.

[0121] VL CD3 The antigen-binding domain of: CDR L 1 (SEQ ID NO: 2): RSSTGAVTTSNYAN CDR L 2 (SEQ ID NO: 3): GTNKRAP CDR L 3 (SEQ ID NO: 4): ALWYSNLWV Includes.

[0122] A preferred sequence for such a linker 1 is SEQ ID NO: 5: GGGSGGGG. Una VH CD123 A preferred sequence for the domain is SEQ ID NO:6: EVQLVQSGAE LKKPGASVKV SCKASGYTFT DYYMKWVRQA PGQGLEWIGD IIPSNGATFY NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCARSH LLRASWFAYW GQGTLVTVSS It is.

[0123] VH CD123 The antigen-binding domain of: CDR H 1 (SEQ ID NO: 7): DYYMK CDR H 2 (SEQ ID NO: 8): DIIPSNGATFYNQKFKG CDR H 3 (SEQ ID NO: 9): SHLLRASWFAY Includes.

[0124] The second polypeptide chain is arranged in an N-terminal to C-terminal direction to bind to the N-terminus of CD123. The VL domain of a monoclonal antibody (VL CD123 ), an intervening linker peptide (e.g. linker 1), the VH domain of a monoclonal antibody capable of binding to CD3 (VH CD3 ), and Such a VL would include the C-terminus. CD123 A preferred sequence for the domain is Column number 10: DFVMTQSPDS LAVSLGERVT MSCKSSQSLL NSGNQKNYLT WYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIK It is.

[0125] VL CD123 The antigen-binding domain of: CDR L 1 (SEQ ID NO: 11): KSSQSLLNSGNQKNYLT CDR L 2 (SEQ ID NO: 12): WASTRES CDR L 3 (SEQ ID NO: 13): QNDYSYPYT It is.

[0126] Such a VH CD3 A preferred sequence for the domain is SEQ ID NO:14: EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSS It is.

[0127] VH CD3 The antigen-binding domain of: CDR H 1 (SEQ ID NO: 15): TYAMN CDR H 2 (SEQ ID NO: 16): RIRSKYNNYATYYADSVKD CDR H 3 (SEQ ID NO: 17): HGNFGNSYVSWFAY It is.

[0128] The sequence-optimized CD123×CD3 bispecific diabody of the present invention is are covalently linked to each other via cysteine ​​residues along their length. Such cysteine ​​residues are engineered into the VL and VH domains of the polypeptide. Alternatively, the linker may be introduced into an intervening linker (e.g., linker 1) that separates the linker from the Preferably, the second peptide (Linker 2) is, for example, Each polypeptide chain, either up to the VL domain or from the C-terminus to the VH domain A preferred sequence for such a linker 2 is SEQ ID NO: 18: GGCGGG. do.

[0129] The polypeptide chain can be further engineered to contain polypeptide coils of opposite charge. Thus, in a preferred embodiment, the formation of heterodimers can be promoted by: One of the polypeptide chains is an “E-coil” whose residues form a negative charge at pH 7. The "E-coil" domain (SEQ ID NO: 19: E VAAL E K E VAAL E K E VAAL E K E VAAL E Contains K) The other of the two polypeptide chains is engineered to have a structure in which the residues are The "K-coil" domain (SEQ ID NO: 20) forms a positive charge on the K VAAL K E K VAAL K E K VAAL K E K VAAL K E). Such a charged domain The presence of promotes the association between the first and second polypeptide chains, thus Promotes heterodimerization.

[0130] It is not important which coil is provided in the first or second polypeptide chain. However, certain preferred sequence-optimized CD123×CD3 bispecific DNA fragments of the present invention are The ear body ("DART-A") has the following sequence (SEQ ID NO:21): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGEV QLVQSGAELK KPGASVKVSC KASGYTFTDY YMKWVRQAPG QGLEWIGDII PSNGATFYNQ KFKGRVTITV DKSTSTAYME LSSLRSEDTA VYYCARSHLL RASWFAYWGQ GTLVTVSSGG CGGGEVAALE KEVAALEKEV AALEKEVAAL EK The first polypeptide chain has the following structure:

[0131] Strand 1 of DART-A is: SEQ ID NO:1 - SEQ ID NO:5 - SEQ ID NO:6 - SEQ ID NO:18 - SEQ ID NO: No. 19. The polynucleotide encoding the first polypeptide chain of DART-A is The code is SEQ ID NO:22: caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg acatgcagat ccagcacagg cgcagtgacc acatctaact acgccaattg ggtgcagcag aagccaggac aggcaccaag gggcctgatc gggggtacaa acaaaagggc tccctggacc cctgcacggt tttctggaag tctgctgggc ggaaaggccg ctctgactat taccggggca caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc gggggtggca caaaactgac tgtgctggga gggggtggat ccggcggcgg aggcgaggtg cagctggtgc agtccggggc tgagctgaag aaacccggag cttccgtgaa ggtgtcttgc aaagccagtg gctacacctt cacagactac tatatgaagt gggtcaggca ggctccagga cagggactgg aatggatcgg cgatatcatt ccttccaacg gggccacttt ctacaatcag aagtttaaag gcagggtgac tattaccgtg gacaaatcaa caagcactgc ttatatggag ctgagctccc tgcgctctga agatacagcc gtgtactatt gtgctcggtc acacctgctg agagccagct ggtttgctta ttggggacag ggcaccctgg tgacagtgtc ttccggagga tgtggcggtg gagaagtggc cgcactggag aaagaggttg ctgctttgga gaaggaggtc gctgcacttg aaaaggaggt cgcagccctg gagaaa It is.

[0132] The second polypeptide chain of DART-A has the following sequence (SEQ ID NO:23): DFVMTQSPDS LAVSLGERVT MSCKSSQSLL NSGNQKNYLT WYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIKGGGSGG GEVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKDR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSSGGCG GGKVAALKEK VAALKEKVAA LKEKVAALKE has.

[0133] Strand 2 of DART-A is SEQ ID NO:10 - SEQ ID NO:5 - SEQ ID NO:14 - SEQ ID NO:18 - SEQ ID NO: The polynucleotide encoding the second polypeptide chain of DART-A is The tide is SEQ ID NO:24: gacttcgtga tgacacagtc tcctgatagt ctggccgtga gtctggggga gcggtgact atgtcttgca agagctccca gtcactgctg aacagcggaa atcagaaaaa ctatctgacc tggtaccagc agaagccagg ccagccccct aaactgctga tctattgggc ttccaccagg gaatctggcg tgcccgacag attcagcggc agcggcagcg gcacagattt taccctgaca atttctagtc tgcaggccga ggacgtggct gtgtactatt gtcagaatga ttacagctat ccctacactt tcggccaggg gaccaagctg gaaattaaag gaggcggatc cggcggcgga ggcgaggtgc agctggtgga gtctggggga ggcttggtcc agcctggagg gtccctgaga ctctcctgtg cagcctctgg attcaccttc agcacatacg ctatgaattg ggtccgccag gctccaggga aggggctgga gtgggttgga aggatcaggt ccaagtacaa caattatgca acctactatg ccgactctgt gaaggataga ttcaccatct caagagatga ttcaaagaac tcactgtatc tgcaaatgaa cagcctgaaa accgaggaca cggccgtgta ttactgtgtg agacacggta acttcggcaa ttcttacgtg tcttggtttg cttatgggg acaggggaca ctggtgactg tgtcttccgg aggatgtggc ggtggaaaag tggccgcact gaaggagaaa gttgctgctt tgaaagagaa ggtcgccgca cttaaggaaa aggtcgcagc cctgaaagag It is.

[0134] DART-A expresses CD123 and CD41 as expressed by human and cynomolgus cells. The administration of DART-A induces T cell activation. It mediates the reduction of blasts, promotes T cell expansion, induces T cell activation, and inhibits the proliferation and proliferation of target cancer cells. It was found to cause target-switching killing (Table 4).

[0135] [Table 4]

[0136] More specifically, DART-A exhibited 50% of its maximal activity (EC 50 ) necessary to achieve At concentrations in the sub-ng / mL range, it exhibited potent target-directed killing capacity, which was due to its ability to target CD123-expressing A target cell line with a high level of expression (Kasumi-3 (EC 50 = 0.01ng / mL), CD A target cell line with moderate 123 expression (Molm13(EC 50 =0.18ng / mL ) and THP-1 (EC 50 = 0.24ng / mL), and the level of CD123 expression was moderate. The target cell line (TF-1(EC 50 =0.46ng / m L) and RS4-11 (EC 50 CD3 epitope binding specificity at 0.5 ng / mL Similarly, DART-A targeted translocation killing is independent of the donor origin. It was also observed in multiple target cell lines, including T cells, and was not detected in cell lines that do not express CD123. No targeted conversion kill was observed. The results are summarized in Table 5.

[0137] [Table 5]

[0138] Furthermore, human T cells and tumor cells (Molm13 or RS4-11) were combined to When subcutaneously injected into OD / SCID gamma (NSG) knockout mice, MOLM13 Tumors were cultured at doses of 0.16, 0.5, 0.2, 0.1, 0.02, and 0.004 mg / kg. The effect of MOLM13 on the expression of β-lactamase was significantly inhibited at doses of 0.004 mg / kg or higher. The MOLM13 model was more active than the RS4-11 model. Lower doses of DART-A associated with inhibition of tumor growth in MOLM1 Study demonstrating that RS4-11 cells have higher CD123 expression levels than RS4-11 cells Consistent with the in vitro data, this indicates that DART- This correlated with increased sensitivity to A-mediated cytotoxicity.

[0139] DART-A is a method to analyze primary AML specimens (bone marrow mononuclear cells (BMNC) and peripheral blood) from AML patients. Primary AML bone marrow samples were treated with DART-A and Co-incubation with IgG4+ / -100% of T cells resulted in a concomitant expansion of residual T cells (both CD4 and CD8). The time course of leukemia was associated with hypertension and induction of T cell activation markers (CD25 and Ki-67). This resulted in depletion of disease cell populations. Upregulation of immunoglobulin B and perforin levels was observed. Primary AML bone marrow samples were treated with DART. Incubation with -A significantly increased the time course of DART compared to the untreated control, i.e., control DART. This resulted in depletion of the leukemic cell population. T cells were counted (CD8 and CD4 staining) and activated When assayed for CD25 staining, DA RT-A expanded and activated T cells. DART-A also activated human PBMCs and It was found that it can mediate the depletion of pDC cells in both macaque PBMCs and crab Monkey pDCs were depleted just 4 days after infusion of just 10 ng / kg DART-A. The cytokines interferon gamma, TNF alpha, IL6, IL5, and I No increase in L4 and IL2 levels was observed in animals treated with DART-A. These data suggest that DART-A-mediated target cell killing is mediated by granzyme B and perforin. These results indicate that the effect was mediated via the α-amino acid pathway.

[0140] No activity was observed with CD123-negative targets (U937 cells) or control DART, which The observed T cell activation was strongly dependent on target cell binding and DART‐ These results indicate that monovalent binding of CD3 by A was insufficient to trigger T cell activation. is doing.

