Bispecific CD112x CD3 diabodies for treatment of hematological malignancies

Abstract

The present invention relates to methods of treating hematological malignancies, such as acute myeloid leukemia (AML) or spinal dysplasia syndrome (MDS), including hematological malignancies that are refractory to chemotherapeutic agents and / or demethylating agents. Methods involve administering to a patient a CD123x CD3 bispecific binding molecule in an amount effective to stimulate cells that kill the hematological malignancy in the patient. The invention is further directed to embodiments of such methods in which a cell sample from a patient demonstrates the expression of one or more target genes relative to the baseline expression level of such genes, e.g., in a reference population of individuals suffering from hematological malignancies, such as, for example, the expression level of such genes, e.g., in the reference population of individuals suffering from hematological malignancies, or in the reference population of individuals suffering from hematological malignancies. Or the expression level relative to the reference gene is increased.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201980087139.5, application date October 29, 2019, and invention name “Bispecific CD123 x CD3 dual antibody for treating hematological malignancies”. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. patent application serial numbers 62 / 878,368 (filed on July 25, 2019; pending), 62 / 769,078 (filed on January 19, 2018; pending), and 62 / 752,659 (filed on October 30, 2018; pending), each of which is incorporated herein by reference in its entirety. References to sequence listings

[0003] In accordance with 37 CFR 1.821 et seq., this application includes one or more sequence listings, which are disclosed in a computer-readable medium (file name: 1301_0155PCT_ST25.txt, created on September 26, 2019, and 31,244 bytes in size), which is incorporated herein by reference in its entirety. Technical Field

[0004] The present invention relates to a method for treating hematological malignancies such as acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), including hematological malignancies that are refractory to chemotherapeutic agents and / or demethylating agents. The method involves administering a CD123 x CD3 bispecific binding molecule to a patient in an amount that effectively stimulates killing of cells of the hematological malignancies in the patient. The present invention is further directed to an embodiment of this method, wherein a cell sample from a patient demonstrates that the expression of one or more target genes is relative to the baseline expression level of such genes, for example, the baseline expression level of such genes in a reference population of individuals suffering from hematological malignancies, or relative to the expression level of a reference gene. Background Art I.CD123

[0005] CD123 (interleukin 3 receptor alpha, IL-3Ra) is a 40 kDa molecule and is part of the interleukin 3 receptor complex (Stomski, FC et al. (1996) "Human Interleukin-3 (IL-3) Induces Disulfide-Linked IL-3 Receptor Alpha-And Beta-Chain Heterodimerization, Which Is Required For Receptor Activation But Not High-Affinity Binding," Mol. Cell. Biol. 16 (6): 3035-3046). Interleukin 3 (IL-3) drives the early differentiation of pluripotent stem cells into erythroid progenitors, myeloid progenitors, and lymphoid progenitors. CD123 is expressed on CD34+ directed progenitor cells (Taussig, DC et al. (2005) "Hematopoietic Stem Cells Express Multiple Myeloid Markers: Implications For The Origin And Targeted Therapy Of Acute Myeloid Leukemia," Blood 106: 4086-4092), but not by CD34+ / CD38- normal hematopoietic stem cells. CD123 is expressed by basophils, mast cells, plasmacytoid dendritic cells, some by monocytes, macrophages and eosinophils, and low or no expression by neutrophils and megakaryocytes. Some non-hematopoietic tissues (placenta, interstitial cells of the testis, some brain cell components and some endothelial cells) express CD123; however, expression is mostly cytoplasmic.

[0006] It is reported that CD123 is expressed by leukemic blasts and leukemic stem cells (LSC) (Jordan, CT et al. (2000) "The Interleukin-3 Receptor Alpha Chain Is A Unique Marker For Human Acute Myelogenous Leukemia Stem Cells," Leukemia 14: 1777-1784; Jin, W. et al. (2009) "Regulation Of Th17 Cell Differentiation And EAE Induction By MAP3K NIK," Blood 113: 6603-6610). In human normal precursor populations, CD123 is expressed by a subtype of hematopoietic progenitor cells (HPCs), but not by normal hematopoietic stem cells (HSCs). CD123 is also expressed by plasmacytoid dendritic cells (pDCs) and basophils, and to a lesser extent by monocytes and eosinophils (Lopez, AF et al. (1989) "Reciprocal Inhibition Of Binding Between Interleukin 3 And Granulocyte-Macrophage Colony-Stimulating Factor To Human Eosinophils," Proc. Natl. Acad. Sci. (USA) 86:7022-7026; Sun, Q. et al. (1996) "Monoclonal Antibody 7G3 Recognizes The N-Terminal Domain Of The Human Interleukin-3 (IL-3) Receptor Alpha Chain And Functions As A Specific IL-3 Receptor Antagonist," Blood 87:83-92; L. et al. (2001) "Interleukin-3Receptor Alpha Chain(CD123)Is WidelyExpressed In Hematologic Malignancies," Haematologica 86(12):1261-1269; Masten, BJ et al. (2006) "Characterization Of Myeloid And Plasmacytoid Dendritic Cells InHuman Lung," J. Immunol. 177:7784-7793; Korpelainen, EI et al. (1995) "Interferon-GammaUpregulates Interleukin-3(IL-3)Receptor Expression In Human Endothelial CellsAnd Synergizes With IL-3In Stimulating Major Histocompatibility Complex ClassII Expression And Cytokine Production," Blood 86:176-182).

[0007] CD123 has been reported to be overexpressed on malignant cells in a wide range of hematological malignancies including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). L. et al. (2001) "Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed In Hematologic Malignancies," Haematologica 86 (12): 1261-1269). Overexpression of CD123 is associated with a poor prognosis in AML (Tettamanti, MS et al. (2013) "Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel CD123-Specific Chimeric Antigen Receptor," Br. J. Haematol. 161: 389-401). II.CD3

[0008] CD3 is a T cell co-receptor composed of four different chains (Wucherpfennig, KW et al. (2010) "Structural Biology Of The T-Cell Receptor: Insights Into Receptor Assembly, Ligand Recognition, And Initiation Of Signaling," Cold Spring Harb. Perspect. Biol. 2 (4): a005140; Pages 1-14). In mammals, the complex contains a CD3γ chain, a CD3δ chain, and two CD3ε chains. These chains associate with a molecule called the T cell receptor (TCR) to generate an initiation signal in T lymphocytes. In the absence of CD3, the TCR cannot be properly assembled and is degraded (Thomas, S. et al. (2010) "Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer," Immunology 129 (2): 170–177). CD3 is found to be bound to the membranes of all mature T cells and to almost no other cell types (see, Janeway, CA et al. (2005) In: Immunobiology: The Immune System In Health And Disease," 6th edition. Garland Science Publishing, NY, pp. 214-216; Sun, ZJ et al. (2001) "Mechanisms Contributing To T Cell Receptor Signaling And Assembly Revealed By The Solution Structure Of An Ecto Domain Fragment Of The CD3ε:γHeterodimer," Cell 105(7):913-923; Kuhns, MS et al. (2006) "Deconstructing The Form And Function Of The TCR / CD3 Complex," Immunity. 2006 Feb; 24(2):133-139). II. AML and MDS

[0009] Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are believed to occur in and persist in a small portion of leukemic stem cells (LSCs), which are usually dormant (i.e., not rapidly dividing cells) and are therefore resistant to cell death (apoptosis) and conventional chemotherapeutic agents. LSCs are characterized by high levels of CD123 expression, which is absent in the corresponding normal hematopoietic stem cells in the bone marrow of normal people (Jin, W. et al. (2009) "Regulation Of Th17 Cell Differentiation And EAE Induction By MAP3K NIK," Blood 113: 6603-6610; Jordan, CT et al. (2000) "The Interleukin-3 Receptor Alpha Chain Is A Unique Marker For Human Acute Myelogenous Leukemia Stem Cells," Leukemia 14: 1777-1784). CD123 is expressed in 45%-95% of AML, 85% of hairy cell leukemia (HCL) and 40% of acute B-lymphocytic leukemia (B-ALL). CD123 expression is also associated with a variety of other malignancies / pre-malignancies: chronic myeloid leukemia (CML) progenitor cells (including blast crisis CML), Hodgkin's Reed Sternberg (RS) cells, transformed non-Hodgkin lymphoma (NHL), some chronic lymphocytic leukemia (CLL) (CD11c+), a subtype of acute T-lymphocytic leukemia (T-ALL) (16%, the most immature, mostly adults), plasmacytoid dendritic cells (pDC) DC2 malignancies and CD34+ / CD38- myelodysplastic syndrome (MDS) myeloid malignancies.

[0010] AML is a clonal disease characterized by the proliferation and accumulation of myeloid progenitor cells transformed in the bone marrow, which ultimately leads to hematopoietic dysfunction. The incidence of AML increases with age, and older patients generally have a poorer treatment effect than younger patients (Robak, T. et al. (2009) "Current And Emerging Therapies For Acute Myeloid Leukemia," Clin. Ther. 2: 2349-2370). Unfortunately, now, most adults with AML die from their disease.

[0011] The treatment of AML initially focuses on induction remission (induction therapy). Once remission is reached, treatment is switched to consolidation of this remission (post-remission or consolidation therapy), and in some cases, switched to maintenance therapy. The standard remission induction paradigm for AML is chemotherapy with anthracycline / cytarabine combination, followed by consolidation chemotherapy (usually with higher doses of the same drugs used during the induction period) or human stem cell transplantation, depending on the ability of the patient to tolerate intensive treatment and the possibility of being cured by chemotherapy alone (see, e.g., Roboz, GJ (2012) "Current Treatment Of Acute Myeloid Leukemia," Curr. Opin. Oncol. 24: 711-719).

[0012] Frequently used agents in induction therapy include cytarabine and anthracyclines. Cytarabine (also known as AraC) kills cancer cells (and other rapidly dividing normal cells) by interfering with DNA synthesis. Side effects associated with AraC treatment include decreased resistance to infection, a result of decreased leukocyte production; bleeding due to decreased platelet production; and anemia due to a potential decrease in red blood cells. Other side effects include nausea and vomiting. Anthracyclines (e.g., daunorubicin, doxorubicin, and idarubicin) have several modes of action, including inhibition of DNA and RNA synthesis, destruction of higher order structures of DNA, and generation of oxygen free radicals that damage cells. The most serious adverse response of anthracyclines is cardiotoxicity, which considerably limits the lifetime dose administered and, to a certain extent, limits their usefulness.

[0013] Stem cell transplantation has been established as the most effective form of anti-leukemic therapy in patients with AML who are in first or subsequent remission (Roboz, GJ (2012) "Current Treatment Of Acute Myeloid Leukemia," Curr. Opin. Oncol. 24:711-719). Unfortunately, however, despite significant advances in the treatment of newly diagnosed AML, 20% to 40% of patients do not achieve remission with standard induction chemotherapy, and 50% to 70% of patients who enter first complete remission are expected to relapse within 3 years. The optimal strategy at relapse or for patients with resistant disease remains uncertain (see, Tasian, SK (2018 "Acute Myeloid Leukemia Chimeric Antigen Receptor T-Cell Immunotherapy: 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 Myeloid Leukemia" Future Oncol. 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 Malignancies: Plasma Cell Disorders," Am. Soc. Clin. Oncol. Educ. Book. 37: 561-568; Kroschinsky, 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). Therefore, new therapeutic strategies are needed. IV. Bispecific Molecules

[0014] Non-monospecific molecules (e.g., bispecific antibodies, bispecific diabodies, Antibodies, etc.) provide significant advantages over monospecific molecules such as natural antibodies: the ability to co-connect and co-localize cells expressing different epitopes. Therefore, bispecific molecules have a wide range of applications, including therapy and immunodiagnosis. Bispecific allows great flexibility in designing and engineering double antibodies in various applications, providing enhanced affinity for multimeric antigens, cross-linking of different antigens, and directional targeting of specific cell types dependent on the presence of two target antigens. Of particular importance is the co-connection of different cells, for example, effector cells such as cytotoxic T cells and tumor cells cross-linking (Staerz et al. (1985) "Hybrid Antibodies Can Target Sites For Attack By T Cells," Nature 314: 628-631, and Holliger et al. (1996) "Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody," Protein Eng. 9: 299-305).

[0015] In order to provide molecules with greater capabilities than natural antibodies, various recombinant bispecific antibody formats have been developed (see, e.g., PCT Application Nos. WO 2008 / 003116, WO 2009 / 132876, WO 2008 / 003103, WO 2007 / 146968, WO 2009 / 018386, WO 2012 / 009544, WO 2013 / 070565), most of which use linker peptides to fuse further binding proteins (e.g., scFv, VL, VH, etc.) to or within the antibody core (IgA, IgD, IgE, IgG or IgM), or to fuse multiple antibody binding portions (e.g., two Fab fragments or scFv) to each other. Alternative formats use linker peptides to fuse binding proteins (e.g., scFv, VL, VH, etc.) to dimerization domains such as CH2-CH3 domains, or alternative polypeptides (WO 2005 / 070966, WO 2006 / 107786, WO 2006 / 107617, WO 2007 / 046893) and other formats in which the CL and CH1 domains are switched from their respective natural positions and / or the VL and VH domains have been diversified (WO 2008 / 027236, WO 2010 / 108127) to allow them to bind more than one antigen.

[0016] The art has additionally noted the ability to generate diabodies capable of binding to two or more different epitope species (see, e.g., Holliger et al. (1993) "'Diabodies': Small Bivalent And Bispecific Antibody Fragments," Proc. Natl. Acad. Sci. (USA) 90:6444-6448. Stable, covalently bonded heterodimeric non-monospecific diabodies have been described (see, e.g., WO 2006 / 113665; WO / 2008 / 157379; WO 2010 / 080538; WO2012 / 018687; WO / 2012 / 162068; Johnson, S. et al. (2010) "Effector Cell Recruitment With Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis And In Vivo B-Cell Immunofluorescence ... Depletion," J. Molec. Biol. 399(3):436-449; Veri, MC et al. (2010) "Therapeutic Control Of B Cell Activation Via Recruitment Of Fcgamma Receptor IIb (CD32B) Inhibitory Function With A NovelBispecific Antibody Scaffold," Arthritis Rheum.62(7):1933-1943; Moore, PA et al. (2011) "Application Of Dual Affinity Retargeting Molecules To Achieve OptimalRedirected T-Cell Killing Of B-Cell Lymphoma," Blood 117(17):4542-4551). Such diabodies incorporate one or more cysteine ​​residues into each polypeptide species employed. For example, the addition of a cysteine ​​residue at the C-terminus of such a construct has been shown to allow disulfide bonds to form between the polypeptide chains, stabilizing the resulting heterodimers without interfering with the binding properties of the bivalent molecule. In addition, trivalent molecules comprising diabody-like domains have been described (see, e.g., WO 2015 / 184203 and WO 2015 / 184207).The diabody epitope binding domains can also be directed against any surface determinant of immune effector cells, such as CD3, CD16, CD32, or CD64 expressed on T lymphocytes, natural killer (NK) cells, or other monocytes. In many studies, it was also found that diabodies bind to effector cell determinants, such as Fcγ receptors (FcγRs) to activate effector cells (Holliger et al. (1996) "Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody," Protein Eng. 9:299-305; Holliger et al. (1999) "Carcinoembryonic Antigen (CEA)-Specific T-cell Activation In Colon Carcinoma Induced By Anti-CD3 x Anti-CEA Bispecific Diabodies And B7xAnti-CEA Bispecific Fusion Proteins," Cancer Res. 59:2909-2916; WO 2006 / 113665; WO 2008 / 157379; WO 2010 / 080538; WO 2012 / 018687; WO 2012 / 162068). Typically, effector cell activation is triggered by the binding of an antigen-binding antibody to an effector cell via an Fc-FcγR interaction; therefore, in this regard, diabody molecules can exhibit Ig-like functionality regardless of whether they include an Fc domain (e.g., as determined in any effector function assay known in the art or exemplified herein (e.g., ADCC assay)). By cross-linking tumor and effector cells, diabodies not only bring effector cells to the vicinity of tumor cells, but also lead to effective tumor killing (see, e.g., Cao et al. (2003) "Bispecific Antibody Conjugates In Therapeutics," Adv. Drug. Deliv. Rev. 55: 171-197).

[0017] Several bispecific molecules targeting CD123 and CD3 that are capable of mediating T cell redirected cell killing of CD123-expressing malignant cells are under development (see, e.g., Vey, N., et al. (2017) "Interim Results From APhase 1 First-In-Human Study Of Flotuzumab, a CD123 x CD3 Bispecific DART Molecule In AML / MDS," Annals of Oncology, 28(S5)5, mdx373.001; Godwin, CD, et al. (2017) "Bispecific Anti-CD123 x Anti-CD3 Adaptor TM Molecules APVO436 and APVO437Have Broad Activity Against Primary Human AML Cells In Vitro” Blood.130(S1):2639; Forslund, A., et al. (2016) “Ex Vivo Activity Profile of the CD123xCD3 Antibody JNJ-63709178Against Primary Acute Myeloid Leukemia Bone Marrow Samples” Blood 128(22):2875). However, efforts to employ bispecific binding molecules capable of targeting T cells to the location of hematological malignancies have not been entirely successful. Therefore, there remains a need to develop new strategies to treat hematological malignancies using CD123xCD3 bispecific binding molecules. The present invention directly addresses this need and other needs, as described below. Summary of the invention

[0018] The present invention relates to a method for treating hematological malignancies such as acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), including hematological malignancies that are refractory to chemotherapeutic agents and / or demethylating agents. The method involves administering a CD123 x CD3 bispecific binding molecule to a patient in an amount that effectively stimulates killing of cells of the hematological malignancies in the patient. The present invention is further directed to an embodiment of this method, wherein a cell sample from a patient demonstrates that the expression of one or more target genes is increased relative to the baseline expression level of such genes, for example, the baseline expression level of such genes in a reference population of individuals suffering from hematological malignancies, or relative to the expression level of a reference gene.

[0019] In detail, the present invention provides a method of treating a chemotherapy-refractory hematological malignancy in a patient, wherein the method comprises administering to the patient a therapeutic dose of a CD123 x CD3 bispecific molecule effective to stimulate killing of cells of the patient's hematological malignancy, thereby treating such malignancy.

[0020] The present invention further provides embodiments of such methods, which additionally include evaluating the expression of one or more target genes and / or reference genes in a cell sample from a patient before and / or after administration of the CD123 x CD3 bispecific molecule. The present invention further provides embodiments of such methods, wherein the method includes evaluating the expression of such one or more target genes and / or such one or more reference genes before administration of the CD123 x CD3 bispecific molecule. The present invention also provides embodiments of such methods, wherein the method includes evaluating the expression of such one or more target genes and / or such one or more reference genes after administration of the CD123 x CD3 bispecific molecule.

[0021] The present invention further provides a method for determining whether a patient is a suitable responder to treatment of a hematological malignancy with a CD123 x CD3 bispecific molecule, wherein the method comprises: (a) assessing the expression of one or more target genes in a cell sample from the patient prior to administration of the CD123 x CD3 bispecific molecule relative to the expression of one or more target genes and / or reference genes; and (b) identifying the patient as a suitable responder for treatment with the CD123 x CD3 bispecific molecule if the expression of the one or more target genes is found to be increased relative to the expression of the one or more target genes and / or reference genes.

[0022] The invention further provides embodiments of this method, wherein the method assesses: (i) expression of one or more target genes; and (ii) one or more reference genes whose expression is not characteristically associated with a hematological malignancy.

[0023] The invention further provides embodiments of such methods, which include assessing the expression of one or more target genes relative to a baseline expression of one or more reference genes in the patient.

[0024] The present invention further provides embodiments of such methods, comprising evaluating the expression of one or more target genes in a patient relative to the expression of one or more target genes in an individual or a population of such individuals who are suffering from a hematological malignancy. The present invention further provides embodiments of such methods, wherein the expression of one or more target genes in such a patient is greater than the first quartile (i.e., greater than the bottom 25%), greater than the second quartile (i.e., greater than the bottom 50%), or greater than the third quartile (i.e., greater than the bottom 75%) of the expression level of such target genes in such an individual or a population of such individuals who are suffering from a hematological malignancy.

[0025] The present invention further provides embodiments of such methods, comprising evaluating the expression of one or more target genes in a patient relative to the expression of one or more target genes in an individual who has not been successfully treated for a hematological malignancy using the methods and compositions of the present invention (e.g., an individual who has not successfully responded to treatment of a hematological malignancy using a CD123 x CD3 bispecific molecule), or a population of such individuals. The present invention further provides embodiments of such methods, wherein the expression of one or more target genes in such patient is greater than the first quartile (i.e., greater than the bottom 25%), greater than the second quartile (i.e., greater than the bottom 50%), or greater than the third quartile (i.e., greater than the bottom 75%) of the expression level of such target genes in such individual or a population of such individuals who have not been treated. The present invention further provides embodiments of such methods, wherein the expression of one or more target genes in such patient has a logarithm of at least about 0.4, at least about 0.5, at least about 0.6, or greater relative to the expression of such target genes in such individual or such population of individuals who have not been successfully treated. 2 Fold change.

[0026] The invention further provides embodiments of such methods, comprising evaluating the expression of one or more target genes in a patient relative to the expression of one or more target genes in an individual previously successfully treated for a hematological malignancy using the methods and compositions of the invention (e.g., an individual who successfully responded to treatment of a hematological malignancy using a CD123 x CD3 bispecific molecule) or a population of such individuals. The invention further provides embodiments of such methods, wherein the expression of one or more target genes in such patient is within the first quartile (i.e., within the bottom 25%), the second quartile (i.e., between the bottom 25% and 50%), or the third quartile (i.e., between the bottom 50% and 75%) of the expression level of such target gene in such individual or such population of successfully treated individuals.

[0027] The invention further provides embodiments of this method, wherein the relative expression level of one or more target genes in a population is established by averaging the gene expression levels in cell samples obtained from a population of individuals.

[0028] The present invention further provides embodiments of this method, wherein the patient exhibits an expression level of at least one of the target genes: (a) greater than the first quartile of expression levels of such target gene in a population of individuals suffering from a hematological malignancy; or (b) greater than the first quartile of expression levels of such target gene in a population of individuals who have not successfully responded to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a logarithmic difference of at least about 0.4 relative to the expression level of such target gene in a population of individuals who have not successfully responded to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule. 2 Fold change; or (d) within at least the first quartile of the expression level of such target gene in a population of individuals who successfully respond to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule.

[0029] The present invention further provides embodiments of this method, wherein the patient exhibits an expression level of at least one of the target genes: (a) greater than the second quartile of expression levels of such target gene in a population of individuals suffering from a hematological malignancy; or (b) greater than the second quartile of the expression level of such target gene in a population of individuals who have not successfully responded to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a logarithmic difference of at least about 0.4 relative to the expression level of such target gene in a population of individuals who have not successfully responded to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule. 2 Fold change; or (d) within at least the second quartile of the expression level of such target gene in a population of individuals who successfully responded to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule.

[0030] The present invention further provides embodiments of this method, wherein the patient exhibits an expression level of at least one of the target genes: (a) greater than the third quartile of the expression level of such target gene in a population of individuals suffering from said hematological malignancy; or (b) greater than the third quartile of the expression level of such target gene in a population of individuals who have not responded successfully to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a logarithmic difference of at least about 0.6 relative to the expression level of such target gene in a population of individuals who have not responded successfully to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule. 2 Fold change.

[0031] The present invention further provides a method for treating a hematological malignancy, wherein the method comprises: (a) using the method of any one of the above embodiments to determine whether the patient is a suitable responder for using a CD123xCD3 bispecific molecule to treat a hematological malignancy; (b) administering to the patient a therapeutic dose of a CD123 x CD3 bispecific molecule if the patient is determined to be a suitable responder to such treatment; Administration of a CD123 x CD3 bispecific molecule stimulates cell killing of the patient's hematological malignancies.

[0032] The invention further provides embodiments of this method which additionally comprises assessing the expression of such one or more target genes in a cell sample obtained from the patient one or more times after initiation of treatment.

[0033] The present invention further provides an embodiment of this method, wherein the cell sample is a bone marrow or blood sample. In particular, an embodiment of this method, wherein the cell sample is a bone marrow sample.

[0034] The present invention further provides an embodiment of this method, which further comprises detecting the expression level of one or more target genes in a sample of the patient's bone marrow. The present invention further provides an embodiment of this method, which further comprises detecting the expression level of one or more reference genes.

[0035] The present invention further provides an embodiment of this method comprising detecting the expression level of such one or more target genes and / or such one or more reference genes in a sample of the patient's bone marrow, in particular before administering the CD123 x CD3 bispecific molecule.

[0036] The present invention further provides embodiments of this method, wherein assessing expression or determining whether a patient is a suitable responder to treatment of a hematological malignancy with a CD123 x CD3 bispecific molecule is performed by: (a) determining the gene expression level of each target gene in one or more cell samples using a gene expression platform; and (b) comparing the target gene expression level with the expression level of one or more reference genes.

[0037] The present invention further provides embodiments of this method, wherein assessing expression or determining whether a patient is a suitable responder to treatment of a hematological malignancy with a CD123 x CD3 bispecific molecule is performed by: (a) measuring the raw RNA level of each target gene in one or more cell samples in a gene expression platform; The gene expression platform includes a reference genome of housekeeping genes; and (b) Using the measured RNA levels of an internal reference gene, a relative expression value is assigned to each measured raw RNA level of the target gene.

[0038] The present invention further provides an embodiment of this method, wherein the one or more target genes include: (a) one or more of the following: CXCL9, CXCL10, CXCL11 and STAT1; and or (b) one or more of the following: CCL5, CD27, CD274, CD276, CD8A, CMKLR1, CXCL9, CXCR6, HLA-DQA1, HLA-DRB1, HLA-E, IDO1, LAG3, NKG7, PDCD1LG2, PSMB10, STAT1, and TIGIT; and / or (c) one or more of the following: AREG, CSF3, CXCL1, CXCL2, CXCL3, CCL20, FOSL1, IER3 (NM_003897.4), IL6, and PTGS2; and / or (d) one or more of the following: CCL2, CCL3 / L1, CCL4, CCL7 and CCL8; and / or (e) one or more of the following: MAGEA3 / A6, MAGEA1, MAGEA12, MAGEA4, MAGEB2, MAGEC1 and MAGEC2; and / or (f) one or more of the following: APOL6, DTX3L, GBP1, IFI16, IFI27, IFI35, IFI6, IFIH1, IFIT1, IFIT2, IFIT3, IFITM1, IFITM2, IRF1, IRF9, ISG15, MX1, OAS1, OAS2, PARP9, PSMB9, STAT2, TMEM140, and TRIM21; and / or (g) one or more of the following: PSMB8, PSMB9 and PSMB10; and / or (h) IL-10; and or (i) CD274; and / or (j) PDCD1LG2.

[0039] The present invention further provides an embodiment of this method, wherein the one or more target genes further include IFNG (NM_000619.2).

[0040] The present invention further provides an embodiment of this method, wherein the one or more reference genes include one or more of the following: ABCF1, G6PD, NRDE2, OAZ1, POLR2A, SDHA, STK11IP, TBC1D10B, TBP and UBB.

[0041] The present invention further provides an embodiment of this method, wherein a gene signature score is determined for one or more target genes. In a specific embodiment of the present invention, this gene signature score is determined from the raw RNA level of each target gene by a method comprising: (a) measuring the raw RNA level of each target gene in a cell sample using a gene expression platform that includes a reference genome of housekeeping genes, (b) normalizing each measured raw RNA level to the geometric mean of this housekeeping gene, and optionally further normalizing each RNA value to a standard, (c) Perform log transformation on each normalized RNA value, (d) multiplying each log-transformed RNA value by the corresponding weight factor to generate a weighted RNA value, and (e) Add the weighted RNA values, and optionally an adjustment factor constant, to generate a single gene signature score.

[0042] Preferably, the target genes provided in Tables 6 and 12A-12G are used to determine the gene signature. In certain embodiments of the invention, the weight factors are those provided in Tables 6 and 12A-12G. In certain embodiments of the invention, an adjustment factor is added to each score. In a particular embodiment, the adjustment factor is those provided in Tables 6 and 12B-12G.

[0043] In particular, the invention provides embodiments of this method, wherein the gene signature score is determined for one or more of: (a) IFNγ signaling signature; (b) tumor inflammation signature; (c) myeloid inflammatory signature; (d) inflammatory chemokine signature; (e) MAGE tag; (f) IFN downstream signaling signature; (g) Immunoproteasome labeling; (h) IL-10 labeling; (i) PD-L1 label; and / or (j) PD-L2 label.

[0044] The present invention further provides an embodiment of this method, wherein the patient gene signature score is: (a) greater than the first quartile of a score of a gene signature calculated from the expression levels of one or more target genes in a population of individuals suffering from a hematological malignancy; or (b) greater than the first quartile of a score for a gene signature calculated from the expression levels of one or more target genes in a population of individuals who have not responded successfully to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a log score of at least about 0.4 relative to the expression level of one or more target genes calculated from a population of individuals who did not successfully respond to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule 2 Fold change; or (d) is within at least the first quartile of a score for a gene signature calculated from the expression levels of one or more target genes in a population of individuals who have successfully responded to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule, Indicating a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule.

[0045] The present invention further provides an embodiment of this method, wherein the patient gene signature score is: (a) greater than the second quartile of a gene signature calculated from the expression levels of one or more target genes in a population of individuals suffering from a hematological malignancy; or (b) greater than the second quartile of a gene signature calculated from the expression levels of one or more target genes in a population of individuals who have not successfully responded to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a log score of at least about 0.5 relative to the expression level of one or more target genes in a population of individuals who did not successfully respond to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule. 2 Fold change; or (d) is within at least the second quartile of a score for a gene signature calculated from the expression levels of one or more target genes in a population of individuals who have successfully responded to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule, Indicating a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule.

[0046] The present invention further provides an embodiment of this method, wherein the patient gene signature score is: (a) greater than the third quartile of the score of the gene signature calculated from the expression levels of one or more target genes in a population of individuals suffering from a hematological malignancy; or (b) a gene signature score greater than the third quartile calculated from the expression levels of one or more target genes in a population of individuals who have not responded successfully to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a log score of at least about 0.6 relative to the expression level of one or more target genes calculated from a population of individuals who did not successfully respond to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule 2 Fold change, Indicating a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule.