[0141] In summary, DART-A binds to the CD3ε subunit of the TCR and mediates several T cells expressing CD123, an antigen that is upregulated in hematological malignancies, DART-A is an antibody-based molecule that targets and reprograms lymphocytes in humans and cynomolgus monkeys. They bind antigens with similar affinity to target T cells from both species to CD123+ cells. The dose of DART-A was increased weekly and administered 4 or 7 days a week. The monkeys showed depletion of circulating CD123+ T cells 72 hours after the start of treatment, which The reduction in circulating T cells was sustained throughout the 4 weeks of treatment, regardless of dosing schedule. Although a small number of patients with rheumatoid arthritis occurred, the 4-day dosing schedule did not improve baseline outcomes before the subsequent infusion in monkeys. The recovery was consistent with DART-A-mediated immobilization. increased circulating PD1+ T cells but not TIM-3+ T cells; In vitro analysis of T cells from these monkeys demonstrated that lysis of transduced target cells was unaltered. The cytotoxicity was observed at DAR 0.1–0.25, indicating that exhaustion was not occurring. Following the first infusion of T‐A, there was limited and minimal cytokine release, but subsequent administrations After increasing the dose, minimal red blood cell mass was observed with a reduction in CD123+ myeloid progenitors. Even if a reversible decrease occurred, it was not limited.

[0142] E. Additional Bispecific Diabody Molecules Another version of DART-A includes an Fc region and has the general structure shown in FIG. The Fc domain is described in U.S. Patent Publication No. 2016-0200827. A preferred polypeptide containing the CH2 and CH3 domains of the ribosome is represented by the sequence (SEQ ID NO:25). ) ("knob-bearing" Fc domain): APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGX (where X is K or absent); and the sequence (SEQ ID NO:26) ("hole-bearing" Fc domain): APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPGX (where X is K or absent) has.

[0143] The first polypeptide of an exemplary DART-A w / Fc construct comprises, from the N-terminus to the C-terminus: At the N-terminus, the VL domain of a monoclonal antibody capable of binding to CD123 (VL CD 123 ), an intervening linker peptide (Linker 1), and a monoclonal antibody capable of binding to CD3 The VH domain (VH CD3 ), linker 2, E coil domain, linker 5, peptide 1 A polypeptide comprising the CH2 and CH3 domains of the Fc domain, and a C-terminal A preferred linker 5 has the sequence: GGG. A preferred peptide 1 has the sequence: The sequence is DKTHTCPPCP (SEQ ID NO: 29). Thus, such a DART-A w / F The first polypeptide of the version 1 construct is: SEQ ID NO:10 - SEQ ID NO:5 - SEQ ID NO:1 4 - SEQ ID NO:18 - SEQ ID NO:19 - GGG - SEQ ID NO:29 - SEQ ID NO:25 (where X is K (which is

[0144] Preferred first polypeptides of such DART-A w / Fc version 1 constructs include The desired sequence is the sequence (SEQ ID NO:27): DFVMTQSPDS LAVSLGERVT MSCKSSQSLL NSGNQKNYLT WYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIKGGGSGG GEVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKDR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSSGGCG GGEVAALEKE VAALEKEVAA LEKEVAALEK GGGDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPSREE MTKNQVSLWC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK It is.

[0145] The second chain of such a DART-A w / Fc version 1 construct is At the N-terminus, the VL domain of a monoclonal antibody capable of binding to CD3 (VL CD 3) an intervening linker peptide (linker 1), a monoclonal antibody capable of binding to CD123 The VH domain of the CD123 ), linker 2, the K coil domain, and the C-terminus Therefore, the second polyclonal antibody of such a DART-Aw / Fc version 1 construct The peptide consists of: SEQ ID NO:1-SEQ ID NO:5-SEQ ID NO:6-SEQ ID NO:18-SEQ ID NO:20 Such a polypeptide may have the sequence (SEQ ID NO:28): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGEV QLVQSGAELK KPGASVKVSC KASGYTFTDY YMKWVRQAPG QGLEWIGDII PSNGATFYNQ KFKGRVTITV DKSTSTAYME LSSLRSEDTA VYYCARSHLL RASWFAYWGQ GTLVTVSSGG CGGGKVAALK EKVAALKEKV AALKEKVAAL KE has.

[0146] The third polypeptide chain of such a DART-A w / Fc version 1 construct is The preferred polypeptides include the CH2 and CH3 domains of the gG Fc domain. The peptides were peptide 1 (DKTHTCPPCP; SEQ ID NO: 29) and the CH2 and CH3 domains of the Fc domain. main (SEQ ID NO:26, where X is K) and SEQ ID NO:30: DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPGK The sequence is:

[0147] Additional CD123 × CD3 bispecific DNA containing another optimized anti-CD3 binding domain The ear body is disclosed in U.S. Patent Application No. 62 / 631,043 (filed February 15, 2018); and U.S. Patent Application No. 62 / 738,632 (filed September 28, 2018) (which are all incorporated herein by reference in their entirety). (which is incorporated herein by reference).

[0148] III. Pharmaceutical Preparations The compositions of the present invention can be used in the manufacture of bulk drug compositions (e.g., unit doses) useful in the manufacture of pharmaceutical compositions. Impure or non-sterile compositions that can be used to prepare formulations (i.e., compositions suitable for administration to a subject or patient) Such compositions contain a prophylactically or therapeutically effective amount of a CD123xCD3 bispecific antibody. and a pharma- ceutically acceptable carrier.

[0149] A preferred pharmaceutical formulation comprises a CD123xCD3 bispecific binding molecule and an aqueous stabilizer, and Optionally, a pharma- ceutically acceptable carrier is included.

[0150] As used herein, the term "pharmaceutically acceptable carrier" refers to a "Easily acceptable carrier" refers to a carrier that is used to transport animals, more specifically humans. approved by a regulatory agency or listed in the United States Pharmacopoeia or other publications as suitable for delivery of The term "pharmaceutical product" refers to any diluent, adjuvant (e.g., furan), etc., as described in another generally recognized pharmacopoeia. Intended to refer to adjuvants (complete and incomplete), excipients, or vehicles Such pharmaceutical carriers may be sterile liquids, such as water and oils, petroleum-, animal-, vegetable-, or synthetic-derived, e.g., peanut oil, soybean oil, mineral oil; Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, as well as aqueous solutions of dextrose and glycerol, are also useful liquids, particularly for parking solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, and the like. Sugar, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, molybdenum Glycerol stearate, talc, sodium chloride, dried skim milk, glycerol Examples of the solvent include ethylene glycol, propylene, glycol, water, and ethanol. The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. It can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation, etc. Cut.

[0151] Generally, the components of the compositions of the invention are packaged, for example, in a vial, ampoule or container indicating the quantity of active agent. may be sold separately as a liquid formulation in an airtight container such as a sachet, as a lyophilized powder or as an anhydrous concentrate. The composition is administered by infusion. In this case, the composition is administered using an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is to be administered by injection, it may be dispensed in a syringe so that the ingredients can be mixed prior to administration. Ampoules of sterile water for injection or saline can be provided.

[0152] The present invention also relates to the use of a CD123×CD3 bispecific binding molecule, either alone or as a stabilizer or drug. A pharmaceutical pack comprising one or more containers containing a pharmaceutical agent in combination with a pharmaceutical acceptable carrier. Additionally, one or more other prophylactic or therapeutic agents useful in treating the disease are also provided. The present invention also relates to a pharmaceutical composition of the present invention. A pharmaceutical pack or capsule comprising one or more containers filled with one or more of the ingredients. Optionally, such one or more containers may be used to store a pharmaceutical or biological product. Related to notices in a form prescribed by a government agency that regulates the manufacture, use, or sale of Such notification may be made by an agency of manufacture, use, or sale for human administration. This reflects approval.

[0153] IV. Kit The present invention relates to CD123×CD3 bispecific binding molecules (e.g., storage, dosage, indications, Explanatory material relating to side effects, counter-indications, etc., as well as, optionally, a stable The present invention provides a kit comprising a CD123×CD 3. The bispecific binding molecules are preferably contained in airtight containers such as ampoules, vials, sachets, etc. The airtight container is preferably packaged in a container that indicates the amount of the molecule contained therein. The container may be made of any pharma- ceutically acceptable material such as glass, resin, or plastic. The CD123×CD3 bispecific binding molecules of such kits may preferably be is supplied as a liquid solution in an airtight container, a sterile lyophilized powder, or a water-free concentrate; For example, it can be reconstituted with water or saline to the appropriate concentration for administration to a subject. Such liquid or lyophilized materials should be stored at 2-8°C in their original containers and The above materials are preferably used within 12 hours, more preferably within 6 hours, 5 hours, or 3 hours after reconstitution. The kit includes, in one or more containers, and / or or further comprising one or more cytotoxic antibodies that bind to one or more cancer antigens associated with the cancer. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic agent. In some embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic agent. may further include instructions for use, or other printed information.

[0154] Additionally, one or more other prophylactic or therapeutic agents useful in the treatment of a disease may also be included in the pharmaceutical pack or capsule. The present invention also relates to a pharmaceutical composition comprising one or more of the components of the pharmaceutical composition of the present invention. Also provided is a pharmaceutical pack or kit comprising one or more containers filled with the plurality of Such one or more containers may be used to manufacture, use, or sell a pharmaceutical or biological product. may be associated with a notice in a form prescribed by a regulating government agency, Notification reflects approval by an agency of manufacture, use, or sale for human administration. do.

[0155] V. Method of Administration The CD123×CD3 bispecific binding molecule pharmaceutical formulation of the invention comprises an effective amount of the molecule of the invention, or administering to a subject a pharmaceutical composition comprising the fusion protein or conjugate molecule of the present invention. administering to the subject a therapeutically effective amount of a compound in the composition of the present invention to treat, prevent, or cure one or more symptoms associated with a disease, disorder, or infection. In a preferred embodiment, the composition is substantially purified (i.e., and is substantially free of substances that limit the effectiveness of the composition or produce undesirable side effects. In a specific embodiment, the subject is an animal, preferably a non-primate (e.g., bovine, Equine, feline, canine, rodent, etc.) or primate (e.g. monkeys such as cynomolgus monkeys, humans etc. In a preferred embodiment, the subject or patient is a human.

[0156] Methods for administering the CD123×CD3 bispecific binding molecule pharmaceutical formulations of the present invention include non- Oral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous) In a specific embodiment, the CD123xCD3 bispecific binding molecule is The composition may be administered by any convenient route, for example, by infusion. It may well be administered together with other biologically active agents.