[0047] The present invention further provides an embodiment of this method, wherein: (a) the gene signature is an IFNγ signaling signature, and a patient gene signature score of at least about 2.5 indicates a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule and / or (b) the gene signature is a tumor inflammation signature, and a patient gene signature score of at least about 5.5 indicates a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule; and / or (c) the gene signature is an IFN downstream signaling signature, and a patient gene signature score of at least about 4.5 indicates a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule.

[0048] The present invention further provides an embodiment of this method, wherein the gene signature is an IFNγ signaling signature, a tumor inflammation signature, or an IFN downstream signaling signature, and the patient gene signature score is: (a) greater than the first quartile of a score of a gene signature calculated from the expression levels of one or more target genes in a population of individuals suffering from a hematological malignancy; or (b) greater than the first quartile of a score for a gene signature calculated from the expression levels of one or more target genes in a population of individuals who have not responded successfully to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a log score of at least about 0.4 relative to the expression level of one or more target genes calculated from a population of individuals who did not successfully respond to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule 2 Fold change; or (d) is within at least the first quartile of a score for a gene signature calculated from the expression levels of one or more target genes in a population of individuals who have successfully responded to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule, Indicating a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule.

[0049] The present invention further provides an embodiment of this method wherein the gene signature is an IFNγ signaling signature, a tumor inflammation signature, or an IFN downstream signaling signature, and the patient gene signature score is: (a) greater than the second quartile of a score for a gene signature calculated from the expression levels of one or more target genes in a population of individuals suffering from a hematological malignancy; or (b) greater than the second quartile of the score of the gene signature calculated from the expression levels of one or more target genes in a population of individuals who have not responded successfully to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a logarithmic difference of at least about 0.5 relative to the expression level of one or more target genes calculated from a population of individuals who did not successfully respond to treatment of a hematological malignancy with the CD123 x CD3 bispecific molecule. 2 Fold change; or (d) within at least the second quartile of a score of a gene signature calculated from the expression levels of one or more target genes in a population of individuals who have successfully responded to hematological malignancy treatment using the CD123 x CD3 bispecific molecule; Indicating a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule.

[0050] The present invention also provides embodiments of this method, wherein patients displaying a gene expression signature characterized by an immune-enriched and IFNγ-dominant tumor microenvironment are shown to be more favorable patients responding to treatment with a CD123 x CD3 bispecific molecule.

[0051] The present invention further provides an embodiment of this method, wherein the CD123 x CD3 bispecific molecule is a bispecific antibody or a bispecific molecule comprising a scFv.

[0052] The present invention further provides an embodiment of this method, wherein the CD123 x CD3 bispecific molecule is JNJ-63709178, XmAb14045 or APVO436.

[0053] The invention further provides embodiments of this method wherein the CD123 x CD3 bispecific molecule is a covalently bonded bispecific diabody having two, three or four polypeptide chains.

[0054] The present invention further provides an embodiment of this method, wherein the CD123 x CD3 bispecific molecule is a diabody comprising: (a) a first polypeptide chain having an 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 first and second polypeptide chains are covalently bonded to each other via a disulfide bond.

[0055] The present invention further provides an embodiment of this method, wherein the patient's hematological malignancy is selected from the group consisting of: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), blast crisis CML, Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B lymphocytic leukemia (B-ALL), acute T lymphocytic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), Richter syndrome, Richter transformation of CLL, hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin lymphoma (NHL) including mantle cell lymphoma (MCL) and small lymphocytic lymphoma (SLL), Hodgkin lymphoma, systemic mastocytosis and Burkitt lymphoma.

[0056] The present invention further provides embodiments of this method, wherein the patient's hematological malignancy is ML, MDS, BPDCN or T-ALL.

[0057] The invention further provides embodiments of this method, wherein the patient's hematological malignancy is refractory to chemotherapy (CTX), such as refractory to cytarabine / anthracycline-based cytotoxic chemotherapy or refractory to hypomethylating agent (HMA) chemotherapy.

[0058] The present invention further provides embodiments of this method, further comprising determining the level of CD123 expression by blast cells (cancer cells) compared to a corresponding baseline level of CD123 expressed by normal peripheral blood mononuclear cells (PBMCs).

[0059] The present invention further provides an embodiment of this method, wherein the expression level is determined by measuring the cell surface expression of CD123. The present invention further provides an embodiment of this method, wherein the cell surface expression of CD123 increases by at least about 20% relative to the baseline expression level. The present invention further provides an embodiment of this method. The increase in CD123 expression makes the patient more responsive to treatment with the CD123 x CD3 bispecific molecule.

[0060] The present invention further provides embodiments of this method, wherein the effective dose of the CD123 x CD3 bispecific molecule is selected from the group consisting of 30, 100, 300 and 500 ng / kg patient weight / day.

[0061] The present invention further provides embodiments of all of the above methods, wherein the therapeutic dose is administered as a continuous infusion. The present invention further provides embodiments of this method, wherein the therapeutic dose is 30 ng / kg / day administered by continuous infusion for 3 days, followed by a therapeutic dose of 100 ng / kg / day administered by continuous infusion for 4 days. The present invention further provides embodiments of this method, wherein the therapeutic dose further comprises administering 500 ng / kg / day administered by continuous infusion.

[0062] The present invention further provides embodiments of all of the above methods, wherein the patient is a human patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figures 1A-1C The overall structure of an exemplary diabody molecule is illustrated. Figure 1A The structures of the first and second polypeptide chains of a two-chain CD123 x CD3 bispecific diabody ("DART-A," also known as flotetuzumab) having two epitope-binding domains, a heterodimer-promoting domain, and a cysteine ​​containing linker are provided. Figure 1B-1C The overall structure of a CD123 x CD3 bispecific diabody with two epitope-binding domains composed of three polypeptide chains is provided. The two polypeptide chains possess 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 connector. Cysteine ​​residues may be present in the connector ( Figure 1A and 1B ) neutralizing and / or heterodimer-promoting domain ( Figure 1C ). VL and VH domains that recognize the same epitope are shown using the same shading or fill pattern.

[0064] Figure 2Unsupervised hierarchical clustering of 46 IO360 signatures or cell types generated from baseline bone marrow biopsies obtained from patients who were refractory to conventional chemotherapy (e.g., patients who were refractory to treatment with cytarabine in combination with daunorubicin (7+3 induction therapy (RefCTX) regimen) or to treatment with the demethylating agents decitabine and azacitidine (RefHMA and including patients with secondary AML), and who all relapsed prior to treatment with vortuzumab (relapsers) are illustrated. Patients' responses to vortuzumab for CD123 x The response of CD3 bispecific binding molecule therapy. This response is annotated as anti-leukemia response (anti-leuk (A)), which includes that the patient shows complete remission (CR), complete remission but hematology is not completely improved (CRi), morphology without leukemia (MLF), other anti-leukemia benefits (OB), or partial response (PR)), or as no response (NR, which includes progressive disease / treatment failure (PD) and stable disease (SD)). Each IO 360 label score is readjusted to -3 to +3 scopes within the score of the group to facilitate comparison across labels. Immune exhaustion and immune enrichment gene labels are framed in cluster 2 and cluster 3 columns respectively.

[0065] Figure 3A-3O Patients refractory to chemotherapy and HMA were shown to have different expression of multiple gene signatures. In particular, the gene expression profiles of relapsed patients showed features of immune exhaustion, while the profiles of HMA-refractory (including HMA-refractory and secondary AML) patients showed features of immune failure 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 with CTX-refractory patients. Figure 3A is a forest plot of the fold change differences between relapsed patients who changed from all refractory (CTX and HMA). Figure 3B is a forest plot of the fold change differences between HMA-refractory patients who changed from relapse; Figure 3C is a forest plot of the fold change differences between HMA-refractory patients and CTX-refractory patients. Figure 3A and 3C The gene signatures for cluster 2 immune depletion (C2) and cluster 3 immune enrichment (C3) are indicated in Figure 2. Figure 3D ), macrophages ( Figure 3E ), neutrophils ( Figure 3F ), B-cells ( Figure 3G ), IFNγ (IFN-γ, Figure 3H )、PD-L1( Fig. 3I )、TIGIT( Figure 3J )、CTLA-4( Figure 3K )、Th1( Figure 3L )、CTL( Figure 3M ), CD8 T cells ( Figure 3N ) and cytotoxicity ( Fig.3O ) Gene signature scores are plotted as immunodepleted (Depl.), immunoenriched (Enriched), and immunodepleted (Exh.) files.

[0066] Figure 4 Shown are the changes in the percentage of bone marrow blasts (relative to baseline) following CD123 x CD3 bispecific binding molecule therapy from 25 patients (relapsed (RL) patients, CTX-refractory (CTx) patients, and HMA-refractory (HMA) patients) and their responses to this therapy (CR, complete remission; mCR, molecular CR; CRi, complete remission with incomplete hematologic improvement; MLF, morphologic leukemia-free status; PR, partial response; SD, stable disease; PD, progressive disease / treatment failure).

[0067] Figures 5A-5C It was shown that the IFNγ signaling signature was increased at baseline in responders to vortuzumab and, therefore, was predictive of a positive response to CD123 x CD3 bispecific binding molecule therapy. Figure 5A Forest plot of baseline fold change differences between OR patients and NR patients showing an increase in IFNγ signaling signature in OR patients' baseline samples (indicating immune-depleted (C2) and immune-enriched (C3) gene signatures). Increases in tumor inflammation signatures and IFN downstream signatures were also seen. Figure 5B The distribution of IFNγ signaling signature scores for the NR and OR populations of patients is shown (2 nd AML; Ref CTX: refractory to CTX; Ref HMA: refractory to HMA, Relapse: primary relapse). Figure 5C Shown is a ROC curve showing the predictive performance of the baseline IFNγ signaling signature score with AUC = 0.819.

[0068] Figure 6 Shown is the expression of gene signatures associated with cytotoxic cells or with CD8+ T cells as detected in RNA from bone marrow samples, either pre-treatment ("Basal") or after the first cycle of treatment with vortuzumab ("Cycle 1").

[0069] Figure 7 Expression of CD123 in patient populations that are chemo-refractory, relapsed, refractory to HMA, or HMA-failed is shown.

[0070] Figure 8 Shown is the correlation between the expression level of PD-L1 in AML blasts of patients at baseline (BL) and whether the patient was an early progressor or responder to CD123 x CD3 bispecific binding molecule therapy. Data are expressed as mean + distribution.

[0071] Fig. 9 The unsupervised hierarchical clustering of 48 IO 360 labels or cell types generated by baseline bone marrow biopsy is illustrated for patients (P) with primary refractory response to conventional chemotherapy, and patients (R) who all relapsed before treatment with vortuzumab. The response of the patient to the CD123 x CD3 bispecific binding molecule therapy with vortuzumab is also indicated. This response is annotated as anti-leukemic response (A, wherein the patient includes showing complete remission (CR), complete remission but incomplete improvement in hematology, morphological leukemia-free state (MLF), other anti-leukemic benefits (OB), or partial response (PR)), or as no response (N, including progressive disease / treatment failure (PD) and stable disease (SD)). Each IO 360 label score is readjusted to -3 to +3 ranges within the score of the group to facilitate comparison across labels. Clusters stratified as immune infiltration and immune consumption are indicated.

[0072] Fig.10 1 is a forest plot showing the difference in baseline fold change in relapsed and refractory patients between CD123 x CD3 bispecific binding molecule therapy with vortuzumab (OR) and non-responders (NR), showing that many signatures were increased in baseline samples from responders, including: IFNγ signaling signature / IFN downstream signature and tumor inflammation signature (each boxed). Gene signatures that constitute the IFN linear module are asterisked and are also increased in baseline samples from responders.

[0073] Figures 11A-11D The distribution of scores for several gene signatures and IFN modules in refractory (Refr.) and relapsed (Rel.) patients is shown, with OR patients indicated by large open circles and NR patients indicated by small solid dots. Paired data were compared using the Mann-Whitney U test. **P<0.01. Fig.11A The distribution of IFNγ signaling signature scores is shown. Fig. 11B The distribution of IFN downstream signaling signature scores is shown. Fig. 11C The distribution of tumor inflammation signature (TIS) scores is shown. Fig.11D The distribution of IFN advantage module (IFN module) scores is shown.

[0074] Figures 12A-12JThe distribution of scores for the nine gene signature scores constituting the IFN dominance module and the tumor inflammation signature (TIS) in non-responders (NR) and responding patients (patients with an anti-leukemic response) (OR) are shown. Fig. 12A FNγ signaling signature scores are shown; Fig. 12B IFN downstream signature scores are shown; Fig. 12C Myeloid inflammation signature scores are shown; Fig.12D Immunoproteasome labeling scores are shown; Fig.12E Inflammatory chemokine signature scores are shown; Fig.12F MAGE tag scores are shown; Figure 12G The PD-L1 signature score is shown; Fig.12H The PD-L2 signature score is shown; Fig.12I IL10 labeling scores are shown; Fig.12J Tumor Inflammation Signature (TIS) scores are shown.

[0075] Figures 13A-13K Shown are ROC curves showing the predictive performance of the baseline score of the nine gene signatures constituting the IFN Dominance Module, the Tumor Inflammation Signature (TIS), and the IFN Dominance Module for a group of 30 refractory / relapsed patients. Fig.13A The ROC curve for the IFNγ signaling signature score with AUC = 0.750 is shown. Fig. 13B The ROC curve for the IFNγ downstream signaling signature score with AUC = 0.755 is shown. Fig. 13C The ROC curve for the myeloid inflammation signature score with AUC = 0.69 is shown. Fig.13D The ROC curve of the immunoproteasome signature score with AUC = 0.505 is shown. Fig.13E The ROC curve for the inflammatory chemokine signature score with AUC = 0.764 is shown. Fig.13F The ROC curve of the MAGE signature score with AUC = 0.736 is shown. Figure 13G The ROC curve of the PD-L1 signature score with AUC = 0.699 is shown. Fig.13H The ROC curve of the PD-L2 signature score with AUC = 0.727 is shown. Fig.13I The ROC curve for the IL10 signature score with AUC = 0.745 is shown. Fig.13J The ROC curve for the TIS score with AUC = 0.852 is shown. Figure 13K The ROC curve for the IFN dominance module score with AUC = 0.806 is shown.

[0076] Figures 14A-14D Tumor inflammation (TIS, Fig.14A), IFNγ signaling ( Fig. 14B ), antigen processing mechanism (APM, Fig. 14C ) and PD-L1( Fig.14D ) Distribution of scores for the gene signature detected in RNA from bone marrow samples, pre-treatment ("Pre") or from bone marrow samples after the first cycle of treatment with vortuzumab ("Post-C1"), OR patients are indicated by large open circles and NR patients are indicated by small solid dots. Paired data were compared using the Mann-Whitney U test. Pre = baseline. C1 = cycle 1. **P < 0.01. ***P < 0.001. DETAILED DESCRIPTION OF THE INVENTION

[0077] The present invention relates to a method for treating hematological malignancies such as acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), including hematological malignancies that are refractory to chemotherapeutic agents and / or demethylating agents. The method involves administering a CD123 x CD3 bispecific binding molecule to a patient in an amount that effectively stimulates killing of cells of the hematological malignancies in the patient. The present invention is further directed to an embodiment of this method, wherein a cell sample from a patient demonstrates that the expression of one or more target genes is increased relative to the baseline expression level of such genes, for example, the baseline expression level of such genes in a reference population of individuals suffering from hematological malignancies, or relative to the expression level of a reference gene.

[0078] As mentioned above, chemotherapy resistance and relapse remain a significant source of mortality in children and adults with acute myeloid leukemia (AML). After receiving conventional chemotherapy, only 26.9% of patients are expected to survive more than 5 years.

[0079] The treatment methods for patients with acute myeloid leukemia (AML) have not changed substantially in more than 30 years. The standard first-line therapy is a two-drug regimen (the so-called 7+3 induction therapy, abbreviated as "CTX" herein) given in combination with cytarabine and daunorubicin. Demethylating agents (abbreviated as "HMA" herein) decitabine and azacitidine are usually administered to elderly patients or patients who are considered unsuitable for the CTX regimen. However, literature estimates show that up to 45% of patients are refractory to standard first-line chemotherapy. Due to the severity of the acute and long-term side effects that these drugs often induce on normal tissues, further intensification of conventional cytotoxic chemotherapy is considered unfeasible (Tasian, SK (2018 "Acute Myeloid Leukemia Chimeric Antigen Receptor T-Cell Immunotherapy: 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 Myeloid Leukemia" Future Oncol. 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 TCell Therapies," Mol. Ther. Methods Clin.Dev.8:210-221; Dhodapkar, MV et al. (2017) "Hematologic Malignancies: Plasma Cell Disorders," Am.Soc. Management,” Crit. Care 14;21(1):89).

[0080] Bispecific antibodies that engage T cells stimulate the release of cytokines that pro-inflammatory responses. These cytokines can increase anti-leukemic efficacy both through direct cytotoxicity and by activating and recruiting immune cells to tumor sites (Hoseini, SS et al. (2017) "Acute Myeloid Leukemia Targets For Bispecific Antibodies," Blood Cancer Journal 7:e522, doi:10.1038 / bcj.2017.2; pp. 1-12. In particular, treatment with vortuzumab, a CD123 x CD3 bispecific binding molecule, is being tested in a Phase 1 / 2 study for relapsed / refractory ("R / R") AML. Although immunotherapy has great potential for selectively targeting cancer cells that cause hematological malignancies (see, e.g., Koch, J. et al. (2017) "Recombinant Antibodies to Arm Cytotoxic Lymphocytes in Cancer Immunotherapy," 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), efforts to employ bispecific binding molecules capable of targeting T cells to the location of hematological malignancies have not been entirely successful.

[0081] Therefore, the discovery of new therapeutic strategies including immunotherapy remains a priority. It has been previously reported that AML patients with an immune-enriched and IFNγ-dominant tumor microenvironment ("TME") experience a significant reduction in relapse-free survival, indicating refractory to standard induction chemotherapy (Vadakekolathu, J. et al. (2017) "Immune Gene Expression Profiling in Children and Adults with Acute Myeloid Leukemia Identifies Distinct Phenotypic Patterns," Blood 130:3942A).

[0082] As used herein, the term "gene expression signature" is intended to refer to a gene expression pattern of a group of genes that is characteristic of a particular cell type and / or biological process (see, e.g., Stenner, F. et al. (2018) "Cancer Immunotherapy and the Immune Response in Follicular Lymphoma," Front. Oncol. 8:219 doi:10.3389 / fonc.2018.00219, pp. 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, pp. 1-11; Shrestha, G. et al. (2016) "The Value Of Genomics In Dissecting The RAS-Network And In Guiding Therapeutics For RAS-Driven Cancers," Semin. Cell Dev.Biol.58:108-117; Gingras, I. et al. (2015) "CCR 20thAnniversary Commentary: Gene-Expression Signature in Breast Cancer--Where DidIt Start and Where Are We Now?," Clin.Cancer Res.21(21):4743-4746; Eberhart, CG (2011) "Molecular Diagnostics In Embryonal Brain Tumors," Brain Pathol. 21(1):96-104; Baylin, SB (2009) "Stem Cells, Cancer, And Epigenetics," StemBook, ed. The Stem Cell Research Community, StemBook, doi / 10.3824 / stembook.1.50.1, pp. 1-14; Asakura, M. et al. (2009) "Global Gene Expression Profiling In The Failing Myocardium,” Circ.J.73(9):1568-1576; Shaffer, AL et al. (2001) "Signatures Of The Immune Response," Immunity 15(3):375-385; Staudt, LM et al. (2005) "The Biology Of Human Lymphoid Malignancies Revealed By Gene Expression Profiling," Adv. Immunol. 87:163-208). The observed gene expression signature and / or changes in the signature caused by an altered (or unaltered) biological process can be used to assess the presence, nature and / or severity of a pathogenic medical condition.

[0083] A central aspect of the invention relates to the recognition that the presence of an IFNγ-dominant AML tumor microenvironment ("TME") predicts a favorable response to treatment with a CD123 x CD3 bispecific binding molecule, including therapy with the CD123 x CD3 bispecific binding molecule, vortuzumab, compared to resistance to standard chemotherapy. The invention arises, in part, from the recognition that certain subpopulations of patients with refractory hematological malignancies (e.g., acute myeloid leukemia) are particularly suitable for treatment with a CD123 x CD3 bispecific binding molecule (e.g., vortuzumab). Members of this subpopulation can be readily identified by their ability to display a gene expression signature characterized by the presence of an immune-enriched and IFNγ-dominant tumor microenvironment. I. Identification of patient populations particularly suitable for treatment with the CD123 x CD3 bispecific binding molecules of the invention A. Methods for Determining "Gene Expression Signatures"

[0084] In order to determine whether a patient exhibits a gene expression signature characterized by the presence of an immune-enriched and IFNγ-dominant tumor microenvironment, thereby being identified as being particularly suitable for the treatment of hematological malignancies using the methods and compositions of the present invention, an RNA sample from a cell sample obtained from the patient is evaluated to determine whether it demonstrates increased expression of one or more "target" genes, whose expression is associated with such a signature. This evaluation can use pre-existing gene expression detection and / or measurement, or can incorporate a step of detecting and / or measuring such gene expression. As used herein, the term "cell sample" refers to a sample containing cells or cell extracts.

[0085] Any cell sample can be used as the source of RNA or protein for determining whether the patient shows a gene expression label characterized by the presence of an immune-enriched and IFNγ advantage tumor microenvironment. However, preferably, the RNA obtained from the bone marrow (BM) sample or blood sample or mother cell (cancer cell) sample of a patient or donor colony is used to carry out this gene expression comparison. When RNA is obtained from this cell of a donor colony to provide a baseline expression level, the mean value (for example, geometric mean value) of the expression level adopted can be used. Many different reference populations can be used for this gene expression comparison. In a particular embodiment, the expression level of at least one target gene exhibited by the patient is compared to the expression level of such target gene exhibited in: a population of individuals suffering from a hematological malignancy; a population of individuals suffering from such a hematological malignancy and who have not successfully responded to treatment of the hematological malignancy at the time of determining such a reference expression level (i.e., a population of individuals who have not successfully responded to treatment of the hematological malignancy with a CD123 x CD3 bispecific molecule); and / or a population of individuals suffering from such a hematological malignancy at the time of determining such a reference expression level and who have subsequently been successfully treated for the hematological malignancy with the methods and compositions of the present invention (i.e., a population of individuals who have successfully responded to treatment of the hematological malignancy with a CD123 x CD3 bispecific molecule). When the comparator population is a population of individuals suffering from a hematological malignancy, such a population preferably includes individuals suffering from the same hematological malignancy as the patient. Such a population may include individuals who have relapsed after prior treatment with a chemotherapeutic agent and / or are refractory (i.e., primary refractory) to treatment with a chemotherapeutic agent. When the comparator population is a population of individuals who have successfully or unsuccessfully responded to treatment of a hematological malignancy with the CD123xCD3 bispecific molecule, such population preferably includes individuals suffering from the same hematological malignancy as the patient.

[0086] As used herein, expression of a gene is referred to as "increased" if its expression is at least about 10% more, at least about 20% more, at least about 30% more, at least about 40% more, 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-fold more, at least about 2-fold more, at least about 2.5-fold more, at least about 3-fold more, at least about 3.5-fold more, at least about 4-fold more, at least about 4.5-fold more, at least about 5-fold more, at least about 5.5-fold more, at least about 6-fold more, at least about 6.5-fold more, at least about 7-fold more, at least about 7.5-fold more, at least about 8-fold more, at least about 8.5-fold more, at least about 9-fold more, at least about 10-fold more, relative to a baseline or other comparator (e.g., expression of such a gene in a population). Alternatively, such an increase is measured according to a "log 2For an increase in expression, a log 2 A fold change is equivalent to approximately 30% more expression; a log of 0.5 2 A fold change is equivalent to approximately 40% more expression; a log 2 A fold change is equivalent to approximately 50% more expression; a log of 0.7 2 A fold change is equivalent to approximately 60% more expression; a log 2 The fold change is equivalent to about 70% more expression; a log 2 A fold change is equivalent to approximately 90% more expression; a log 2 A fold change is equivalent to a 2-fold increase; a log of 1.5 2 The fold change is equivalent to a 2.8-fold increase; 2 log 2 A fold change is equivalent to a 4-fold increase; a log of 2.5 2 The fold change is equivalent to a 5.7-fold increase; a log of 3 2 The fold change is equivalent to an 8-fold increase; a log of 3.5 2 The fold change is equivalent to an 11.3-fold increase; a log of 4 2 A fold change is equivalent to a 16-fold increase, etc. 2 Fold change is often used to compare count and array data and is also applicable to t-tests.

[0087] Alternatively, this increase is described in terms of a "gene signature score", where the expression of each of the target gene clusters is measured, normalized to one or more housekeeping genes and / or internal standards, logarithmically transformed, weighted, and summed to generate a single gene signature score. Methods for calculating such scores are known in the art and specific methods are provided herein (see, Example 1 below).

[0088] As used herein, expression of a gene signature (e.g., an IFNγ signaling signature) is referred to as "increased" if the gene signature score is at least about 2, or at least about 2.5, or at least about 3.0, or at least about 3.5, or at least about 4, or at least about 4.5, or at least about 5, or at least about 5, or at least about 5.5, or at least about 5.5, or at least about 6, or greater than about 6.5.

[0089] A patient's gene signature score is also referred to as "increased" if the patient's gene signature score is greater than the first quartile of gene signature scores calculated from the expression levels of such target genes in a population of individuals suffering from hematological malignancies (i.e., greater than the bottom 25%), greater than the second quartile of gene signature scores calculated from the expression levels of such target genes in a population of individuals suffering from hematological malignancies (i.e., greater than the bottom 50%), greater than the third quartile of the gene signature scores (i.e., greater than the bottom 75%), greater than 85%, greater than 90%, or greater than 95% of the gene signature scores calculated from the expression levels of such target genes in a population of individuals suffering from hematological malignancies.

[0090] A patient's gene signature score is greater than the first quartile of gene signature scores calculated from the expression levels of such target genes in a population of individuals who have not successfully responded to treatment for a hematological malignancy (e.g., a population of individuals who have not successfully responded to treatment for a hematological malignancy with a CD123 x CD3 bispecific molecule) (i.e., greater than the bottom 25%), greater than the second quartile of gene signature scores calculated from the expression levels of such target genes in a population of individuals who have not successfully responded to treatment for a hematological malignancy (e.g., a population of individuals who have not successfully responded to treatment for a hematological malignancy with a CD123 x CD3 bispecific molecule) (i.e., greater than the bottom 50%), greater than the third quartile of gene signature scores calculated from the expression levels of such target genes in a population of individuals who have not successfully responded to treatment for a hematological malignancy (e.g., a population of individuals who have not successfully responded to treatment for a hematological malignancy with a CD123 x CD3 bispecific molecule) (i.e., greater than the bottom 75%), greater than the third quartile of gene signature scores calculated from the expression levels of such target genes in a population of individuals who have not successfully responded to treatment for a hematological malignancy (e.g., a population of individuals who have not successfully responded to treatment for a hematological malignancy with a CD123 x CD3 bispecific molecule) (i.e., greater than the bottom 50%), greater than the third quartile of gene signature scores calculated from the expression levels of such target genes in a population of individuals who have not successfully responded to treatment for a hematological malignancy (e.g., a population of individuals who have not successfully responded to treatment for a hematological malignancy with a CD123 x CD3 bispecific molecule) A patient's gene signature score is also referred to as "increased" if the patient's expression level of such target gene is greater than 85%, greater than 90%, or greater than 95% of the gene signature score calculated for the patient's population (i.e., a population of individuals who have not successfully responded to treatment for hematological malignancies with the CD3 bispecific molecule).

[0091] If the patient's gene signature score has a logarithmic difference of at least about 0.4, or at least about 0.5, or at least about 0.6 or more relative to the gene signature score calculated from the expression level of such target gene in a population of individuals who have not successfully responded to treatment for a hematological malignancy (e.g., a population of individuals who have not successfully responded to treatment for a hematological malignancy with a CD123 x CD3 bispecific molecule), 2 The fold change,score of a patient’s gene signature is also referred to as an “increase”.

[0092] A patient's gene signature score is also referred to as "increased" if its gene signature score is within at least the first quartile (i.e., within the bottom 25%) and more preferably, within at least the second quartile (i.e., between the bottom 25% and 50%), within at least the third quartile (i.e., between the bottom 50% and 75%), greater than 85%, greater than 90%, or greater than 95% of the gene signature scores calculated from the expression levels of such target genes in a population of individuals who have previously been successfully treated for hematological malignancies using the methods and compositions of the invention (e.g., a population of individuals who have successfully responded to treatment of hematological malignancies using the CD123 x CD3 bispecific molecule).

[0093] It was found that increased gene signature scores were indicative of a more favorable patient response to treatment of hematological malignancies with the CD123 x CD3 bispecific molecules of the invention.

[0094] In one embodiment, a patient is identified as exhibiting a gene expression signature characterized by the presence of an immune-enriched and IFNγ-dominant tumor microenvironment, and thus particularly suitable for treatment of a hematological malignancy using the methods and compositions of the invention, by determining whether expression of the target gene is "increased" relative to a baseline level of its expression in the patient as assessed when the patient was healthy or before the patient received a diagnosis of a hematological malignancy, or relative to expression of the gene during the course of the patient's chemotherapeutic treatment regimen or during the course of the patient's treatment regimen involving a CD123 x CD3 bispecific binding molecule.

[0095] In a second embodiment, by comparing the expression level of one or more target genes with the average and weighted baseline expression level of such target genes from a population of individuals suffering from hematological malignancies, the patient is identified as exhibiting a gene expression signature characterized by the presence of an immune-enriched and IFNγ-dominant tumor microenvironment, and is therefore particularly suitable for treating hematological malignancies using the methods and compositions of the present invention. Target genes whose expression is greater than this average or weighted baseline level are referred to as exhibiting an "increased" expression level, and the methods and compositions of the present invention are particularly suitable for treating hematological malignancies in such patients. For example, the methods and compositions of the present invention are particularly suitable for patients who exhibit an "increased" target gene expression level greater than the first quartile (i.e., greater than the bottom 25%) of the expression level of such target genes in a population of individuals suffering from hematological malignancies. The methods and compositions of the present invention are particularly suitable for patients who exhibit an "increased" target gene expression level greater than the second quartile (i.e., greater than the bottom 50%) of the expression level of such target genes in a population of individuals suffering from hematological malignancies. The methods and compositions of the present invention are particularly suitable for patients who exhibit an "increased" target gene expression level that is greater than the third quartile (i.e., greater than the bottom 75%) of the expression level of such target gene in a population of individuals suffering from a hematological malignancy. The methods and compositions of the present invention are particularly suitable for patients who exhibit an "increased" target gene expression level that is greater than 85%, greater than 90%, or greater than 95% of the expression level of such target gene in a population of individuals suffering from a hematological malignancy.