[0157] Administration by infusion is preferably accomplished using an infusion pump. A fusion pump is a device that delivers fluids into a patient's body in a controlled manner, especially at a predetermined rate for an extended period of time. An infusion pump is a medical device that delivers fluid over a range of More preferably, they are powered by electricity. Some infusion pumps are "stationary" is a "onary" infusion pump designed for use at the patient's bedside. Other infusion pumps, called "ambulatory" infusion pumps, are portable. Or designed to be wearable. "Syringe" pumps are An infusion pump in which the fluid to be delivered is held in a reservoir (e.g., a syringe) in a chamber and a movable piston is used to control the volume of the chamber and therefore the delivery of fluid. In an "elastomeric" infusion pump, the fluid is pumped through a stretchy balloon reservoir. The balloon is held within a cavity and fluid delivery is achieved by pressure exerted by the balloon's flexible walls. In a "peristaltic" infusion pump, a set of rollers is flexible. It clamps the tubing and moves down its length, forcing the fluid out. The i-channel infusion pump can deliver fluid from multiple reservoirs at multiple rates. A "smart pump" is a computer-controlled fluid delivery system. Depending on the risk of adverse drug interactions or the power of the pump, A warning can be issued if a parameter is set beyond a specified limit. Examples of infusion pumps are known in the art, see, for example, [author unknown] 2002 “General-Purpose Infusion Pumps.” mps,” Health Devices 31(10):353-387; and U.S. Patent No. 10,029,051, U.S. Patent No. 10,029,047, U.S. Patent No. 10,029,045, U.S. Patent No. 10, No. 10,022,495, U.S. Pat. No. 10,022,494, U.S. Pat. No. 10,016,55 No. 9, U.S. Patent No. 10,006,454, U.S. Patent No. 10,004,846, U.S. Patent No. 9,993,600, U.S. Patent No. 9,981,082, U.S. Patent No. 9,974, No. 901, U.S. Patent No. 9,968,729, U.S. Patent No. 9,931,463, U.S. Patent This is provided in Patent No. 9,927,943, etc.

[0158] The CD123×CD3 bispecific binding site of the present invention is used to allow patients to move through their treatment regimen. The combined molecular pharmaceutical formulation is administered by infusion facilitated by one or more ambulatory pumps. The CD123×CD3 bispecific binding molecule pharmaceutical formulation of the present invention is preferably administered by continuous infusion. In a preferred embodiment, the 7-day continuous infusion regimen is administered in a 3-day infusion. A therapeutic dose of approximately 30 ng / kg patient body weight / day for 4 days followed by A therapeutic dosage of about 100 ng / kg / day (e.g., 30 ng / kg of patient body weight for 3 days) / day treatment dose, followed by 100ng / kg / day treatment dose for 4 days) In a particularly preferred embodiment, such a 7-day consecutive infusion regimen is followed by 21 consecutive days of infusion. A drop regimen was followed, where a treatment dosage of 500 ng / kg / day was administered for the 21-day regimen described above. The treatment dose was administered on days 1-4 of each week, with no treatment dose administered on days 5-7 of each week. Alternatively, a 7-day continuous infusion regimen is followed by a 21-day continuous infusion regimen, where: A therapeutic dose of 500 ng / kg / day is administered daily for 21 days.

[0159] In any of the above treatment courses, the proportion of CD8+ T lymphocytes in the tumor microenvironment was further Such monitoring may be performed prior to administration of the CD123×CD3 bispecific binding molecule, During the course of CD123×CD3 binding molecule therapy and / or This may be performed after completion of one cycle.

[0160] VI. Uses of the Compositions of the Invention The CD123xCD3 bispecific binding molecules of the present invention are associated with the expression of CD123 or can be used to treat any disease or condition characterized by this. The CD123xCD3 bispecific binding molecules can be used to treat hematological malignancies. The CD123×CD3 bispecific binding molecules of the invention are useful in treating chemotherapy-refractory hematological malignancies, including As used herein, chemotherapeutic agents are particularly suitable for use in the treatment of hematological malignancies. Refractory hematologic malignancies are those that are refractory to 2 or more induction trials, with a first CR of 6 or more. < 2 months or failure after 2 or more cycles of treatment with hypomethylating agents; It is a malignant tumor.

[0161] Thus, without being limited thereto, such molecules include: acute myeloid leukemia (AML) ( including primary chemotherapy-refractory AML); blast crisis of CML, and Abel syndrome associated with CML Chronic myeloid leukemia (CML); myeloid variants, including the son oncogene (Bcr-ABL translocation) Myeloblastic syndrome (MDS); acute B-lymphoblastic leukemia (B-ALL); acute T-lymphoblastic leukemia Hematologic malignancies (T-ALL); Richter's syndrome, or transformation of Richter's syndrome in CLL , chronic lymphocytic leukemia (CLL); hairy cell leukemia (HCL); blastic plasmacytoid dendritic cell BPDCN; mantle cell lymphoma (MCL) and small lymphocytic lymphoma ( Non-Hodgkin's lymphoma (NHL), including SLL; Hodgkin's lymphoma, systemic mastocytosis; The CD123×CD3 combination of the present invention can also be used for the diagnosis or treatment of Burkitt's lymphoma. The polyspecific binding molecules can further be used in the manufacture of a medicament for the treatment of the above mentioned conditions.

[0162] The CD123×CD3 bispecific binding molecules of the present invention are useful in the treatment of acute myeloid leukemia (AML, primary chemotherapy-refractory acute myeloid leukemia), blood myelodysplastic syndrome (MDS), blastoid leukemia Plasmocytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL), or acute T-lymphoma It is particularly suitable for use in the treatment of myocardial infarction and lymphoblastic leukemia (T-ALL).

[0163] VII. Specific Embodiments of the Invention The present invention has been generally described above, but the following numbered embodiments ("E1" to "E6") The present invention will be more readily understood by reference to the following embodiments. The embodiments are provided by way of example only and are not intended to be limitations of the invention unless otherwise specified. It is not intended to be.

[0164] E1. A method of treating a chemotherapy-refractory hematological malignancy in a patient, the method comprising: administering to the patient a therapeutic dosage of a CD123×CD3 bispecific molecule, The dosage provides a therapeutic effect by stimulating the killing of cells of the hematological malignancy in the patient. The method is effective for treating said malignant tumor.

[0165] E2. The method further comprises administering, before and / or after the administration of the CD123×CD3 bispecific molecule, A method for evaluating the expression of one or more target and / or reference genes in a cell sample from said patient. The method of E1, further comprising the step of:

[0166] E3. The method further comprises administering, prior to administration of the CD123xCD3 bispecific molecule, one or more Evaluating the expression of the target gene and / or the one or more reference genes. 2. The method according to claim 2.

[0167] E4. The method further comprises administering the CD123×CD3 bispecific molecule followed by one or more Evaluating the expression of the target gene and / or the one or more reference genes. 2. The method according to claim 2.

[0168] E5. A patient is receiving treatment for the use of a CD123×CD3 bispecific molecule to treat a hematological malignancy. 1. A method for determining whether a patient is a favorable responder to a therapeutic use, the method comprising: (a) determining whether the CD123×C and detecting one or more target genes in a cell sample from the patient prior to administration of the D3 bispecific molecule. assessing expression; and (b) determining whether the expression of the one or more target genes is related to the one or more target and / or reference genes; If the expression of CD123×CD 3. Identifying Favorable Responders to Treatment with Bispecific Molecules A method comprising:

[0169] E6. The above method is: (i) expression of one or more target genes; and (ii) one or more reference genes whose expression is not characteristically associated with hematological malignancies The method according to any one of E2 to E6, wherein the

[0170] E7. The method comprises: determining whether the patient has a baseline expression of the one or more reference genes; Any one of E2 to E6, comprising a step of evaluating the expression of one or more target genes. The method described above.

[0171] E8. The method comprises administering to an individual suffering from the hematological malignancy, or to a group of a plurality of such individuals, the expression of said one or more target genes of said patient for said one or more target genes of said group; The method according to any one of E2 to E7, comprising a step of evaluating expression of.

[0172] E9. The method comprises administering a CD123×CD3 bispecific antibody to treat the hematological malignancy. An individual or a group of individuals who have not responded well to the use of the molecule. for expression of said one or more target genes, The method of any one of E2 to E7, comprising evaluating the present.

[0173] E10. The method includes administering a CD123×CD3 bispecific antibody to treat the hematological malignancy. A review of an individual or a group of several such individuals who have responded well to the use of the therapeutic molecule. determining the expression of said one or more target genes in said patient for said one or more target genes; The method of any one of E2 to E7, comprising an evaluating step.

[0174] E11. The relative expression levels of the one or more target genes in the population are determined by the individual It is established by averaging gene expression levels in cell samples obtained from a population. A method according to any one of E7 to E10.

[0175] E12. The patient is monitored for expression levels of at least one of the target genes: (a) detecting the expression level of said target gene in a population of individuals suffering from said hematological malignancy; higher than the first quartile of the (b) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies A first quartile of expression levels of the target genes in a population of individuals who were not good responders. higher than the quantile; or (c) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies For the expression level of the target gene in a population of individuals who were not favorable responders, have a log2 fold change of at least about 0.4; or (d) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies At least one third of the expression level of the target gene in a population of individuals who had a favorable response Within one quartile, The method according to any one of E2 to E11, which indicates the expression level.

[0176] E13. The patient is monitored for expression levels of at least one of the target genes: (a) detecting the expression level of said target gene in a population of individuals suffering from said hematological malignancy; higher than the second quartile of the (b) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies A second quartile of expression levels of the target genes in a population of individuals who were not favorable responders. higher than the quantile; or (c) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies For the expression level of the target gene in a population of individuals who were not favorable responders, have a log2 fold change of at least about 0.5; or (d) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies At least one third of the expression level of the target gene in a population of individuals who had a favorable response Within the 2nd quartile, The method according to any one of E2 to E11, which indicates the expression level.

[0177] E14. The patient is monitored for expression levels of at least one of the target genes: (a) detecting the expression level of said target gene in a population of individuals suffering from said hematological malignancy; higher than the third quartile of the (b) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies The third and fourth fold changes in the expression levels of the target genes in a population of individuals who were not good responders. higher than the quantile; or (c) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies For the expression level of the target gene in a population of individuals who were not favorable responders, having a log2 fold change of at least about 0.6, The method according to any one of E2 to E11, which shows CD123×CD3 expression level.

[0178] E15. A method of treating a hematological malignancy, the method comprising: (a) The method according to any one of the methods described in any one of E6 to E14, Favorable response to the use of CD123×CD3 bispecific molecules to treat malignant tumors determining whether the person is a (b) administering a therapeutic dose if the patient is determined to be a favorable responder to the treatment. administering to said patient an amount of said CD123xCD3 bispecific molecule Including, The administration of the CD123×CD3 bispecific molecule inhibits the hematological malignancy in the patient. A method for stimulating the killing of tumor cells.

[0179] E16. The method further comprises administering to said patient one or more target proteins obtained from said patient after initiation of said treatment. The method of E15, further comprising assessing expression of the gene one or more times.

[0180] E17. A method of treating a hematological malignancy comprising: (a) administering an effective therapeutic dosage of a CD123×CD3 bispecific molecule; (b) administering to the patient at one or more time points following administration of the CD123×CD3 bispecific molecule. The expression of one or more target genes in a cell sample obtained from the above CD123×CD 3. Determine expression levels relative to corresponding baseline levels obtained prior to administration of the bispecific molecule. Steps to be followed; (c) determining whether said expression of said one or more target genes is greater than or equal to said corresponding baseline expression level; determining whether said gene expression is increased relative to said The patient is then identified as suitable for treatment with the CD123×CD3 bispecific molecule. identifying said patients as potential responders; and (d) an adjusted or additional effective therapeutic dose of the CD123×CD3 bispecific molecule as described above. to said patient who is any of said favorable responders. A method comprising:

[0181] E18. The method according to any one of E2 to E17, wherein the cell sample is a blood sample.