[0096] In a third embodiment, by comparing the expression level of one or more target genes with the average or weighted baseline expression level of such target genes in a population of individuals who have not been successfully treated for hematological malignancies using the methods and compositions of the present invention (e.g., a population of individuals who have not successfully responded to the treatment of hematological malignancies using CD123 xCD3 bispecific molecules), the patient is identified as exhibiting a gene expression signature characterized by the presence of an immune-enriched and IFNγ-dominant tumor microenvironment, and is therefore particularly suitable for the treatment of hematological malignancies using the methods and compositions of the present invention. Target genes whose expression is equal to or greater than this average or weighted baseline level are referred to as exhibiting an "increased" expression level, and the methods and compositions of the present invention are particularly suitable for treating hematological malignancies in such patients. The methods and compositions of the present invention are particularly suitable for patients who exhibit an "increased" target gene expression level greater than the first quartile (i.e., greater than the bottom 25%) of the expression level of such target genes in the population of individuals who have not been successfully treated. The methods and compositions of the present invention are particularly suitable for patients who exhibit an "increased" target gene expression level that is greater than the second quartile (i.e., greater than the bottom 50%) of the expression level of the target gene in the population of individuals who have not been successfully treated. The methods and compositions of the present invention are particularly suitable for patients who exhibit an "increased" target gene expression level that is greater than the third quartile (i.e., greater than the bottom 75%) of the expression level of the target gene in the population of individuals who have not been successfully treated. The methods and compositions of the present invention are particularly suitable for patients who exhibit an "increased" target gene expression level that is greater than 85%, greater than 90%, or greater than 95% of the expression level of the target gene in the population of individuals who have not been successfully treated.

[0097] In a fourth embodiment, by comparing the expression level of one or more target genes with the average or weighted baseline expression level of such target genes in a population of individuals who have previously been successfully treated for hematological malignancies using the methods and compositions of the present invention (e.g., a population of individuals who have successfully responded to the treatment of hematological malignancies using the CD123 x CD3 bispecific molecule), the patient is identified as exhibiting a gene expression signature characterized by the presence of an immune-enriched and IFNγ-dominant tumor microenvironment, and is therefore particularly suitable for the treatment of hematological malignancies using the methods and compositions of the present invention. Target genes whose expression is equal to or greater than this average or weighted baseline level are referred to as exhibiting an "increased" expression level, and the methods and compositions of the present invention are particularly suitable for treating hematological malignancies in such patients. The methods and compositions of the present invention are particularly suitable for patients who exhibit an "increased" target gene expression level within at least the first quartile (i.e., within the bottom 25%) of the expression level of such target genes in such a population of successfully treated individuals. The methods and compositions of the present invention are particularly applicable to patients who exhibit an "increased" target gene expression level that is within at least the second quartile of the expression level of the target gene in the population of successfully treated individuals (i.e., between the bottom 25% and 50%). The methods and compositions of the present invention are particularly applicable to patients who exhibit an "increased" target gene expression level that is within at least the third quartile of the expression level of the target gene in the population of successfully treated individuals (i.e., between the bottom 50% and 75%). The methods and compositions of the present invention are even more particularly applicable to patients who exhibit an "increased" target gene expression level that is within at least the fourth quartile of the expression level of the target gene in the population of previously treated individuals (i.e., above the bottom 75%).

[0098] However, it is preferred to determine whether the expression of the target gene is "increased" by comparing the expression level of the target gene with the expression level of one or more genes ("reference" genes) that are not related to the disease or do not show increased expression due to the disease state. Since reference genes are usually expressed at different levels, the geometric mean of the expression of the reference genes can be used to calculate the proportionality factor. The geometric mean is obtained by multiplying each gene of each sample value in the data set and then taking the nth root (where n is the digital count in the group). The geometric mean is similar to the arithmetic mean because it represents the central tendency of a group of numbers. However, unlike the arithmetic mean, the geometric mean is less sensitive to changes in the amplitude of the count level between probes. In order to compare the biological labels of the entire sample group, the geometric mean from a group of "reference" genes can be used to standardize the individual samples of the entire data set so that comparisons can be made between biological genes without being affected by technical differences such as sample quality input and sample quality.

[0099] Preferably, a "reference" gene is constitutively expressed at the same level in normal and malignant cells. Housekeeping genes (Eisenberg, E. et al. (2003) "Human Housekeeping Genes Are Compact," Trends in Genetics. 19(7): 362-365; kon Butte, AJ et al. (2001) "Further Defining Housekeeping, 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; Eisenberg, E. et al. (2013) "Human Housekeeping Genes, Revisited," Trends in Genetics. 29(10): 569-574), such as genes required for maintaining basic cellular functions, are a preferred class of reference genes.

[0100] In a further embodiment, determining whether a patient is specifically suitable for treatment with a CD123 x CD3 binding molecule therapy further comprises: (a) assessing the expression level of CD123 in the blastocytes (cancer cells) of the patient. This level can be compared with the expression of CD123 in the blastocytes of this patient before or during the course of the chemotherapy treatment regimen of this patient, or with respect to the corresponding baseline level CD123 of the expression of CD123 in the blastocytes of the population of individuals with HMA therapy relapse or refractory to HMA therapy. The discovery that CD123 expression increases further indicates that the patient is suitable for receiving CD123 x CD3 binding molecule therapy suitable for receiving hematological malignancies. The determination of CD123 expression can be completed by assessing the presence of CD123-encoding mRNA or by assessing the presence of CD123 in cell lysates or extracts. Alternatively, the determination of CD123 expression can be completed by assessing the presence of CD123 molecules arranged on the cell surface of CD123 expressing cells (e.g., blastocytes). An increase in CD123 expression so determined (e.g., an increase in expression of at least 10%, an increase in expression of at least 15%, an increase in expression of at least 20%, an increase in expression of at least 25%, an increase in expression of at least 30%, or an increase in expression of at least 40%) indicates that the patient is suitable for receiving CD123 x CD3 binding molecule therapy for a hematological malignancy. and / or (b) Assessing the expression level of PD-L1 in the patient's blasts (cancer cells). In certain embodiments, the PD-L1 expression level of the entire sample of the patient's blasts is assessed, thereby assessing the percentage of blasts expressing PD-L1. In other embodiments, the PD-L1 expression level in the patient's blasts is compared with the PD-L expression in the blasts of this patient during the course of the chemotherapy treatment regimen of this patient. Therefore, the expression level of PD-L1 in the patient's cells can be assessed before any administration of the CD123 x CD3 binding molecule and / or after such administration. The finding of low expression of PD-L1 indicates that the patient is suitable for receiving CD123 x CD3 binding molecule therapy for hematological malignancies. The finding of high expression of PD-L1 indicates that the patient is suitable for receiving CD123 x CD3 binding molecule therapy in combination with a PD-1 / PD-L1 axis antagonist for hematological malignancies. The determination of PD-L1 expression can be accomplished by assessing the presence of PD-L1-encoding mRNA or by assessing the presence of PD-L1 in a cell lysate or extract. Alternatively, determination of PD-L1 expression can be accomplished by assessing the presence of PD-L1 molecules arranged on the cell surface of PD-L1-expressing cells (e.g., PBMCs). An increase in PD-L1 expression so determined (e.g., at least 10% of the mother cells express, at least 15% of the mother cells express, at least 20% of the mother cells express, at least 25% of the mother cells express, at least 30% of the mother cells express, or at least 40% of the mother cells express) indicates that the patient is suitable for receiving CD123x CD3 binding molecule therapy in combination with PD-1 / PD-L1 axis antagonist therapy for hematological malignancies (see, e.g., WO 2017 / 214092).

[0101] In a further embodiment, during and / or after administration of the CD123 x CD3 bispecific molecule, an increase in the proportion of CD8+ T-lymphocytes in the tumor microenvironment is monitored.

[0102] In a further embodiment, the CD123 x CD3 binding molecule therapy of the present invention may further include administering an anti-human PD-L1 binding molecule, such as an anti-human PD-L1 antibody, or a diabody having a human PD-L1 binding domain. Anti-human PD-L1 binding molecules that can be used according to this embodiment include atezolizumab, avelumab, and durvalumab (see, e.g., U.S. Patent Nos. 9,873,740; 8,779,108). The amino acid sequences of the complete heavy and light chains of atezolizumab (WHO Drug Information, 2015, Recommended INN: List 74, 29(3):387), durvalumab (WHO Drug Information, 2015, Recommended INN: List 74, 29(3):393-394) and avelumab (WHO Drug Information, 2016, Recommended INN: List 74, 30(1):100-101) are known in the art.

[0103] In an alternative further embodiment, the CD123 x CD3 binding molecule therapy of the present invention may further include the administration of an anti-human PD-1 binding molecule, such as an anti-human PD-1 antibody, or a diabody having a human PD-1 binding domain. Anti-human PD-1 binding molecules that can be used according to this embodiment include: nivolumab (also known as 5C4, BMS-936558, ONO-4538, MDX-1106, and is available as a ), pembrolizumab (formerly lambrolizumab, also known as MK-3475, SCH-900475 and marketed by Merck as ), EH12.2H7 (commercially available from BioLegend), pidilizumab (CAS Reg. No. 1036730-42-3 also known as CT-011, CureTech), hPD-1 mAb7(1.2)IgG4(P), and DART-I (disclosed in WO 2017 / 019846), (see also, e.g., U.S. Pat. Nos. 5,952,136; 7,488,802; 7,521,051; 8,008,449; 8,088,905; 8,354,509; 8,552,154; 8,779,105; 8,900,587; 9,084,776; PCT Patent Publications WO 2004 / 056875; WO 2006 / 121168; WO 2008 / 156712; WO2012 / 135408; WO 2012 / 145493; WO 2013 / 014668; WO 2014 / 179664; WO 2014 / 194302; WO2015 / 112800; WO 2017 / 019846 and WO 2017 / 214092). 1. Exemplary “target” genes

[0104] IFNγ stimulates the gene expression of more than 200 genes, including primary response genes such as IRF, Fc-γ receptor (FCGR), GBP (guanylate binding protein), class I and class II molecules of the major histocompatibility complex (MHC), proteins involved in antigen presentation, antiviral proteins such as PKR and OAS proteins, etc. (Boehm, U et al. (1997) "Cellular Responses To Interferon-γ," Annu. Rev. Immunol. 15:749-795; Schroder, K. et al. (2003) "Interferon-Gamma: An Overview Of Signals, Mechanisms And Functions," J. Leukoc. Biol. 75(2):163-189).

[0105] Table 1 discloses exemplary target genes and representative, non-limiting Accession numbers (see, 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), the documents of which are incorporated herein by reference.

[0106] As provided herein, a highly preferred gene expression signature for determining whether a patient has an immune-enriched and IFNγ-dominant tumor microenvironment is referred to herein as an "interferon (IFN)γ signaling signature". The genes of the IFNγ signaling signature are: CXCL9, CXCL10, CXCL11, and STAT1 (Table 6). The IFNγ signaling signature may further comprise IFNG (see, e.g., representative NCBI sequence accession number: NM_000619.2). Increased expression of the IFNγ signaling signature is particularly associated with a patient's suitability for CD123 x CD3 bispecific binding molecule therapy.

[0107] Other suitable target genes can be added. This other target gene can be easily identified as the downstream regulatory gene of IFNγ using the INTERFEROME database (Samarajiwa1, SA et al. (2009) "INTERFEROME: The Database Of Interferon Regulated Genes," Nucleic Acids Research 37: D852-D857). In particular, preferred other genes are PDCD1 (also referred to herein as PDL1), PDCD1LG2 (also referred to herein as PDL2), IL10, CTLA4 (Table 13) and / or one or more of those genes present in the following gene tags: "Interferon (IFN) downstream tag" (genes listed in Table 12B); "Myeloid inflammation tag" (genes listed in Table 12C); "Inflammatory chemokine tag" (genes listed in Table 12D); "MAGES tag" (genes listed in Table 12E) and / or "immunoproteasome tag" (genes listed in Table 12F), provided in the following examples.

[0108] In particular, the expression of multiple genes and tags can be evaluated in a cluster as a "module" to evaluate the patient's suitability for CD123 x CD3 bispecific binding molecule therapy. A particularly preferred module that can be used to determine whether a patient exhibits an immune-infiltrated (immune-enriched) IFN-dominant tumor microenvironment is referred to herein as an "IFN advantage module". Target genes associated with the IFN advantage module include: PDL1, PDL2, IL10, CTLA4, and genes present in each of the following gene expression tags: IFNγ signaling tag, interferon downstream tag, myeloid inflammatory tag, inflammatory chemokine tag, MAGE tag, and immunoproteasome tag (Table 10).

[0109] An IFN advantage module is referred to as "increased" if the module score is 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 about 30, at least about 31, at least about 32, at least about 33, or at least about 35.

[0110] A patient's IFN Dominance Module score is also referred to as "increased" if the patient's IFN Dominance Module score is greater than the first quartile of IFN Dominance Module scores calculated from the expression levels of such target gene in a population of individuals suffering from hematological malignancies (i.e., greater than the bottom 25%), greater than the second quartile of IFN Dominance Module scores calculated from the expression levels of such target gene in a population of individuals suffering from hematological malignancies (i.e., greater than the bottom 50%), greater than the third quartile of IFN Dominance Module scores calculated from the expression levels of such target gene in a population of individuals suffering from hematological malignancies (i.e., greater than the bottom 75%), greater than 85%, greater than 90%, or greater than 95% of IFN Dominance Module scores calculated from the expression levels of such target gene in a population of individuals suffering from hematological malignancies.

[0111] If the patient's IFN Dominance Module score is greater than the first quartile (i.e., greater than the bottom 25%) of the IFN Dominance Module score calculated from the expression level of such target gene in a population of individuals who have not successfully responded to treatment for a hematological malignancy (e.g., a population of individuals who have not successfully responded to treatment with a CD123 x CD3 bispecific molecule for a hematological malignancy), greater than the second quartile (i.e., greater than the bottom 50 ... bottom quartile (i.e., greater than the bottom 50%) of the IFN Dominance Module score calculated from the expression level of such target gene in a population of individuals who have not successfully responded to treatment for a hematological malignancy (e.g., a population of individuals who have not successfully responded to treatment with a CD123 x CD3 bispecific molecule for a hematological malignancy), The IFN Dominance Module score of a patient is also referred to as "increased" if the patient's IFN Dominance Module score is greater than the third quartile (i.e., greater than the lower 75%) of the IFN Dominance Module score calculated from the expression level of such target gene in a population of individuals who have not successfully responded to treatment with a hematological malignancy (e.g., a population of individuals who have not successfully responded to treatment with a CD123 x CD3 bispecific molecule for a hematological malignancy), greater than 85%, greater than 90%, or greater than 95% of the IFN Dominance Module score calculated from the expression level of such target gene in a population of individuals who have not successfully responded to treatment with a CD123 x CD3 bispecific molecule for a hematological malignancy.

[0112] A patient's IFN Dominance Module score is also referred to as "increased" if the patient's IFN Dominance Module score is within at least the first quartile (i.e., within the bottom 25%) and more preferably, within at least the second quartile (i.e., between the bottom 25% and 50%), within at least the third quartile (i.e., between the bottom 50% and 75%), greater than 85%, greater than 90%, or greater than 95% of IFN Dominance Module scores calculated from the expression levels of such target genes in a population of individuals previously successfully treated for hematological malignancies using methods and compositions of the invention (e.g., a population of individuals who successfully responded to treatment of hematological malignancies using the CD123 x CD3 bispecific molecule).

[0113] Gene signatures associated with the IFN advantage module (CD274, PDCD1LG2, IL10, CTLA4, IFNγ signaling signature, interferon downstream signature, myeloid inflammation signature, inflammatory chemokine signature, MAGES signature, and immunoproteasome signature) can be assessed individually to evaluate the suitability of patients for CD123 x CD3 bispecific binding molecule therapy.

[0114] As further provided herein, another set of highly preferred target genes that can be used to determine whether a patient exhibits a gene expression signature associated with suppression of an adaptive immune response within a tumor (also referred to herein as a "tumor inflammation signature, or simply "TIS") includes the genes: CCL5, CD27, CD274, CD276, CD8A, CMKLR1, CXCL9, CXCR6, HLA-DQA1, HLA-DRB1, HLA-E, IDO1, LAG3, NKG7, PDCD1LG2, PSMB10, STAT1 and / or TIGIT (Table 12A). Increased expression of the tumor inflammation signature is particularly associated with the patient's suitability for CD123 x CD3 bispecific binding molecule therapy. 2. Exemplary “reference” genes

[0115] Housekeeping genes that are constitutively expressed at the same level in normal and malignant cells include preferred types of reference genes. Housekeeping genes include genes involved in general gene expression (such as genes encoding transcription factors, repressors, RNA splicing factors, translation factors, tRNA synthetases, RNA binding proteins, ribosomal proteins, mitochondrial ribosomal proteins, RNA polymerases, protein processing factors, heat shock proteins, histones, cell cycle regulators, apoptosis, oncogenes, DNA repair / replication, etc.), metabolism (such as genes encoding the following enzymes: carbohydrate metabolism, citric acid cycle, lipid metabolism, amino acid metabolism, NADH dehydrogenase, cytochrome C oxidase, ATPase, lysosomal enzymes, proteasome proteins, ribonucleases, sulfur reductases, etc.), cell structural integrity (such as genes encoding cytoskeletal proteins, proteins involved in organelle synthesis, mitochondrial proteins, etc.) and cell surface proteins (such as genes encoding cell adhesion proteins, ion channels and transporters, receptors, HLA / immunoglobulin / cell recognition proteins, etc.), kinases / signaling proteins (such as growth factors, tissue necrosis factor, casein kinase, etc.). Reference genes suitable for this purpose include those encoding: Sterol regulatory component binding proteins (e.g., ATF1, ATF2, ATF4, ATF6, ATF7, ATF7, BTF3, E2F4, ERH, HMGB1, ILF2, IER2, JUND, TCEB2, etc.); Repressors (e.g., PUF60, etc.); RNA splicing proteins (e.g., BAT1, HNRPD, HNRPK, PABPN1, SRSF3, etc.); Translation factors (e.g., EIF1, EIF1AD, EIF1B, EIF2A, EIF2AK1, EIF2AK3, EIF2AK4, EIF2AK1, EIF2B2, EIF2B3, EIF2B4, EIF2S2, EIF3A, EIF3B, EIF3D, EIF3G, EIF3I, EIF3H, EIF3J, EIF3K, EIF3L, EIF3M, EIF3S5, EIF3S8, EIF4A1, EIF4A2, EIF4A3, EIF4E2, EIF4G1, EIF4G2, EIF4G3, EIF4H, EIF5, EIF5, EIF5A, EIF5AL1, EIF5B, EIF6, TUFM, etc.); tRNA synthases (e.g., AARS, AARS2, AARSD1434, CARS, CARS2, DARS, DARS2, EARS2614, FARS2, FARSA, FARSB, GARS, HARS, HARS2, IARS, IARS2, KARS, LARS2, MARS, MARS2, NARS, NARS2, QARS, RARS, RARS2, SARS, TARS, VARS2, WARS2, YARS, YARS2436, etc.); RNA binding proteins (e.g., ELAVL1, etc.); Ribosomal proteins (e.g., RPL5, RPL8, RPL9, RPL10A, RPL11, RPL14, RPL25, RPL26L1, RPL27, RPL30, RPL32, RPL34, RPL35, RPL35A, RPL36AL, RPS5, RPS6, RPS6KA3, RPS6KB1, RPS6KB2, RPS13, RPS19BP1, RPS20, RPS23, RPS24, RPS27, RPN1, etc.); Mitochondrial ribosomal proteins (e.g., MRPL9, MRPL1, MRPL10, MRPL11, MRPL12, MRPL13, MRPL14, MRPL15, MRPL16, MRPL17, MRPL18, MRPL19, MRPL2, MRPL20, MRPL21, MRPL22, MRPL23, MRPL24, MRPL27, MRPL28, MRPL3, MRPL30, MRPL32, MRPL33, MRPL35, MRPL36, MRPL37, MRPL38, MRPL4, MRPL40, MRPL41, MRPL42, MRPL43, MRPL44, MRPL45, MRPL46, MRPL47, MRPL48, MRPL49, MRPL50, MRPL51, MRPL52, MRPL53, MRPL54, MRPL55, MRPL56, MRPL57, MRPL58, MRPL59, MRPL60, MRPL61, MRPL62, MRPL63, MRPL64, MRPL65, MRPL66 PL47, MRPL48, MRPL49, MRPL50, MRPL51, MRPL52, MRPL53, MRPL54, MRPL55, MRPL9, MRPS10, MRPS11, MRPS12, MRPS14, MRPS15, MRPS16, MRPS17, MRPS18A, MRPS18B , MRPS18C, MRPS2, MRPS21, MRPS22, MRPS23, MRPS24, MRPS25, MRPS26, MRPS27, MRPS28, MRPS30, MRPS31, MRPS33, MRPS34, MRPS35, MRPS5, MRPS6, MRPS7, MRPS9, etc.); RNA polymerase (e.g., POLR1C, POLR1D, POLR1E, POLR2A, POLR2B, POLR2C, POLR2D, POLR2E, POLR2F, POLR2G, POLR2H, POLR2I, POLR2J, POLR2K, POLR2L, POLR3C, POLR3E, POLR3GL, POLR3K, etc.); Protein processing proteins (e.g., PPID, PPIE, PPIF, PPIG, PPIH, CANX, CAPN1, CAPN7, CAPNS1, NACA, NACA2, 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, H2AFY, H2AFZ, etc.); Cell cycle proteins (e.g., ARHGAP35, ARHGAP5, ARHGDIA, ARHGEF10L, ARHGEF11, ARHGEF40, ARHGEF7, RAB10, RAB11A, RAB11B, RAB14, RAB18, RAB1A, RAB1B, RAB21, RAB22A, RAB2A, RAB2B380, RAB3GAP1, RAB3GAP2, RAB40C, RAB4A, RAB5A, RAB5B, RAB5C, RAB6A, RAB7A, RAB9A, RABEP1, RABEPK, RABGEF1, RABGGTA, RABGGTB, CENPB, CTBP1, CCNB1IP1, CCNDBP1 , CCNG1, CCNH, CCNK402, CCNL1, CCNL2, CCNY, PPP1CA, PPP1CC, PPP1R10, PPP1R11, PPP1R15B, PPP1R37, PPP1R7, PPP1R8, PPP2CA, PPP2CB552, PPP2R1A, PPP2R2A, PPP2R2D, PPP2R3C, PPP2R4, PPP2R5A, 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, B4GALT5, B4GALT7, GSK3A, GSK3B, TPI1, PGK1, PGAM5, 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 dehydrogenase (e.g., NDUFA2, NDUFA3, NDUFA4, NDUFA5, NDUFA6, NDUFA7, NDUFA8, NDUFA9, NDUFA10, NDUFA11, NDUFA12, NDUFA13, NDUFAF2, NDUFAF3, NDUFAF4, NDUFB2, NDUFB3, NDUFB4, NDU FB5, NDUFB6, NDUFB7, NDUFB10, NDUFB11, NDUFB8, NDUFB9, NDUFC1, NDUFC2, NDUFC2, NDUFS5, NDUFV2, NDUFS2, NDUFS3, NDUFS4, NDUFS5, NDUFS6, NDUFS7, NDUFS8, NDUFV1, NDUFV2, etc.); Cytochrome C oxidase (e.g., COX4I1, COX5B, COX6B1, COX6C, COX7A2, COX7A2L, COX7C, COX8, COX8A, COX11, COX14, COX15, COX16, COX19617, COX20, CYC1, UQCC, UQCR10, UQCR11, UQCRB, UQCRC1, UQCRC2, UQCRHL591, UQCRQ, ATPase, ATP2C1, ATP5A1, ATP5B, ATP 5C1, ATP5D, ATP5F1, ATP5G2, ATP5G3, ATP5H, ATP5J, ATP5J2, ATP5J2, ATP5L, ATP5O, ATP5S, ATP5SL, ATP6AP1, ATP6V0A2, A TP6V0B, ATP6V0C, ATP6V0D1, ATP6V0E1, ATP6V1C1, ATP6V1D, ATP6V1E1, ATP6V1F, ATP6V1G1, ATP6V1H, ATPAF2, ATPIF1, etc.); Lysosomal proteins (e.g., CTSD, CSTB, LAMP1, LAMP2, M6PR, etc.); Proteasome proteins (e.g., PSMA1, PSMA2, PSMA3, PSMA4, PSMA5, PSMA6, PSMA7, PSMB1, PSMB2, PSMB3, PSMB4, PSMB5, ​​PSMB6, PSMB7, PSMC2, PSMC3, PSMC4, PSMC5, PSMC6, PSMD1, PSMD10, PSMD11, PSMD12, PSMD13, PSMD14, PSMD2, PSMD3, PSMD4, PSMD5, PSMD6, PSMD7, PSMD8, PSMD9, PSME2, ​​PSME3, PSMF1, PSMG2, PSMG3, PSMG4591, UBA1, UBA2, UBA3, UBA5, UBA52, UBAC2, UBALD1, UBAP1, UBAP2L, UBALD2 BB, UBC, UBE2A, UBE2B, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2F, UBE2G2, UB E2H, UBE2I, UBE2J1, UBE2J2, UBE2K, UBE2L3, UBE2M, UBE2N, UBE2NL989, UBE2Q1, UBE2R2, UBE2 V1, UBE2V2, UBE2W, UBE2Z, UBE3A, UBE3B, UBE3C, UBE4A, UBE4B, USP10, USP14, USP16, USP19, USP22, USP25, USP27X073, USP33, USP38, USP39, USP4, USP47, USP5, USP7, USP8, USP9X590, etc.); Ribonucleases (e.g., RNH, etc.); Sulfur reductases (e.g., TXN2, TXNDC11, TXNDC12, TXNDC15, TXNDC17, TXNDC9, TXNL1, TXNL4A, TXNL4B, TXNRD1, cytoskeleton, ANXA6, ANXA7, ARPC1A, ARPC2, ARPC5L, CAPZA2, CAPZB, RHOB, RHOT1, RHOT2, TUBB, WDR1, etc.); Proteins involved in organelle synthesis (e.g., BLOC1S1, BLOC1S2, BLOC1S3, BLOC1S4, BLOC1S6, AP1G1, AP1M1, AP2A1, AP2A2, AP2M1, AP2S1, AP3B1, AP3D1, AP3M1, AP3S1, AP3S2, AP4B1, AP5M1, ANXA6, ANXA7, AP1B1, CLTA, CLTB, CLTC, etc.); Mitochondrial proteins (e.g., MTX2, etc.); Cell surface proteins (e.g., AP2S1, CD81, GPAA1, LGALS9, MGAT2, MGAT4B, VAMP3, etc.); Cell adhesion proteins (e.g., CTNNA1, CTNNB1, CTNNBIP1, CTNNBL1, CTNND1458, etc.); Ion channel and transporter proteins (e.g., ABCB10, ABCB7, ABCD3, ABCE1, ABCF1, ABCF2, ABCF3, CALM1, MFSD11, MFSD12, MFSD3, MFSD5, SLC15A4, SLC20A1, SLC25A11, SLC25A26, SLC25A28, SLC25A3, SLC25A32, SLC25A38, SLC25A39, SLC25A44, SLC25A45 6. SLC25A5, SLC27A4, SLC30A1, SLC30A5, SLC30A9, SLC35A2, SLC35A4, SLC35B1, SLC35B2, SLC35C2, SLC35E1, SLC35E3, SLC35F5, SLC38A2, SLC39A1, SLC39A3, SLC39A7, SLC41A3, SLC46A3, SLC48A1, receptor, 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, MAPKAPK2, MAP2K2, MAP3K11, PITPNM, RAC1, RAP1B, RAGA, STK19, STK24, STK25, YWHAB, YWHAH, YWHAQ, YWHAZ, etc.); growth factors (e.g., AIF1, HDGF, HGS, LTBP4, VEGFB, ZFP36L1, tissue necrosis factor, CD40, casein kinase, CSNK1E, CSNK2B, etc.); and miscellaneous proteins (e.g., ALAS1, ARHGEF2, ARMET, AES, BECN1, BUD31, CKB, CPNE1, ENSA, FTH1, GDI2, GUK1, HPRT, IFITM1, JTB, MMPL2, NME2, NONO, P4HB, PRDX1, PTMA, RPA2, SULT1A3, SYNGR2, TTC1, C11Orf13, C14orf2, C21orf33, SPAG7, SRM, TEGT, DAZAP2, MEA1, etc.).

[0116] Preferred housekeeping genes include those listed in Table 2. Representative, non-limiting NCBI accession numbers for each gene are also provided in Table 2. Any combination or subcombination of such genes (and / or splice variants thereof) may be employed.

[0117] The following reference genes are particularly preferred: ABCF1, G6PD, NRDE2, OAZ1, POLR2A, SDHA, STK11IP, TBC1D10B, TBP and UBB. 3. Exemplary Methods for Assessing Expression of Target and Reference Genes

[0118] To reveal the expression level of the target gene relative to the baseline or reference gene, the amount of mRNA in the cell sample corresponding to each target gene evaluated is determined and normalized to the mRNA expression corresponding to the baseline or reference gene. Any suitable method can be used to accomplish this analysis. A preferred method uses Analysis system (NanoString Technologies, Inc.). In the analytical system, RNA from a sample is incubated in the presence of a collection of gene-specific reporter probes and capture probes under conditions sufficient to allow hybridization of the sample RNA to the probes. Each reporter probe carries a fluorescent barcode, and each capture probe contains a biotin moiety that can fix the hybridization complex to a solid support for data collection. After hybridization, excess probe is removed and the support is scanned by an automated fluorescence microscope. A barcode is calculated for each target molecule. Data analysis preferably uses 4.0 analysis software (NanoString Technologies, Inc.). The data shown in Example 1 were analyzed using PanCancer IO360TM The Gene Expression Panel kit (NanoString Technologies, Inc.) was obtained, which contains probe sets for 770 different genes (750 genes cover key pathways at the tumor interface, tumor microenvironment, and immune response) and 20 internal reference genes that can be used for data normalization (Table 5). The gene signature score is calculated as follows: The raw data counts for each gene were normalized to the geometric mean of the housekeeping (HK) genes (e.g., ABCF1, NRDE2, G6PD, OAZ1, POLR2A, SDHA, STK11IP, TBC1D10B, TBP, UBB) selected for each sample. • The HK normalized data is then normalized to the IO 360 grouping standards, preferably to those standards run on the same cartridge as the samples tested. Each normalized gene count was then log-transformed. Once normalized and log-transformed, each gene was multiplied by its weight value. Each of these weighted counts is summed to generate a single score. An adjustment factor, i.e. a constant, is added to the final calculated score (e.g. +7). The adjustment factor can be derived from the lowest observed score (from TCGA and / or cell line analysis) so that the score range is above 0.