[0182] E19. The method according to any one of E2 to E17, wherein said cell sample is a bone marrow sample.

[0183] E20. Detection of one or more target genes and / or one or more of the target genes in a bone marrow sample of the patient. Any of E2 to E17, further comprising a step of detecting the expression level of the above reference gene. The present invention further comprises the step of detecting the expression level of one or more reference genes. In accordance with another aspect of the present invention, there is provided an embodiment of the above method, further comprising:

[0184] E21. Assessment of said expression or said patient is receiving CD123 therapy for treating a hematological malignancy. The determination of whether a patient is a favorable responder to the use of the xCD3 bispecific molecule is: (a) detecting, using a gene expression platform, each of the target genes in one or more cell samples; determining the gene expression level of (b) comparing the expression level of said target gene with the expression level of one or more reference genes; Tep The method according to any one of E2 to E20, wherein the method is carried out by

[0185] E22. Assessment of said expression or said patient is administered CD123 The determination of whether a patient is a favorable responder to the use of the xCD3 bispecific molecule is: (a) detecting, using a gene expression platform, each of the target genes in one or more cell samples; determining the raw RNA level of the gene expression platform, a reference gene set of mouse-keeping genes; and (b) using the measured RNA level of the internal reference gene to measure the RNA level of the target gene; assigning a relative expression value to each of said determined raw RNA levels. The method according to any one of E2 to E21, wherein the method is carried out by

[0186] E23. One or more of the target genes is: (a) one or more of CXCL9, CXCL10, CXCL11, and STAT1; and / or (b)CCL5, CD27, CD274, CD276, CD8A, CMKLR1, CX CL9, CXCR6, HLA‐DQA1, HLA‐DRB1, HLA‐E, IDO1, L AG3, NKG7, PDCD1LG2, PSMB10, STAT1, and TIGIT one or more of the following: (c)AREG, CSF3, CXCL1, CXCL2, CXCL3, CCL20, FO One or more of SL1, IER3 (NM_003897.4), IL6, and PTGS2 above; and / or (d) one or more of CCL2, CCL3 / L1, CCL4, CCL7, and CCL8 above; and / or (e) MAGEA3 / A6, MAGEA1, MAGEA12, MAGEA4, MAGE B2, MAGEC1, and MAGEC2; and / or (f) APOL6, DTX3L, GBP1, IFI16, IFI27, IFI35, I FI6, IFIH1, IFIT1, IFIT2, IFIT3, IFITM1, IFITM 2, IRF1, IRF9, ISG15, MX1, OAS1, OAS2, PARP9, PS one or more of MB9, STAT2, TMEM140, and TRIM21; and / or teeth (g) one or more of PSMB8, PSMB9, and PSMB10; and / or (h) IL-10; and / or (i) CD274; and / or (j)PDCD1LG2 The method according to any one of E2 to E22, comprising:

[0187] E24. The method of E23, wherein the one or more target genes further include IFNG.

[0188] E25. One or more of the reference genes are ABCF1, G6PD, NRDE2, OAZ1. , POLR2A, SDHA, STK11IP, TBC1D10B, TBP, and UBB The method of any one of E2 to E24, including one or more of the above.

[0189] E26. Determining a gene signature score for the one or more target genes. The method according to any one of claims 1 to 25.

[0190] E27. The above gene signature score is: (a) Gene expression platform including a reference gene set of housekeeping genes The raw RNA level for each of the target genes in one or more cell samples is calculated using measuring; (b) comparing each of the measured raw RNA levels with the geometric mean of the housekeeping genes; normalizing to the mean and optionally further normalizing each RNA value to a standard; (c) log-transforming each normalized RNA value; (d) Each log-transformed RNA value was multiplied by the corresponding weighting factor to obtain the weighted RNA value. generating (e) The weighted RNA values ​​are summed together, optionally with an adjustment constant, to produce a single gene Steps for generating child signature scores The method of claim E26, wherein the method is determined by a process comprising:

[0191] E28. The gene signature score is calculated by adding one or more of the target genes to the gene signature score. Determined using the weights and, optionally, the adjustment factors provided in Tables 6 and 12A-12G, E 26 or E27.

[0192] E29. The above gene signature score is: (a) IFN gamma signaling signature; (b) tumor inflammation signature; (c) bone marrow inflammatory signature; (d) inflammatory chemokine signature; (e) MAGE signature; (f) IFN downstream signaling signature; (g) immune proteasome signature; (h) IL‐10 signature; (i) CD274 signature; and / or (j) PDCD1LG2 signature A gene signature score determined for one or more of E26 to E28. The method according to any one of the above.

[0193] E30. A patient gene signature score comprising: (a) detecting one of the target genes in a population of individuals suffering from the hematological malignancy; The first quartile of the score for the above gene signature calculated from expression levels of greater than; or (b) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies The expression of one or more of the target genes in a population of individuals who were not good responders greater than the first quartile of the score for the above gene signature calculated from the current level; or (c) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies The expression of one or more of the target genes in a population of individuals who were not good responders At least about 0.4 for the score for the gene signature calculated from the current level or (d) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies The expression levels of one or more of the target genes in a population of individuals who had a favorable response The score for the gene signature calculated from the above gene signature is at least within the first quartile. be, The patient gene signature score was used to predict the efficacy of treatment with the CD123×CD3 bispecific molecule. The method according to any one of E26 to E29, which is an indication of a relatively favorable patient response to method.

[0194] E31. A patient gene signature score comprising: (a) detecting one of the target genes in a population of individuals suffering from the hematological malignancy; The score for the above gene signature calculated from expression levels of greater than; or (b) Good results for the treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression levels of one or more of the target genes in a population of non-responsive individuals or greater than the second quartile of the score for the gene signature calculated from the (c) A successful treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression levels of one or more of the target genes in a population of non-responsive individuals A score for the gene signature calculated from the log of at least about 0.5 have an og2 fold change; or (d) Use of CD123×CD3 bispecific molecules for the treatment of hematological malignancies The expression levels of one or more of the target genes in a population of individuals who were responsive The score for the gene signature calculated from the above is at least within the second quartile. , The patient gene signature score was used to predict the efficacy of treatment with the CD123×CD3 bispecific molecule. The method according to any one of E26 to E29, which is an indication of a relatively favorable patient response to method.

[0195] E32. A patient gene signature score comprising: (a) detecting one or more of the target genes in a population of individuals suffering from a hematological malignancy; greater than the third quartile of the score for the above gene signature calculated from the expression levels above loud; or (b) Good results for the treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression levels of one or more of the target genes in a population of non-responsive individuals or greater than the third quartile of the score for the gene signature calculated from the (c) A successful treatment of hematological malignancies using CD123×CD3 bispecific molecules The expression levels of one or more of the target genes in a population of non-responsive individuals A score for the gene signature calculated from the log of at least about 0.6 with og2 fold change, The patient gene signature score was used to predict the efficacy of treatment with the CD123×CD3 bispecific molecule. The method according to any one of E26 to E29, which is an indication of a relatively favorable patient response to method.

[0196] E33. (a) the gene signature is an IFN gamma signaling signature, A patient gene signature score of approximately 2.5 was obtained for the CD123×CD3 bispecific molecule. is indicative of a relatively favorable patient response to the treatment used; and / or (b) the gene signature is a tumor inflammation signature, and is at least about 5.5 The gene signature score is a predictor of response to treatment with the CD123×CD3 bispecific molecule. and / or is indicative of a relatively favorable patient response; (c) the gene signature is an IFN downstream signaling signature, and comprises at least A patient gene signature score of approximately 4.5 was obtained using the CD123×CD3 bispecific molecule. is an indicator of a relatively favorable patient response to a given treatment. The method according to claim E28 or E29.

[0197] E34. The above gene signature is an IFN-gamma signaling signature, a tumor inflammation signature. or an IFN downstream signaling signature, A patient genetic signature score comprising: (a) detecting one of the target genes in a population of individuals suffering from the hematological malignancy; greater than the first quartile of the score of the gene signature calculated from expression levels of or (b) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies The expression of one or more of the target genes in a population of individuals who were not good responders greater than the first quartile of the score for the gene signature calculated from the current level; or (c) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies The expression of one or more of the target genes in a population of individuals who were not good responders At least about 0.4 for the score for the gene signature calculated from the current level or (d) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies The expression levels of one or more of the target genes in a population of individuals who had a favorable response The score for the gene signature calculated from the above gene signature is at least within the first quartile. be, The patient gene signature score was used to predict the efficacy of treatment with the CD123×CD3 bispecific molecule. The method of claim 8, wherein the method is indicative of a relatively favorable patient response to the

[0198] E35. The above gene signature is an IFN-gamma signaling signature, a tumor inflammation signature. or an IFN downstream signaling signature, A patient genetic signature score comprising: (a) detecting one of the target genes in a population of individuals suffering from the hematological malignancy; greater than the second quartile of the score of the above gene signature calculated from expression levels of or (b) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies The expression of one or more of the target genes in a population of individuals who were not good responders greater than the second quartile of the score for the above gene signature calculated from the current level; or (c) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies The expression of one or more of the target genes in a population of individuals who were not good responders At least about 0.5 for the score for the gene signature calculated from the current level or (d) Use of CD123×CD3 bispecific molecules in the treatment of the above hematological malignancies The expression levels of one or more of the target genes in a population of individuals who had a favorable response The score for the gene signature calculated from the above gene signature is at least within the second quartile. be, The patient gene signature score was used to predict the efficacy of treatment with the CD123×CD3 bispecific molecule. The method of claim 8, wherein the method is indicative of a relatively favorable patient response to the

[0199] E36. The above gene signature is an IFN-gamma signaling signature, a tumor inflammation signature. or an IFN downstream signaling signature, A gene signature score for a patient, comprising: (a) detecting one of the target genes in a population of individuals suffering from the hematological malignancy; The third quartile of the score for the above gene signature calculated from expression levels of greater than; or (b) for the treatment of the above hematological malignancies using CD123×CD3 bispecific molecules one or more of the target genes in a population of individuals who did not have a good response Greater than the third quartile of the score for the above gene signature calculated from the expression levels or (c) for the treatment of the above hematological malignancies using the CD123×CD3 bispecific molecules one or more of the target genes in a population of individuals who did not have a good response A score of at least about 0. with a log2 fold change of 6, The gene signature score of the patient was evaluated for predicting the outcome of treatment with the CD123×CD3 bispecific molecule. The method according to E28 or E29, wherein the IL-16 expression level is indicative of a more favorable patient response to the treatment.

[0200] E37. IFN dominance module scores are determined and IFN dominance of at least approximately 25 patients is determined. The efficacy module score was compared to treatment with the CD123×CD3 bispecific molecule. The method of E29 which is indicative of a relatively favorable patient response.

[0201] The E38.IFN dominance module score was determined, The patient's IFN dominance module score: (a) detecting one of the target genes in a population of individuals suffering from the hematological malignancy; From the first quartile of the score of the above IFN-dominant module calculated from the expression levels of Large; or (b) for the treatment of the above hematological malignancies using CD123×CD3 bispecific molecules one or more of the target genes in a population of individuals who did not have a good response greater than the first quartile of the score of the above IFN-dominant module calculated from expression levels; or (c) for the treatment of the above hematological malignancies using the CD123×CD3 bispecific molecules The expression levels of one or more of the target genes in a population of individuals who had a good response were At least within the first quartile of the IFN-dominant module score calculated from the baseline be, The IFN-dominant module score of the patient was determined using the CD123×CD3 bispecific molecule described above. The method of E29, wherein the IL-10 level is indicative of a relatively favorable patient response to the treatment.

[0202] E39.IFN dominance module score determined The patient's IFN dominance module score: (a) detecting one of the target genes in a population of individuals suffering from the hematological malignancy; From the second quartile of the score of the above IFN-dominant module calculated from the expression level of Large; or (b) for the treatment of the above hematological malignancies using CD123×CD3 bispecific molecules one or more of the target genes in a population of individuals who did not have a good response greater than the second quartile of the score of the above IFN-dominant module calculated from expression levels; or (c) for the treatment of the above hematological malignancies using the CD123×CD3 bispecific molecules The expression levels of one or more of the target genes in a population of individuals who had a good response were The IFN-dominant module score calculated from the baseline was at least within the second quartile. R, The IFN-dominant module score of the patient was determined using the CD123×CD3 bispecific molecule described above. The method of E29, wherein the IL-10 level is indicative of a relatively favorable patient response to the treatment.

[0203] E40. Gene expression signatures characteristic of immune-enhanced and IFN-gamma-dominant tumor microenvironments The relative preference for treatment with the CD123×CD3 bispecific molecule was observed in patients who exhibited the above-mentioned The method according to any one of E26 to E39, which is an indication of a favorable patient response.

[0204] E41. The CD123×CD3 bispecific molecule is a bispecific antibody or The method of any one of E1 to E40, wherein the antibody is a bispecific molecule.

[0205] E42. The above CD123×CD3 bispecific molecule is JNJ‐63709178, Xm The method according to E41, wherein the method is Ab14045 or APVO436.

[0206] E43. The CD123×CD3 bispecific molecule is a fusion protein comprising two, three or four polypeptides. Any one of E1 to E40, which is a covalent bispecific diabody having a tide chain. The method according to

[0207] E44. The CD123×CD3 bispecific molecule is: (a) a first polypeptide chain having the amino acid sequence of SEQ ID NO:21; and (b) a second polypeptide chain having the amino acid sequence of SEQ ID NO:23. is a diabody comprising The first polypeptide chain and the second polypeptide chain are connected by a disulfide bond. The method according to E43, wherein the

[0208] E45. The hematologic malignancies in the above patient are: acute myeloid leukemia (AML); chronic myeloid leukemia (CML); CML; Blast crisis of CML; Abelson oncogene associated with CML (Bcr -ABL translocation; myelodysplastic syndrome (MDS); acute B-lymphoblastic leukemia (B-ALL) );Acute T-lymphoblastic leukemia (T-ALL);Chronic lymphocytic leukemia (CLL);Richta Richter's syndrome; transformation of Richter's syndrome in CLL; hairy cell leukemia (HCL); blastic form Plasmocytoid dendritic cell neoplasm (BPDCN); mantle cell lymphoma (MCL) and small lymphoma Non-Hodgkin's lymphoma (NHL), including cytoplasmic lymphoma (SLL); Hodgkin's lymphoma, systemic and Burkitt's lymphoma, any of E1 to E44. The method according to any one of the above.

[0209] E46. The method of E45, wherein said hematological malignancy of said patient is AML.

[0210] E47. The method of E45, wherein said hematological malignancy of said patient is MDS.

[0211] E48. The method of E45, wherein said hematological malignancy of said patient is BPDCN.

[0212] E49. The method of E45, wherein said hematological malignancy of said patient is T-ALL.

[0213] E50. The hematological malignancy of the patient is refractory to chemotherapy, E2-E49 2. The method according to claim 1 ,

[0214] E51. The above hematological malignancy in the above patient is treated with cytarabine / anthracycline cytotoxic The method of E1 or E50, which is refractory to chemotherapy.

[0215] E52. The hematological malignancy of the patient is refractory to hypomethylating agent chemotherapy; The method according to E1 or E50.

[0216] E53. The expression level of CD123 in blast cells (cancer cells) was compared with that in normal PBMCs. and determining a corresponding baseline level of CD123 expressed. The method according to any one of E2 to E52.

[0217] E54. The level of expression is determined by measuring cell surface expression of CD123. The method according to claim E56.

[0218] The above cell surface expression of E55.CD123 was less than baseline levels of expression. The method according to claim E54, wherein both of the above-mentioned

[0219] E56. The increase in CD123 expression was achieved using the CD123×CD3 bispecific molecule. The method of claim 5, wherein said patient is responsive to said treatment.

[0220] E57. The therapeutic dosage of the CD123×CD3 bispecific molecule is 30, 100, At least one selected from the group consisting of 300, 500, and 600 ng / kg of patient body weight / day The method according to any one of E1 to E4 and E6 to E56, comprising a dose of

[0221] E58. The method of E57, wherein said therapeutic dosage comprises a dose of 30 ng / kg / day.

[0222] E59. The method of E57, wherein said therapeutic dosage comprises a dose of 100 ng / kg / day.

[0223] E60. The method of E57, wherein said therapeutic dosage comprises a dose of 300 ng / kg / day.

[0224] E61. The method of E57, wherein said therapeutic dosage comprises a dose of 500 ng / kg / day.

[0225] E62. The therapeutic dose is administered by continuous infusion, E1-E4 and E6-E61. 2. The method according to claim 1 ,

[0226] E63. The method of any one of E1-E62, wherein said patient is a human patient. EXAMPLES

[0227] Having thus generally described the present invention, the present invention will be further illustrated by reference to the following examples. These embodiments are provided by way of example only and will be readily understood by those skilled in the art. Unless otherwise stated, they are not intended to be limitations of the invention.

[0228] Example 1 Patient populations particularly suitable for treatment with the CD123×CD3 bispecific binding molecules of the invention Gene expression signatures The expression patterns of genes in patients with hematological malignancies, especially AML, and CD123×C To demonstrate a correlation between the favorable outcome of D3 bispecific binding molecule therapy, Mab (NCT#02152956, an exemplary CD123xCD3 bispecific binding molecule) From 40 relapsed or refractory AML patients enrolled in a Phase 1 / 2 clinical trial, individual patients Seventy-eight bone marrow ("BM") samples (36 at baseline, 36 at first RNA was isolated from 27 after the treatment cycle and 15 after the second treatment cycle. Direct multiplexed mRNA quantification of low-abundance transcripts in a single reaction with high sensitivity and linearity The nCounter™ system (NanoString Technology) Gene expression was assessed using the ELISA kit (Vadakekolathu, J., et al. (201 7) “Immune gene Expression Profiling In Children And Adults With Acute Myeloid Leukemia Identifies Distinct Phenotypic Patterns,” Blood 130:3942-3942; Payton, JE, et al. (2009). “High throughput digital quantification of mRNA abundance in primary human acute myeloid leukemia samples,” J Clin Invest 119:1714-1726 Baseline bone marrow samples from 36 patients were included in the analysis, of which 35 patients were treated with doses of 500ng / kg / day or more. NanoString PanCan cer IO360™ Assay (NanoString Technology s, Inc.) for 14 immune cell types and 32 immune-oncological signatures. Covering key pathways at the interface of the tumor, the tumor microenvironment, and the immune response, including at the tumor-specific levels We compared 750 genes in the NanoString PanCancer IO The 360™ assay uses controls for data normalization as provided below. and the expression profiles of an internal reference gene were compared.

[0229] The expression profiles included gene signatures of the following pathways or cells: proliferation, J AKSTAT loss, endothelial cells, B7-H3, APM loss, glycolytic activity, mast cells, cytotoxicity sex, cytotoxic cells, CD8 T cells, lymphocytes, T cells, Treg cells, CTLA4 , TIS, Th1 cells, TIGIT, NK CD56 dim Cells, NK cells, apoptosis , hypoxia, ARG1, IL‐10, IFN gamma, macrophages, bone marrow cells, neutrophils , PD‐L2, stroma, dendritic cells (DCs), MAGE, IDO1, B cells, PD‐1, NO S2, inflammatory chemokines, PD-L1, CD45, exhausted CD8 T cells, immunoproteins asome, APM, IFN downstream regulatory genes, bone marrow inflammatory genes, MHC2 genes, TGF-β genes Data, MMR loss.

[0230] All IO360 gene signature analyses were performed essentially as described below. nCounter™ System with O360 Reporting Module (NanoSt The experiment was carried out using a 3D printer (Fiber Ring Technologies, Inc.).

[0231] Interferon (including representative and non-restrictive NCBI accession numbers for each gene) The (IFN) gamma signaling signature genes and weighting coefficients are shown in Table 6 below.

[0232] [Table 6]

[0233] To calculate the IFN-gamma signaling signature score, the following steps were performed: do: The raw data for each gene was counted using 10 housekeeping K) genes (ABCF1, NRDE2, G6PD, OAZ1, POLR2A, SDHA, STK11IP, TBC1D10B, TBP, UBB). Next, the HK normalized data was compared with the IO360 panel standard, in this case the same as the cohort samples. Normalize against standards run on the same cartridge. Next, each normalized gene count is log-transformed. After normalization and logarithmic transformation, each gene is multiplied by the weight value in Table 6. These weighted numbers are then summed to produce a single score. The final calculated score is For the IFN-gamma signaling signature, The adjustment factor is 6.457026. The adjustment factor is used to set the score range above 0. In particular, it can be derived from the lowest score observed (from TCGA and cell line analysis).

[0234] In general, the possible range of IFN gamma signaling signature scores is 0-10. For this first cohort, the range is 1 to 5. Scores were calculated for each baseline (score 1000, score 15 ... Cleaning day - 14) Calculate for the sample.

[0235] The genes and weighting factors for the additional signatures examined are provided below.

[0236] As detailed below, for this cohort, the IFN-gamma signaling signature We conducted some analyses comparing the scores (and all IO360 signature scores) The differences in fold changes between different patient groups are presented as forest plots, here In the figure, the size of the box indicates significance, and each line indicates a confidence interval (e.g., Figures 3A-3C and 3C). 5A). The distribution of IFN-gamma signaling signature scores among patient groups was Presented as box plots (see, for example, Figure 5B ).