[0119] In Table 3, genes are classified as follows: Column 1: gene name; Column 2: internal reference gene; Column 3: cell type (B: B cell; CD8: CD8 T cell; Cyto: cytotoxic cell; CD45: CD45-expressing cell; CD56d: NKCD56 dim Column 4: Release of cancer antigens; Column 5: Cancer antigen presentation; Column 6: T cell priming and activation; Column 7: Localization of immune cells to tumors; Column 8: Stromal factors; Column 9: Recognition of cancer cells by T-cells; Column 10: Killing of cancer cells; Column 11: Myeloid cell activity; Column 12: NK cell activity; Column 13: Cell cycle and proliferation; Column 14: Tumor intrinsic factors; Column 15: Immunometabolism; Column 16: General signaling pathways. Genes associated with additional pathways and cell types that make up a particular gene expression signature and methods for calculating gene expression signature scores are provided in the following examples. B. Analysis of “Gene Expression Signatures”

[0120] Gene expression analysis of bone marrow (BM) cell samples at baseline stratified chemotherapy-refractory, HMA-refractory (including secondary AML), and relapsed patients into 3 cluster groups in the immune chain: patients displaying an immune-depleted gene expression signature, patients displaying an immune-exhausted gene expression signature, and patients displaying an immune-enriched gene expression signature.

[0121] As described in more detail below, patients with primary refractory disease (refractory to ≥2 induction attempts, first CR of <6 months, or failure after ≥4 cycles of the demethylating agent HMA) displayed a gene expression signature of an immune-infiltrated tumor microenvironment, as evidenced by their approximately 33% higher levels of inflammatory chemokines relative to levels seen in relapsed patients (3.27±0.22 vs. 2.46±0.07, p=0.026).

[0122] Within this group, chemotherapy-refractory and HMA-refractory patients were further stratified into a first subpopulation that exhibited a gene signature of an immune-exhausted tumor microenvironment (see, Figure 2 , boxed labels indicated for cluster 2) and a second subpopulation that exhibited an immune-enriched tumor microenvironment gene signature including an interferon gamma (also referred to herein as "IFNγ") signaling signature (see, Figure 2, boxed labels indicated for cluster 3). HMA-refractory patients showed characteristics of immune exhaustion and adaptive immune resistance, including an increase of approximately 44% in TIGIT expression relative to chemotherapy-refractory patients (5.55±0.34 vs. 3.85±0.24, p=0.006), an increase of approximately 48% in PD-L1 expression (3.55±0.18 vs. 2.4±0.29, p=0.009), and an increase of approximately 32% in Treg cell-specific expression (4.87±0.23 vs. 3.69±0.19, p=0.0009). HMA-refractory patients also showed a trend toward increasingly exhausted CD8 T cells (measured by their expression of CD244, EOMES, LAG3, and PTGER4) compared to chemotherapy-refractory patients.

[0123] Focusing only on chemotherapy-refractory (i.e., refractory to ≥2 induction attempts, first CR <6 months) and relapsed AML patients (i.e., excluding HMA-refractory patients), further analysis of a broader set of genes (performed by aggregating the scores of the three signature modules as described below) stratified relapsed and refractory AML patient BM samples at baseline into two immune subtypes, referred to herein as "immune-infiltrated" and "immune-depleted" subtypes. II. Exemplary CD123 x CD3 Bispecific Binding Molecules A.JNJ-63709178

[0124] JNJ-63709178 is a humanized IgG4 bispecific antibody with silent Fc function. The antibody was obtained using Genmab JNJ-63709178 is produced by the technology and can bind to CD123 on tumor cells and CD3 on T cells. JNJ-63709178 can recruit T cells to CD123-expressing tumor cells and induce killing of these tumor cells in vitro (MOLM-13, OCI-AML5 and KG-1; EC50 = 0.51-0.91nM). JNJ-63709178 is disclosed in WO 2016 / 036937, Gaudet, F. et al. (2016) "Development of aCD123x CD3 Bispecific Antibody (JNJ-63709178) for the Treatment of Acute Myeloid Leukemia (AML), "Blood 128: 2824; and Forslund, A. et al. (2016) "Ex Vivo Activity Profile of the CD123xCD3 Antibody JNJ-63709178 Against Primary Acute Myeloid Leukemia Bone Marrow Samples,” Blood 128:2875, which document is incorporated herein by reference). The amino acid sequences of the heavy and light chains of JNJ-63709178 and / or related antibodies: 13RB179, 13RB180, 13RB181, 13RB182, 13RB183, 13RB186, 13RB187, 13RB188, 13RB189, CD3B19, 7959, 3978, 7955, 9958, 8747, 8876, 4435 and 5466 are disclosed in WO 2016 / 036937. B.XmAb14045

[0125] XmAb14045 (also known as vibecotamab) is a tumor-targeted antibody containing both a CD123 binding domain and a cytotoxic T-cell binding domain (CD3). The XmAb bispecific Fc domain serves as a scaffold for these two antigen binding domains and confers XmAb14045 with long circulation half-life, stability, and ease of manufacture. Engagement of XmAb14045 with CD3 activates T cells to efficiently and targetedly kill CD123-expressing tumor cells (US Patent Publication 2017 / 0349660; Chu, SY et al. (2014) "Immunotherapy with Long-Lived Anti-CD123 x CD3 Bispecific Antibodies Stimulates Potent T Cell-Mediated Killing of Human AMLCell Lines and of CD123+Cells in Monkeys: A Potential Therapy for AcuteMyelogenous Leukemia," Blood 124(21):2316, which is incorporated herein by reference). The amino acid sequences of the heavy and light chains of XmAb14045 and similar CD123 x CD3 bispecific binding molecules are disclosed in US Patent Publication 2017 / 0349660 and WHO Drug Information, Recommended INN List 120, 2018, 32(4):658-660. C.APVO436

[0126] APVO436 is an ADAPTIR with an anti-CD123 scFv portion and an anti-CD3 scFv portion TMCD123 x CD3 bispecific binding molecule. Each scFv portion is bound to an Fc binding domain that has been modified to eliminate ADCC / CDC effector function. APVO436 is disclosed to have an EC in the low nM range. 50 The values ​​bind to human CD123 and CD3-expressing cells, and demonstrate potent target-specific activity against CD123-expressing tumor cell lines at low effector-to-target ratios. APVO436 is disclosed to be able to effectively induce T-cell priming and proliferation, accompanied by the depletion of CD123-expressing cells in experiments with primary AML subject samples and normal donor samples. APVO436 (see, Comeau, MR et al. (2018) "APVO436, a Bispecific anti-CD123 xanti-CD3ADAPTIR TM Molecule for Redirected T-cell Cytotoxicity,Induces Potent T-cell Activation,Proliferation and Cytotoxicity with Limited CytokineRelease," AACR Annual Meeting April 2018, Abstract 1786; Godwin, CD et al. (2017) "Bispecific Anti-CD123 x Anti-CD3 ADAPTIR TM Molecules APVO436 and APVO437 Have Broad Activity Against Primary Human AML Cells In Vitro,” American Society of Hematology Annual Meeting, December 2017, Blood130:2639; Comeau, MR et al. (2017) “Bispecific anti-CD123 x anti-CD3ADAPTIR TM Molecules for Redirected T-cell Cytotoxicity in Hematological Malignancies,” AACR Annual Meeting April 2017, Abstract 597). The amino acid sequences of the heavy and light chains of the APVO436CD123 x CD3 bispecific binding molecule are disclosed in WO2018 / 057802A1. D.DART-A

[0127] DART-A (also known as vortuzumab, CAS number: 1664355-28-5) is a preferred CD123xCD3 bispecific binding molecule of the present invention. DART-A is a sequence-optimized bispecific diabody that is capable of simultaneously and specifically binding to an epitope of CD123 and an epitope of CD3 ("CD123 x CD3" bispecific diabody) (US Patent Publication No. US2016-0200827, PCT Publication No. WO 2015 / 026892, 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, Vey, N. et al. (2017) "A Phase 1, First-in-Human Study of MGD006 / S80880 (CD123 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). DART-A was found to exhibit enhanced functional activity relative to other non-sequence optimized CD123 x CD3 bispecific diabodies of similar composition and is therefore referred to as a "sequence optimized" CD123 x CD3 bispecific diabody. PCT application PCT / US2017 / 050471 describes a preferred dosing regimen for administering DART-A to a patient and is incorporated herein by reference in its entirety.

[0128] DART-A comprises a first polypeptide chain and a second polypeptide chain ( FIG. 1 ). The first polypeptide chain of the bispecific diabody will comprise, in the N-terminal to C-terminal direction, an N-terminal, a light chain variable domain (VL domain) of a monoclonal antibody capable of binding to CD3 (VL CD3 ), an intervening linker peptide (Linker 1), a heavy chain variable region (VH domain) of a monoclonal antibody capable of binding to CD123 (VH CD123 ) and C-terminus.

[0129] This VL CD3 The preferred sequence of the domain is SEQ ID NO: 1:

[0130] V L CD3 The antigen binding domain of CDR L 1 (SEQ ID NO: 2): RSSTGAVTTSNYAN CDR L 2 (SEQ ID NO:3): GTNCRAP CDR L 3 (SEQ ID NO:4): ALWYSNLWV

[0131] The preferred sequence of this linker 1 is SEQ ID NO:5: GGGSGGGG. CD123 The preferred sequence of the domain is SEQ ID NO:6:

[0132] 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

[0133] The second polypeptide chain will include, in the N-terminal to C-terminal direction, an N-terminal, a VL domain (VL CD123 ), an intervening linker peptide (eg, linker 1), a VH domain of a monoclonal antibody capable of binding to CD3 (VH CD3 ) and C-terminus. This VL CD123 The preferred sequence of the domain is SEQ ID NO: 10:

[0134] V L 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

[0135] This VH CD3The preferred sequence of the domain is SEQ ID NO: 14:

[0136] 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

[0137] The sequence-optimized CD123 x CD3 bispecific diabodies of the present invention are engineered so that the first and second polypeptides are covalently bonded to each other via cysteine ​​residues along their lengths. Such cysteine ​​residues can be introduced into an intervening linker (e.g., linker 1) that separates the VL and VH domains of the polypeptide. Alternatively and more preferably, a second peptide (linker 2) is introduced into each polypeptide chain, for example, at the position of the N-terminus to the VL domain or the C-terminus to the VH domain of the polypeptide chain. The preferred sequence of such linker 2 is SEQ ID NO: 18: GGCGGG.

[0138] The formation of heterodimers can be driven by further engineering such polypeptide chains to contain oppositely charged polypeptide helices. Thus, in a preferred embodiment, one of the polypeptide chains will be engineered to contain an "E-helix" domain (SEQ ID NO: 19: E VAAL E K E VAAL E K E VAAL E K E VAAL E K), whose residues will form a negative charge at pH 7, and the other of the two polypeptide chains is engineered to contain a "K-helix" domain (SEQ ID NO: 20: K VAAL K E K VAAL K E K VAAL K E K VAAL K E), whose residues will form a positive charge at pH 7. The presence of this charged domain promotes the association between the first polypeptide and the second polypeptide, and thus promotes heterodimerization.

[0139] It is not important which helix is ​​provided to the first or second polypeptide chain. However, a preferred sequence-optimized CD123 x CD3 bispecific diabody ("DART-A") of the invention has a first polypeptide chain having the sequence (SEQ ID NO: 21):

[0140] DART-A chain 1 comprises: SEQ ID NO: 1─SEQ ID NO: 5─SEQ ID NO: 6─SEQ ID NO: 18─SEQ ID NO: 19. The polynucleotide encoding the first polypeptide chain of DART-A is SEQ ID NO: 22:

[0141] The second polypeptide chain of DART-A has the sequence (SEQ ID NO:23):

[0142] DART-A chain 2 comprises: SEQ ID NO: 10─SEQ ID NO: 5─SEQ ID NO: 14─SEQ ID NO: 18─SEQ ID NO: 20. The polynucleotide encoding the second polypeptide chain of DART-A is SEQ ID NO: 24:

[0143] As tested by human and cynomolgus monkey cells, DART-A has the ability to bind CD123 and CD3 simultaneously. It was found that providing DART-A can cause T cell priming, mediate blast reduction, drive T cell expansion, induce T cell activation, and cause redirected killing of target cancer cells (Table 4).

[0144] More specifically, in cells with high CD123 expression (Kasumi-3 (EC 50 =0.01 ng / mL)), CD123 expression (Molm13 (EC 50 =0.18 ng / mL) and THP-1 (EC 50 =0.24 ng / mL)) and medium or low CD123 expression (TF-1 (EC 50 =0.46 ng / mL) and RS4-11 (EC 50=0.5ng / mL)) in target cell lines, regardless of CD3 epitope binding specificity, DART-A exhibited a strong redirected killing ability at a concentration required to achieve 50% of maximum activity (EC50) in the sub-ng / mL range. Similarly, DART-A-redirected killing was also observed in multiple target cell lines with T cells from different donors, and redirected killing activity was not observed in cell lines that did not express CD123. The results are summarized in Table 5.

[0145] In addition, when human T cells and tumor cells (Molm13 or RS4-11) are combined and injected subcutaneously into the mice of NOD / SCIDγ (NSG) knockout, MOLM13 tumors are significantly suppressed at 0.16, 0.5, 0.2, 0.1, 0.02 and 0.004mg / kg dosage levels. In the MOLM13 model, 0.004mg / kg and higher dosages are effective (active). Compared with the RS4-11 model, the lower DART-A dosage related to tumor growth inhibition in the MOLM13 model is consistent with the in vitro data, which shows that MOLM13 cells have a higher level of CD123 expression than RS4-11 cells, which is related to the sensitivity of the increased cytotoxicity of DART-A mediation in vitro in MOLM13 cells.

[0146] DART-A is active in primary AML samples (bone marrow mononuclear cells (BMNC) and peripheral blood mononuclear cells (PBMC)) from AML patients. Incubation of primary AML bone marrow samples with DART-A results in the consumption of leukemic cell colonies over time, accompanied by the concomitant expansion of residual T cells (both CD4 and CD8) and the induction of T cell activation markers (CD25 and Ki-67). The upregulation of granzyme B and perforin levels was observed in both CD8 and CD4 T cells. Compared with untreated controls or control DARTs, incubation of primary AML bone marrow samples with DART-A results in the depletion of leukemic cell colonies over time. When counting T cells (CD8 and CD4 staining) and determining activation (CD25 staining), compared with untreated or control DART samples, the T cells in the DART-A samples expand and are activated. It was also found that DART-A was able to mediate the depletion of pDC cells in both human and cynomolgus monkey PBMCs, with cynomolgus monkey pDCs being depleted as early as 4 days after infusion of only 10 ng / kg DART-A. No increase in the levels of cytokines interferon gamma, TNFα, IL-6, IL-5, IL-4, and IL-2 was observed in DART-A treated animals. These data suggest that DART-A mediated target cell killing is mediated through the granzyme B and perforin pathways.