[0237] Baseline expression of the profiled genes was assessed to determine whether patients responded to conventional chemotherapy. Whether or not patients show a refractory response (i.e., cytarabine given in combination with daunorubicin) Patients who showed a refractory response to a treatment regimen (7+3 induction therapy) using CTX or CTX-refractory) or hypomethylating agents (e.g., desitavidin) Patients who showed a refractory response to a treatment regimen using cyclosporine and azacitidine (Ref HMA or HMA-refractory) or relapsed patients Secondary AML (i.e., arising from myelodysplasia or from a previous chemical reaction) was correlated with Patients with AML (AML as a product of AML therapy) were included in the HMA-refractory group for these analyses. Data are included in the CD123×CD3 bispecific binding study using flotetuzumab. The results also correlated with patient response to molecular therapy. Figure 4 shows the results of 25 evaluations of patients treated with the target dose. These responses are based on the objective response ( Patients were scored as having either an OR or no response (NR). In addition, OR was classified as molecular complete response (mCR), complete response with incomplete hematologic improvement (CRi), or complete response with incomplete hematologic improvement (CRi). ), morphological leukemia-free status (MLF), and partial response (PR) were included. In addition to non-responders, NR included patients with progressive disease / treatment failure (PD) and stable disease ( All patients showing SD were included.

[0238] Figure 2 shows the teacher-generated IO360 signatures of 46 cell types generated from the above results. The results are shown in Table 1. Baseline levels of expression of genes were compared for each gene signature evaluated (each in a separate row). To facilitate comparison between signatures, each IO360 signature score is was rescaled to a scale of −3 to +3 within the scores for this cohort.

[0239] Gene expression analysis of baseline BM samples identifies AML patients with a continuum of immunology The primary deficiency was stratified into three clusters in the body: immune depletion, immune exhaustion, and immune enhancement (Figure 2). Patients with refractory disease (relapsed; refractory to 2 or more induction trials, first CR within 6 months Failure after less than 1 or 4 cycles of hypomethylating agent HMAs was primarily due to immune infiltration of tumor microenvironments. showed a TME phenotype, which is characterized by a higher inflammatory chemokine score compared to relapsed patients (3.27±0.22 vs. 2.46±0.07, p=0.026). Among these groups, chemotherapy-refractory and HMA-refractory patients were treated with immune enhancement and immune therapy, respectively. Further stratification into immune exhaustion phenotypes. Gene signatures associated with immune exhaustion and immune enhancement phenotypes The results are listed in Table 7 below and shown in the forest plots in Figures 3A, 3B, and 4A. As shown above.

[0240] [Table 7]

[0241] All refractory and relapsed patients (Figure 3A), HMA refractory and relapsed patients (Figure 3B), H The basis of multiple gene signatures between MA-refractory and CTX-refractory patients ( Fig. 3C ). Forest plot of line fold change differences shows that HMA-refractory patients have a more senescent phenotype Specifically, HMA-refractory patients exhibit immune exhaustion and adaptive immune resistance. The tumors showed a myeloblastic feature, and CD8 T cells (CD244, EOMES) were more abundant than those in CTX-refractory patients. , LAG3, and PTGER4) tended to be increasingly exhausted, while TIGIT (5.55 ±0.34 vs 3.85±0.24, p=0.006), PD‐L1(3.55±0 .18 vs 2.4±0.29, p=0.009) and Treg cells (4.87±0. 23 vs 3.69±0.19, p=0.0009), immune exhaustion and The results show characteristics of adaptive immune resistance (Figures 3A-3C). were either immune-enhanced (cluster 2, Figures 3D-3I) or immune-exhausted (cluster 3, Figures 3J-3O). The gene signature scores associated with the profile are shown in Fig. 3D. phages (Figure 3E), neutrophils (Figure 3F), B cells (Figure 3G), IFN gamma (Figure 3H ), PD‐L1 (Fig. 3I), TIGIT (Fig. 3J), CTLA‐4 (Fig. 3K), Th1 ( L), CTL (Fig. 3M), CD8 T cell (Fig. 3N), and cytotoxicity (Fig. 3O). The gene signature scores were plotted for each cluster (immunodepletion (Depl. ), immune enrichment (Enriched), and immune exhaustion (Exh.), p-value (Kruskal Wallis) reports.

[0242] A comparative analysis of IFN-gamma signaling signature scores was performed in OR patients (CR, complete response). A; mCR, molecular CR; CRi, complete response with incomplete hematologic improvement; MLF, morphological (including all patients who achieved a clinically relevant leukemia-free state; or PR, partial response) and NR patients (SD, or PD, including all patients with progressive disease / treatment failure) Figure 4 shows the CD123xCD3 bispecific binding at a target dose of 500 ng / kg / day. As a measure of response to molecular flotetuzumab, From 25 patients (classified as CTX-resistant or HMA-refractory patients) Changes (vs. baseline) in blast cells present in bone marrow samples are shown. The objective response (OR) rate to the above regimen for patients with glaucoma was 50% (7 / 14). The complete response (CR) rate for primary refractory patients was 35.7% (5 / 14). Ta.

[0243] Figure 5A shows the baseline between OR and NR patients (including PD, SD, TF, and NE). A forest plot of the difference in fold transformation of IFN-gamma signaling is presented. Figure 5A shows that expression of the IL-1 signature was increased in baseline samples of OR patients. Furthermore, the TIS and IFN downstream signatures were significantly higher in OR patients than in NR patients. Figure 5B shows the distribution of IFN-gamma signaling signature scores. In particular, the response to flotetuzumab was increased in OR patients. Responders had significantly higher IFN-gamma signaling at baseline compared with non-responders showed a signature score (3.31±0.32 vs. 2.27±0.11, p=0. 0005). The sensitivity and specificity of the IFN gamma signaling signature score were measured. The diagnostic ability of response was estimated using different threshold cutoffs for the range of data in this cohort. Bootstrapping is performed for all samples using the threshold or sensitivity and specificity values Confidence intervals (CIs) were calculated using bootstrap resampling and averaging methods. For all bootstrap CIs, patients were resampled and corrected curves were constructed. After constructing the model, we calculate the statistics of interest. The screening was performed in a stratified fashion by default. IF of area under the curve (AUC) = 0.819. Receiver operating characteristics (referred to herein as “characteristics”) showing the predictive performance of the N gamma signaling signature score. The area under the curve (abbreviated as ROC in this paper) is plotted in FIG. 5C. To find samples with high or low IFN-gamma signaling signature scores, The true positive rate (TPR) and false positive rate (FPR) achieved using the optimal score cutoff for the cohort The ROC curves for the 1st and 2nd genotypes are plotted along the diagonal. A signature with perfect predictive power would have a curve that reaches the upper left corner. The shaded areas surrounding the lines indicate the confidence intervals. These data are for primary refractoriness. This is consistent with the high frequency of responders among patients, who generally had higher rates of relapse than relapsed patients. Thus, baseline IFN gamma signaling signature was elevated in both groups compared with control. Gamma signaling signature score predicts efficacy of CD123×CD3 bispecific binding molecule therapy The results show a strong correlation with patient response to Flotetuzumab (AUC = 0.919; Figure 5C). Clusters of immune signatures and response rates at baseline. Comparison between clusters Immune Depletion (Cluster 1) and Immune Infiltration (Cluster 2-3) is shown in Table 8. ), and Table 9 (Immune Exhaustion (Cluster 2) and Immune Enhancement (Cluster 3)).

[0244] [Table 8]

[0245] [Table 9]

[0246] A panel of genes related to the stimulation of cytotoxic cells or related to CD8+ T cells Gene expression signatures were generated from pre-treatment bone marrow samples ("base") and after flotetuzumab. In RNA from a bone marrow sample following the first cycle of treatment with The results of this study are shown in Figure 6. The results show that treatment with flotetuzumab: It has been demonstrated that immune cells in the tumor microenvironment were stimulated. Comparison of BM samples with baseline samples demonstrated that treatment with flotetuzumab significantly increased immune cell Elevated infiltration and immune activation scores (which manifest as a higher tumor inflammatory signature) 6.49±0.20 vs 5.93±0.12, p=0.015), and enhanced immune protein asome score (5.72±0.07 vs. 5.23±0.10, p=0.0002) and IFN-gamma signaling signature score (3.38 ± 0.23 vs. 2.53 It was shown that this leads to an increase in the vascular endothelial cell proliferation rate (Human vascular disease) and the vascular endothelial cell proliferation rate (Human vascular disease) (±0.14, p=0.0015). Tumor microenvironment (TME) gene activation induced by izumab represents an immune exhaustion signature rather, it was an indication of an immune-boosting signature.

[0247] As shown in Figure 7, the flotetuzumab-responsive population and, in particular, the over-responsiveness to chemotherapy Patients who were refractory to chemotherapy showed higher expression of CD123.

[0248] AML blast samples collected during screening were screened for PD-1 by flow cytometry. As shown in Figure 8, early onset of flotetuzumab treatment was Patients who progressed to early stage (<15 days) had higher expression of PD-L1 on AML cells than other patients. They had high baseline levels and evidence of response (SD, OB, PR, CR). The results of this study indicate that PD-L1 expression is associated with reduced activity in vivo. PD-1 / PD-L1 antagonist and CD123×CD3 bispecific binding molecule therapy The authors support the use of the method in combination with other methods (see, for example, WO 2017 / 214092).

[0249] Overall, these data suggest that baseline IFN-gamma signaling signaling is showed that β-lactamase correlated with response to CD123×CD3 bispecific binding molecule therapy. Evidence of anti-leukemic activity for CD123×CD3 bispecific binding molecule therapy Most patients (6 / 7; 86%) with BRCA1 had high immune infiltration in the bone marrow. The most sensitive populations are the immune-compromised population. In addition, patients previously treated with HMAs represents an immune-enhanced but exhausted tumor microenvironment (e.g., bone marrow) and expression of CD123× in combination with immune checkpoint blockade. These findings suggest a potential benefit of CD3 bispecific binding molecule therapy. Although not limited by the above, CD123×CD3 bispecific binding molecule therapy is a promising candidate for this cluster. Activates the immune-exhausted tumor microenvironment as demonstrated by 25% anti-leukemic activity in the cohort In particular, treatment with DART-A, a CD123×CD3 bispecific binding molecule, has been reported. The treatment was assessed using immune activation, antigen processing / presentation, and IFN-gamma signaling signature scores. Enhancement was observed.

[0250] Example 2 Gene expression signatures of relapsed chemotherapy-refractory patient populations IFN-gamma signaling signatures, TIS, and AML in cases of immune infiltration Higher expression of genes including, but not limited to, the interferon downstream signature , using bispecific immunotherapeutic agents targeting CD123 × CD3, e.g., flotetuzumab Further analyses were performed to further explore the correlation between the treatment benefit and the The study included 30 patients enrolled in the CP‐MGD006‐01 clinical trial (NCT#02152956). chemotherapy refractory (refractory to ≥2 induction trials, first complete response <6 months) ) or from patients with relapsed AML (essentially as described below) Gene signatures obtained using the PanCancer IO360™ assay This analysis focused on the combination of markers and signatures derived from HMA-refractory patients. samples were excluded, and further samples from previously unanalyzed relapsed and chemotherapy-refractory patients were included. It included:

[0251] This analysis identified three signature modules: IFN-dominant, adaptive, and myeloid scores. By aggregating the data, BM samples from relapsed and refractory AML patients at baseline were were stratified into two immune subtypes, referred to herein as immune infiltration and immune depletion ( Figure 9). The gene signatures associated with these three signature modules are listed in Table 1 below. The module score is the sum of the individual gene signature scores for each sample. (Each gene signature score was calculated as described above.)

[0252] [Table 10]

[0253] Figure 9 shows the results of the CP-MGD006-01 clinical trial (NCT#02152956). Bone marrow (BM) microcirculation in patients with relapsed / refractory AML before receiving izumab immunotherapy Unsupervised hierarchical clustering of immune and biological activity signatures in the human immune system (Eugene, Responders are defined as complete remission (CR), incomplete hematologic recovery (CR), or complete hematologic recovery (CR). CR with recovery (CRi), CR with partial hematologic recovery (CRh), partial remission (PR) ), or “other benefit” (OB; >30% reduction in BM blasts) Non-responders were those who demonstrated an anti-leukemic response, defined as treatment failure (TF), Patients were classified as having stable disease (SD) or progressive disease (PD). Defined as first CR with 2 or more induction attempts or initial CR duration less than 6 months To facilitate comparison between signatures, each IO360 signature score was calculated based on this Within scores on the Hort were rescaled to a scale of -3 to +3.

[0254] Anti-leukemia CD123×CD3 bispecific binding molecule therapy with flotetuzumab BM samples from 92% (11 of 12) of patients with evidence of response were from non-responders had immune infiltrated TME compared with nonresponders (Figure 9).

[0255] FIG. 10 shows the results of responders (CR, CRi) from 30 chemotherapy-refractory or relapsed AML patients. , CRh, PR, and OB) and non-responders (PD, SD, TF) A forest plot of the difference in change is presented, consistent with the analysis provided in Example 1 above. Thus, the IFN gamma signaling signature (encircled in bold in FIG. 10 ), The expression of IFN downstream signatures and tumor inflammatory signatures was significantly higher in responders than in non-responders. Furthermore, the gene signature constituting the IFN-dominant module was increased in the baseline samples. Most were elevated in baseline samples of responders compared to non-responders (asterisks in Figure 10). is at risk).

[0256] IFN-gamma signaling signatures between refractory and relapsed patients ( Fig. 11A ); IFN downstream signature (Figure 11B), tumor inflammation signature (TIS, Figure 11C), and I The distribution of FN-dominant module (Figure 11D) scores is plotted in Figures 11A-11D. The distribution of scores is increased in refractory patients. A nine-gene signature constituting an IFN-dominant module and tumor inflammation in OR The distribution of signature scores is plotted in Figures 12A-12J: IFN gamma signature signaling signature (Fig. 12A); IFN downstream signature (Fig. 12B); bone marrow inflammatory signature necha (Figure 12C); immunoproteasome signature (Figure 12D); inflammatory chemokine signature (Figure 12E); signature (Fig. 12E); MAGE signature (Fig. 12F); PD-L1 signature (Fig. 1 2G); PD-L2 signature (Fig. 12H); IL10 signature (Fig. 12I); Tumor inflammation The distribution of scores for these gene signatures is In particular, as shown in Table 11, the responders were non- Compared with responders, patients had significantly higher IFN-gamma signaling signatures at baseline. The IFN downstream signature, TIS, and IFN dominant module scores were shown. For decisions not involving the same group, comparisons were made using the Mann-Whitney U test.

[0257] [Table 11]

[0258] Essentially as described above, a nine-gene signature constituting the IFN-dominant module was generated. , TIS, and the sensitivity of the scores for the IFN-dominant module in this group of patients ( Response diagnostic ability (ROC AUC) was measured by measuring the accuracy (detection rate) and specificity (false positive rate). The ROC curves showing the prediction performance are presented in Figures 13A-13K. IFN downstream signature (Figure 13A, AUC=0.750); (Figure 13B, AUC=0.755); bone marrow inflammation signature (Figure 13C, AUC=0.69 ); immune proteasome signature (Figure 13D, AUC=0.505); inflammatory chemokines AU signature (Figure 13E, AUC=0.764); MAGE signature (Figure 13F, C=0.736); PD-L1 signature (Fig. 13G, AUC=0.699); PD-L 2 signature (Figure 13H, AUC = 0.727); IL10 signature (Figure 13I, AU C = 0.745); TIS (Figure 13J, AUC = 0.852); and IFN-dominant modules le (Figure 13K, AUC = 0.806).

[0259] On-treatment BM samples (obtained from 19 patients at the end of cycle 1) were showed increased antigen presentation and immune activation against the IgG samples (for unpaired determinations, Comparisons were performed using the Mann-Whitney U test), which was associated with higher TIS scores ( 6.47±0.22 vs 5.93±0.15, p=0.0006, Fig. 14A), antigen Processing machine (APM) signature score (5.67±0.16 vs. 5.31± 0.12, p = 0.002, Figure 14C), IFN-gamma signaling signature score (3.58±0.27 vs 2.81±0.24, p=0.0004, Figure 14B), and and PD-L1 signature score (3.43 ± 0.28 vs. 2.73 ± 0.21, p = 0.0062; FIG. 14D). This result is reflected in AM with immune-infiltrated TME. We demonstrated clinical benefit in patients with L. Supports the nodal effect.

[0260] As described above, A has an immune-enhanced and IFN-gamma-dominant tumor microenvironment ("TME"). Patients with ML have been reported to have significantly shorter recurrence-free survival, which is consistent with standard induction therapy. This suggests that the disease is unresponsive to chemotherapy (Vadakekolathu, J. et al. (2017) “T Immune G ene Expression Profiling in Children and Adults with Acute Myeloid Leukemia Iden tifies distinct phenotypic patterns,” Blood 130:3942A. These data are IFN gamma signaling signature, IFN downstream signature, and I FN dominance module score indicates resistance to standard chemotherapy and CD123×CD 3 shows that the IL-16 expression level strongly correlates with response to bispecific binding molecule therapy. In heavily pretreated individuals (mean 4 prior therapies) who were evaluated, IFN-dominant Most of the gene signatures that make up the tumor inflammation signature (TIS) are These results correlated with the response to 123×CD3 bispecific binding molecule therapy. Each score is calculated based on the proportion of patients with chemotherapy-refractory AML compared to those with relapsed AML at the time of treatment. was significantly higher in patients who responded to chemotherapy and had evidence of anti-leukemic activity compared with non-responders. The strong correlation was reflected in the ROC curve and AUC value. There are.

[0261] Gene signatures The number of IO360 genes was basically calculated using nCounter®s as follows: system (NanoString Technologies, Inc.) RNA (~100 ng / sample) was purified from bone marrow aspirates and hybridized. For detection, the reporter and capture probe mixture was incubated. Count transcripts on the nCounter FLEX Analysis System using resolution settings As described in detail herein, the reporter code number Using the RCC) output file, basically the same as above Using a predefined linear combination (weighted average) of biologically relevant gene sets , and calculate the gene signature score.

[0262] The IFN-gamma signaling signature has been previously described in detail. The abundance signature of the genomic DNA was determined by the following method: Danaher, P., et al., 2017, “Gene Expression Markers of Tumor Infiltrating Leukocytes,” J Immunother Cancer 5, 18. , tumor inflammation signatures are Danaher, P., et al., 2018 (“Pan-cancer Adaptive Immune Recognition sistance as Defined by the Tumor Inflammation Signature (TIS): Results From The Cancer Genome Atlas (TCGA),” J Immunother Cancer. 6(1):63) (Ayers. M., et al. 2017, “IFN-γ-Related mRNA Profile Predicts Clinical Response to PD-1 blockade” J Clin Invest. 127(8):2930-2940 Other signatures include those described by Dana her, P., et al., (2018, “Development of Gene Expression Signatures Characterizi ng The Tumor-Immune Interaction,” J Clin Oncol 36, 205-205. For ease of reference, the selected gene signatures used in these studies are The genes and weighting factors for the marker are provided below.

[0263] Tumor inflammation signature (TIS) genes (representative and non-restrictive NCB for each gene) I registration number), and weighting factors (see, for example, WO 2016 / 094377). ) are shown in Table 12A below.

[0264] [Table 12]

[0265] Interferon (IFN) downstream signature genes (representative and non-regulatory information for each gene) The signatures, including the applicable NCBI accession numbers, and weighting factors are shown in Table 12B below. The adjustment factor for tea is 5.342598.

[0266] [Table 13]

[0267] Inflam chemokines signature genes (each gene The following table lists the NCBI accession numbers (including representative but non-limiting NCBI accession numbers for each gene) and weighting factors: 12C. The adjustment factor for this signature is 6.0968.

[0268] [Table 14]

[0269] MAGE signature genes (representative and non-restrictive NCBI accession numbers for each gene) The weighting factors and the weighting coefficients for this signature are shown in Table 12D below. The adjustment coefficient for this signature is , 3.965625.

[0270] [Table 15]

[0271] Myeloid Inflamation Signature Genes (Representatives for each gene) The gene sequences (including non-limiting NCBI accession numbers) and weighting factors are shown in Table 12E below. The adjustment factor for this signature is 5.41931.

[0272] [Table 16]

[0273] Immunoproteasome signature genes (representative and non-restrictive NCBs for each gene) The ID, registration number, and weighting factor for this signature are shown in Table 12F below. The adjustment factor is 6.096812.

[0274] [Table 17]

[0275] Single gene signature genes (representative and non-restrictive NCBI accession numbers for each gene) The adjustment factors, including the above-mentioned factors, are set forth in Table 12G below.

[0276] [Table 18]

[0277] The signature scores, after normalization and log transformation, are provided for each gene in Tables 12A-12F. Essentially the same as above, except multiplying by the weights indicated and adding the adjustment factors indicated. For single gene signatures (e.g. PDL1), the weights are calculated as Not used, log2 normalized gene expression values ​​are added to the adjustment factors provided in Table 12G. do.

[0278] All publications and patents referred to herein are incorporated by reference in their entirety. Each is specifically and individually indicated to be incorporated by reference in its entirety. The present invention is hereby incorporated by reference to the same extent as if fully set forth herein. While the invention has been described in terms of embodiments, further modifications are possible and it is to be understood that the invention is not limited to the disclosed embodiments. as is within the known method or practice of the art, and applicable to the essential features set forth above. Any variation of the present invention that generally follows the principles of the present invention, including deviations from the present disclosure, It is to be understood that all uses or modifications are intended to be covered.

Claims

1. 1. A method of treating a chemotherapy refractory hematological malignancy in a patient, comprising: The method comprises administering to the patient a therapeutic dosage of a CD123xCD3 bispecific molecule. Including steps, The dosage is effective to stimulate killing of cells of the hematological malignancy in the patient. wherein the method is effective for treating said malignant tumor.

2. The method further comprises administering to the patient a CD123xCD3 bispecific molecule prior to and / or after administration of the CD123xCD3 bispecific molecule. assessing the expression of one or more target and / or reference genes in a cell sample from the subject. The method of claim 1 further comprising:

3. Patients responding to the use of CD123×CD3 bispecific molecules to treat hematological malignancies 1. A method for determining whether a patient is a preferred responder to a test, comprising the steps of: The method comprises: (a) determining whether the CD123×CD3 expression of one or more target genes in a cell sample from said patient prior to administration of the bispecific molecule; evaluating (b) determining whether the expression of the one or more target genes is related to the one or more target and / or reference genes; If the expression of CD123×CD is found to be increased, the patient is 3. Identifying Preferred Responders to Treatment with Bispecific Molecules A method comprising:

4. The method comprises: (i) expression of the one or more target genes; and (ii) one or more reference genes whose expression is not characteristically associated with hematological malignancies The method according to claim 2 or 3, wherein the

5. The method further comprises: determining whether said one or more reference genes are related to a baseline expression of said one or more reference genes in said patient; The method of claim 2 or 3, comprising evaluating the expression of one or more target genes.

6. The method comprises: (a) an individual, or a population of a plurality of such individuals, suffering from said hematological malignancy; or (b) the use of a CD123×CD3 bispecific molecule for treating said hematological malignancies. or a population of such individuals who have not responded well to (c) the use of the CD123×CD3 bispecific molecule for treating said hematological malignancies. An individual or a group of a plurality of such individuals who have had a good response to of the one or more target genes in the patient with respect to the expression of the one or more target genes. The method of any one of claims 2 to 5, comprising the step of assessing expression.

7. The relative expression levels of the one or more target genes in the population are determined by dividing the population of individuals by the The gene expression level is established by averaging the gene expression levels in the obtained cell samples.

7. The method according to claim 5 or 6.

8. The patient has an expression level of at least one of the target genes: (a) detecting the expression level of said target gene in a population of individuals suffering from said hematological malignancy; higher than the first quartile of the (b) The use of CD123×CD3 bispecific molecules in the treatment of said hematological malignancies. A first quartile of expression levels of the target gene in a population of individuals who were not favorable responders. higher than the quantile; or (c) The use of CD123×CD3 bispecific molecules in the treatment of said hematological malignancies. to the expression level of the target gene in a population of individuals who did not have a favorable response At least about 0.4 log 2 have a fold change; or (d) A method for the treatment of said hematological malignancies using CD123×CD3 bispecific molecules. At least one third of the expression level of the target gene in a population of individuals who had a favorable response Within one quartile The method according to any one of claims 2 to 7, which indicates the expression level.

9. 1. A method of treating a hematological malignancy, the method comprising: (a) The method according to any one of claims 3 to 8, wherein a patient is The present invention relates to a method for treating tumors in which the subject is a favorable responder to the use of CD123×CD3 bispecific molecules to treat tumors. determining whether (b) administering a treatment if the patient is determined to be a favorable responder to the treatment. administering to said patient an amount of said CD123xCD3 bispecific molecule Including, The administration of the CD123xCD3 bispecific molecule inhibits the hematological malignancy in the patient. A method for stimulating the killing of tumor cells.

10. The method according to any one of claims 2 to 9, wherein the cell sample is a bone marrow sample.

11. said assessing expression or said patient receiving CD123×CD3 Said determination of whether a patient is a suitable responder to the use of a bispecific molecule comprises: (a) detecting a gene expression profile for each target gene in one or more cell samples using a gene expression platform; determining the gene expression level of (b) comparing the expression level of the target gene with the expression level of one or more reference genes. Tep The method according to any one of claims 2 to 10, which is carried out by

12. said assessing expression or said patient receiving CD123×CD3 Said determination of whether a patient is a suitable responder to the use of a bispecific molecule comprises: (a) detecting a gene expression profile for each target gene in one or more cell samples using a gene expression platform; determining the raw RNA level of the gene expression platform, a reference gene set of mouse-keeping genes; and (b) using the measured RNA level of an internal reference gene to measure the RNA level of said target gene; assigning a relative expression value to each of the determined raw RNA levels. The method according to any one of claims 2 to 11, which is carried out by

13. The one or more target genes include: (a) one or more of CXCL9, CXCL10, CXCL11, and STAT1; and / or (b) CCL5, CD27, CD274, CD276, CD8A, CMKLR1, CX CL9, CXCR6, HLA-DQA1, HLA-DRB1, HLA-E, IDO1, L AG3, NKG7, PDCD1LG2, PSMB10, STAT1, and TIGIT and / or (c) AREG, CSF3, CXCL1, CXCL2, CXCL3, CCL20, FO One or more of SL1, IER3 (NM_003897.4), IL6, and PTGS2 above; and / or (d) one or more of CCL2, CCL3 / L1, CCL4, CCL7, and CCL8 above; and / or (e) MAGEA3 / A6, MAGEA1, MAGEA12, MAGEA4, MAGE B2, MAGEC1, and MAGEC2; and / or (f) APOL6, DTX3L, GBP1, IFI16, IFI27, IFI35, I FI6, IFIH1, IFIT1, IFIT2, IFIT3, IFITM1, IFITM 2, IRF1, IRF9, ISG15, MX1, OAS1, OAS2, PARP9, PS one or more of MB9, STAT2, TMEM140, and TRIM21; and / or teeth (g) one or more of PSMB8, PSMB9, and PSMB10; and / or (h) IL-10; and / or (i) CD274; and / or (j) PDCD1LG2 The method according to any one of claims 2 to 12, comprising:

14. The one or more reference genes are ABCF1, G6PD, NRDE2, OAZ1, POL One of R2A, SDHA, STK11IP, TBC1D10B, TBP, and UBB The method according to any one of claims 2 to 13, comprising one or more.

15. A gene signature score is determined for the one or more target genes according to claims 2 to 1.

5. The method according to any one of claims 4 to 4.

16. The gene signature score is: (a) A gene expression platform comprising a reference gene set of housekeeping genes. to determine the raw RNA levels for each of the target genes in one or more cell samples. measuring; (b) comparing each of the measured raw RNA levels to the geometric mean of the housekeeping gene; normalizing to the mean and optionally further normalizing each RNA value to a standard; (c) log-transforming each normalized RNA value; (d) multiplying each log-transformed RNA value by a corresponding weighting factor to obtain a weighted RNA value generating (e) adding the weighted RNA values ​​together, optionally with an adjustment coefficient constant, to obtain a single gene Steps for generating child signature scores The method of claim 15, wherein the determination is made by a process comprising:

17. The gene signature score is calculated by dividing the number of gene signatures by one or more of the target genes, a scoring weight, and and optionally using the adjustment factors provided in Tables 6 and 12A-12G. Or the method according to claim 16.

18. The gene signature score is: (a) IFN gamma signaling signature; (b) tumor inflammation signature; (c) bone marrow inflammation signature; (d) inflammatory chemokine signature; (e) MAGE signature; (f) IFN downstream signaling signature; (g) immune proteasome signature; (h) IL-10 signature; (i) a PD-L1 signature; and / or (j) PD-L2 signature 17. The method according to claim 15, wherein the gene signature score is determined for one or more of the following:

2. The method according to claim 1 .

19. A patient genetic signature score, comprising: (a) detecting one of the target genes in a population of individuals suffering from the hematological malignancy; The first quartile of the score for the gene signature calculated from the expression levels of greater than; or (b) The use of CD123×CD3 bispecific molecules in the treatment of said hematological malignancies. The expression of one or more of the target genes in a population of individuals who did not have a favorable response to the greater than the first quartile of the score for said gene signature calculated from the current level; or (c) The use of CD123×CD3 bispecific molecules in the treatment of said hematological malignancies. The expression of one or more of the target genes in a population of individuals who did not have a favorable response to the at least about 0.4 for the score for said gene signature calculated from the current level Log of 2 have a fold change; or (d) A method for the treatment of said hematological malignancies using CD123×CD3 bispecific molecules. The expression levels of one or more of the target genes in a population of individuals who had a favorable response At least within the first quartile of the score for the gene signature calculated from the be, The patient gene signature score is a predictor of treatment with the CD123xCD3 bispecific molecule. The method according to any one of claims 15 to 18, which is indicative of a relatively favorable patient response to How to.

20. (a) the gene signature is an IFN-gamma signaling signature, A patient gene signature score of about 2.5 identifies the CD123×CD3 bispecific molecule. is indicative of a relatively favorable patient response to the treatment used; and / or (b) the gene signature is a tumor inflammation signature, and is at least about 5.5 The gene signature score is a predictor of response to treatment with the CD123xCD3 bispecific molecule. and / or is indicative of a relatively favorable patient response; (c) the gene signature is an IFN downstream signaling signature, A patient gene signature score of about 4.5 was obtained using the CD123×CD3 bispecific molecule. is an indicator of a relatively favorable patient response to a given treatment.

19. The method according to claim 17 or 18.

21. The gene signature may be an IFN-gamma signaling signature, a tumor inflammation signature, or IFN downstream signaling signature according to any one of claims 17 to 19. Method of posting.

22. Showing gene expression signatures characteristic of immune-enhanced and IFN-gamma-dominant tumor microenvironments The patient is a relatively favorable patient for treatment with the CD123xCD3 bispecific molecule. The method according to any one of claims 17 to 21, wherein the method is an indicator of a subject's response.

23. The CD123xCD3 bispecific molecule may be a bispecific antibody or a bispecific The method according to any one of claims 1 to 22, wherein the molecule is an isomeric molecule.

24. The CD123xCD3 bispecific molecule is JNJ-63709178, XmAb14 045, or APVO436.

25. The CD123xCD3 bispecific molecule comprises: (a) a first polypeptide chain having the amino acid sequence of SEQ ID NO:21; and (b) a second polypeptide chain having the amino acid sequence of SEQ ID NO:

23. and wherein the covalent bispecific diabody comprises The first polypeptide chain and the second polypeptide chain are connected by a disulfide bond. The method of any one of claims 1 to 22, wherein the molecules are covalently bonded to each other.

26. The hematological malignancy of the patient is: acute myeloid leukemia (AML); chronic myeloid leukemia (CML); ML); blast crisis of CML; Abelson oncogene associated with CML (Bcr-ABL translocation); myelodysplastic syndrome (MDS); acute B-lymphoblastic leukemia (B-ALL); acute T-lymphoblastic leukemia (T-ALL); chronic lymphocytic leukemia (CLL); Richter's syndrome ; Richter's syndrome transformation in CLL; Hairy cell leukemia (HCL); Blastic plasmacytoid Dendritic cell neoplasm (BPDCN); mantle cell lymphoma (MCL) and small lymphocytic lymphoma Non-Hodgkin's lymphoma (NHL), including non-Hodgkin's lymphoma (SLL); Hodgkin's lymphoma, systemic lymphoma and Burkitt's lymphoma.

2. The method according to claim 1.

27. 27. The method of claim 26, wherein the hematological malignancy in the patient is AML.

28. The method according to any one of claims 2 to 27, wherein the hematological malignancy in the patient is refractory to chemotherapy. The method according to any one of claims 1 to 5.

29. The therapeutic dosage of the CD123xCD3 bispecific molecule is 30, 100, 300, and 500 ng / kg of patient body weight / day. The method of any one of claims 1 to 4 and 6 to 28, comprising:

30. Any of claims 1, 2 and 4-29, wherein the therapeutic dosage is administered by continuous infusion.

2. The method according to claim 1.

31. The method of any one of claims 1 to 30, wherein the patient is a human patient.

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