[0147] No activity was observed against CD123-negative targets (U937 cells) or control DARTs, indicating that the observed T cell activation was strictly dependent on target cell engagement and that monovalent engagement of CD3 by DART-A was not sufficient to trigger T cell priming.

[0148] In summary, DART-A is an antibody-based molecule that engages the CD3ε subunit of TCR to redirect T lymphocytes to cells expressing CD123 (antigens upregulated in several hematological malignancies). DART-A binds to human and cynomolgus monkey antigens with similar affinity, and redirects T cells from both species to kill CD123+ cells. Monkeys infused with weekly increasing doses of DART-A for 4 or 7 days a week, regardless of the dosing regimen, showed consumption of circulating CD123+ cells 72 hours after treatment initiation, which lasted throughout the 4 weeks of treatment. A reduction in circulating T cells also occurred, but the 4-day dose regimen returned to baseline before subsequent infusions in monkeys, consistent with DART-A-mediated mobilization. DART-A administration increased circulating PD1+, but did not increase TIM-3+T cells; in addition, in vitro analysis of T cells from treated monkeys showed unchanged redirected target cell lysis, indicating no exhaustion. Toxicity was limited to minimal transient release of cytokines after the first infusion of DART-A, but not after subsequent administrations, even at escalating doses, and minimal reversible reductions in erythrocyte mass accompanied by reductions in CD123+ myeloid progenitors. E. Additional Bispecific Diabody Molecules

[0149] comprising an Fc region and having Figure 1B An alternative version of DART-A of the general structure shown in is described in US2016-0200827. A preferred polypeptide containing the CH2 and CH3 domains of an Fc domain has the sequence (SEQ ID NO: 25) (the "knob-bearing" Fc domain): Where X is K or not present and sequence (SEQ ID NO:26) ("hole-bearing" Fc domain): Where X is K or not present

[0150] The first polypeptide of the exemplary DART-A w / Fc construct includes, in the N-terminal to C-terminal direction, an N-terminal, a VL domain (VL CD123 ), an intervening linker peptide (Linker 1), a VH domain of a monoclonal antibody capable of binding to CD3 (VHCD3 ), Linker 2, E-coil domain, Linker 5, Peptide 1, a polypeptide containing the CH2 and CH3 domains of the Fc domain, and a C-terminus. A preferred Linker 5 has the sequence: GGG. A preferred Peptide 1 has the sequence: DKTHTCPPCP (SEQ ID NO: 29). Thus, the first polypeptide of this DART-A w / Fc version 1 construct includes: SEQ ID NO: 10 ─ SEQ ID NO: 5 ─ SEQ ID NO: 14 ─ SEQ ID NO: 18 ─ SEQ ID NO: 19 ─ GGG ─ SEQ ID NO: 29 ─ SEQ ID NO: 25 (wherein X is K).

[0151] A preferred sequence for the first polypeptide of this DART-A w / Fc version 1 construct has the sequence (SEQ ID NO:27):

[0152] The second chain of this DART-A w / Fc version 1 construct will include, in the N-terminal to C-terminal direction, an N-terminal, a VL domain (VL CD3 ), an intervening linker peptide (Linker 1), a VH domain of a monoclonal antibody capable of binding to CD123 (VH CD123 ), connector 2, K-coil domain and C-terminus. Thus, the second polypeptide of this DART-A w / Fc version 1 construct comprises: SEQ ID NO: 1─SEQ ID NO: 5─SEQ ID NO: 6─SEQ ID NO: 18─SEQ ID NO: 20. This polypeptide has the sequence (SEQ ID NO: 28):

[0153] The third polypeptide chain of this DART-A w / Fc version 1 will include the CH2 and CH3 domains of the IgG Fc domain. Preferred polypeptides include peptide 1 (DKTHTCPPCP; SEQ ID NO: 29) and the CH2 and CH3 domains of the Fc domain (SEQ ID NO: 26, wherein X is K) and have a sequence of SEQ ID NO: 30:

[0154] Additional CD123 x CD3 bispecific diabodies comprising alternative optimized anti-CD3 binding domains are provided in U.S. Appl. Nos.: 62 / 631,043 (filed February 15, 2018); and 62 / 738,632 (filed September 28, 2018) (all of which are incorporated herein). III. Pharmaceutical Preparations

[0155] The compositions of the present invention include bulk pharmaceutical compositions (e.g., impure or non-sterile compositions) for use in the manufacture of pharmaceutical compositions and pharmaceutical compositions that can be used for the preparation of unit dosage forms (i.e., compositions suitable for administration to a subject or patient). Such compositions include a prophylactically or therapeutically effective amount of a CD123 x CD3 bispecific binding molecule and a pharmaceutically acceptable carrier.

[0156] Preferred pharmaceutical formulations include the CD123 x CD3 bispecific binding molecule and an aqueous stabilizer and optionally a pharmaceutically acceptable carrier.

[0157] As used herein, the term "pharmaceutically acceptable carrier" is intended to refer to diluents, adjuvants (e.g., Freund's adjuvant (complete and incomplete)), excipients or vehicles that are approved by regulatory agencies or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeias and are suitable for delivery to animals and more particularly to humans. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain a small amount of wetting or emulsifier or pH buffer. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

[0158] In general, the components of the composition of the present invention are supplied separately or mixed together in a unit dosage form, such as a liquid formulation, a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container such as a vial, an ampoule or a pouch indicating the amount of active agent. Where the composition is administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water or saline for injection can be provided so that the components can be mixed prior to administration.

[0159] The present invention also provides a pharmaceutical package or kit comprising one or more containers containing a CD123 x CD3 bispecific binding molecule alone or with a stabilizer and / or a pharmaceutically acceptable carrier. In addition, one or more other preventive or therapeutic agents for treating a disease may also be included in the pharmaceutical package or kit. The present invention also provides a pharmaceutical package or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical composition of the present invention. Optionally associated with such a container may be a notification in a form specified by a government agency regulating the manufacture, use, and sale of a pharmaceutical preparation or biological product, which reflects the approval of the government agency for manufacture, use, or sale for human administration. IV. Kit

[0160] The present invention provides a kit comprising a CD123 x CD3 bispecific binding molecule, instructional materials (e.g., related to storage, dosage, indications, side effects, contraindications, etc.), and optionally a stabilizer and / or carrier that can be used in the above method. In such a kit, the CD123 x CD3 bispecific binding molecule is preferably packaged in an airtight sealed container such as an ampoule, a vial, a pouch, etc., preferably indicating the amount of the molecule contained herein. The container can be formed of any pharmaceutically acceptable material, such as glass, resin, plastic, etc. The CD123 x CD3 bispecific binding molecule of such a kit is preferably supplied in an airtight sealed container as a liquid solution, a dry sterilized freeze-dried powder, or a water-free concentrate that can be reconstituted, for example, with water or saline to a concentration suitable for administration to a subject. Such a liquid or freeze-dried material should be stored between 2 and 8° C. in its source container and the material should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1 hour after reconstitution. The kit may further include one or more other preventive and / or therapeutic agents for treating cancer in one or more containers; and / or the kit may further include one or more cytotoxic antibodies that bind to one or more cancer antigens associated with cancer. In certain embodiments, the other preventive or therapeutic agent is chemotherapeutic. In other embodiments, the preventive or therapeutic agent is biological or hormonal therapeutic. The kit may further include instructions for use, or other printed information.

[0161] In addition, one or more other preventions or therapeutic agents for the treatment of disease may also be included in pharmaceutical packaging or test kits. The present invention also provides pharmaceutical packaging or test kits comprising one or more containers of one or more compositions filled with pharmaceutical composition of the present invention. Optionally, relevant to this container may be a notification of the form specified by a governmental agency for the manufacture, purposes and sale of a regulating and controlling pharmaceutical preparation or biological product, which notification reflects the approval of a governmental agency for the manufacture, purposes or sale used by a person. V. Methods of Administration

[0162] The CD123 x CD3 bispecific binding molecule pharmaceutical preparation of the present invention can be provided for treating, preventing and improving one or more symptoms associated with a disease, disorder or infection by administering an effective amount of a molecule of the present invention or a pharmaceutical composition comprising a fusion protein or conjugated molecule of the present invention to a subject. In a preferred aspect, such a composition is substantially purified (i.e., substantially free of substances that limit its effect or produce undesirable side effects). In a specific embodiment, the subject is an animal, preferably a mammal such as a non-primate (e.g., cattle, horses, cats, dogs, rodents, etc.) or a primate (e.g., monkeys such as cynomolgus monkeys, humans, etc.). In a preferred embodiment, the subject or patient is a human.

[0163] Methods of administering the CD123 x CD3 bispecific binding molecule pharmaceutical preparations of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous). In a specific embodiment, the CD123 x CD3 bispecific binding molecule is administered intravenously. The composition can be administered by any convenient route, for example, by infusion, and can be administered with other biologically active agents.

[0164] Administration by infusion is preferably accomplished using an infusion pump. An "infusion pump" is a medical device that delivers fluid to the patient's body in a controlled manner, especially at a limited rate and for a prolonged period of time. An infusion pump can be powered mechanically, but is more preferably powered electrically. Some infusion pumps are "fixed" infusion pumps and are designed to be used at the patient's bedside. Others are called "ambulatory" infusion pumps, which are designed to be portable or wearable. A "syringe" pump is an infusion pump in which the fluid to be delivered is maintained in a reservoir of a chamber (e.g., a syringe) and a movable piston is used to control the volume of the chamber and the delivery of the fluid. In an "elastomeric" infusion pump, the fluid is maintained in a stretchable balloon reservoir, and the pressure from the elastic wall of the balloon drives the fluid delivery. In a "peristaltic" infusion pump, a group of rollers squeeze downward on a section of flexible tube, pushing the fluid forward. In a "multi-channel" infusion pump, fluid can be delivered at multiple rates from multiple reservoirs. "Smart pump" refers to an infusion pump that is equipped with a computer-controlled fluid delivery system so as to be able to respond to the risk of adverse drug interactions or to sound an alarm when the pump's parameter settings exceed specified limits. Examples of infusion pumps are well known and are provided, for example, in [Anonymous] 2002 "General-Purpose Infusion Pumps," Health Devices 31(10):353-387 and in U.S. Pat. Nos. 10,029,051, 10,029,047, 10,029,045, 10,022,495, 10,022,494, 10,016,559, 10,006,454, 10,004,846, 9,993,600, 9,981,082, 9,974,901, 9,968,729, 9,931,463, 9,927,943, etc.

[0165] Preferred CD123 x CD3 bispecific binding molecule pharmaceutical preparations of the present invention are administered by one or more mobile pumps to facilitate infusion so that the patient will be mobile during the therapeutic regimen. Preferred CD123 x CD3 bispecific binding molecule pharmaceutical preparations of the present invention are administered by continuous infusion. In a preferred embodiment, a 7-day continuous infusion regimen includes a therapeutic dose of about 30 ng / kg patient weight / day for 3 days, followed by a therapeutic dose of about 100 ng / kg / day for 4 days (e.g., a therapeutic dose of 30 ng / kg patient weight / day for 3 days, followed by a therapeutic dose of 100 ng / kg / day for 4 days, etc.). In a particularly preferred embodiment, this 7-day continuous infusion regimen is followed by a 21-day continuous infusion regimen, wherein a therapeutic dose of 500 ng / kg / day is administered during 1-4 days per week of this 21-day regimen and a therapeutic dose is not administered during 5-7 days per week. Alternatively, this 7-day continuous infusion regimen is followed by a 21-day continuous infusion regimen, wherein a therapeutic dose of 500 ng / kg / day is administered every day for 21 days.

[0166] During any of the above treatments, the proportion of CD8+ T-lymphocytes in the tumor microenvironment may additionally be monitored. Such monitoring may occur prior to administration of the CD123 x CD3 bispecific binding molecule, during the course of CD123 x CD3 binding molecule therapy, and / or after completion of a cycle of CD123 x CD3 binding molecule therapy. VI. Uses of the Compositions of the Invention

[0167] The CD123 x CD3 bispecific binding molecules of the invention can be used to treat any disease or condition associated with or characterized by CD123 expression. In particular, the CD123 x CD3 bispecific binding molecules of the invention can be used to treat hematological malignancies. The CD123 x CD3 bispecific binding molecules of the invention are particularly suitable for treating hematological malignancies, including chemotherapy-refractory hematological malignancies. As used herein, a chemotherapy-refractory hematological malignancy is a hematological malignancy that is refractory to two or more induction attempts, a first CR of less than 6 months, or a failure after two or more cycles of treatment with a hypomethylating agent.

[0168] Thus, without limitation, such molecules may be used in the diagnosis or treatment of acute myeloid leukemia (AML) (including primary chemotherapy-refractory AML), chronic myeloid leukemia (CML) including blast crisis CML and Abelson oncogene (Bcr-ABL translocation) associated with CML, myelodysplastic syndrome (MDS), acute B lymphocytic leukemia (B-ALL), acute T lymphocytic leukemia (T-ALL), chronic lymphocytic leukemia (CLL) including Richter syndrome or Richter transformation of cells, hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin lymphoma (NHL) including mantle cell lymphoma (MCL) and small lymphocytic lymphoma (SLL), Hodgkin lymphoma, systemic mastocytosis and Burkitt lymphoma. The CD123 x CD3 bispecific binding molecules of the present invention may additionally be used to prepare medicaments for the treatment of the above-mentioned conditions.

[0169] The CD123 x CD3 bispecific binding molecules of the present invention are particularly suitable for the treatment of acute myeloid leukemia (AML, including primary chemotherapy-refractory acute myeloid leukemia), hematological myelodysplastic syndrome (MDS), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL) or acute T-lymphocytic leukemia (T-ALL). VII. Specific embodiments of the present invention

[0170] Having now generally described the invention, the invention will be more readily understood by reference to the following numbered embodiments ("E1" - "E60"), which are provided by way of illustration only and are not intended to limit the invention unless otherwise specified: E1. A method of treating a chemotherapy-refractory hematological malignancy in a patient, wherein said method comprises administering to said patient a therapeutic dose of a CD123 x CD3 bispecific molecule effective to stimulate killing of cells of said hematological malignancy in said patient and thereby treat said malignancy. E2. The method according to E1, wherein said method further comprises assessing the expression of one or more target genes and / or reference genes in a cell sample from said patient before and / or after said administering of said CD123xCD3 bispecific molecule. E3. The method according to E2, wherein said method comprises assessing the expression of said one or more target genes and / or said one or more reference genes prior to said administering said CD123 x CD3 bispecific molecule. E4. The method according to E2, wherein said method comprises, subsequent to said administering of said CD123 x CD3 bispecific molecule, assessing the expression of said one or more target genes and / or said one or more reference genes. E5. A method of determining whether a patient is a suitable responder to a CD123 x CD3 bispecific molecule for treatment of a hematological malignancy, wherein the method comprises: (a) assessing the expression of one or more target genes in a cell sample from said patient prior to administration of said CD123xCD3 bispecific molecule relative to the expression of one or more target genes and / or reference genes; and (b) identifying the patient as a suitable responder for treatment with the CD123 x CD3 bispecific molecule if expression of said one or more target genes is found to be increased relative to said expression of said one or more target genes and / or a reference gene. E6. The method according to any one of E2-E6, wherein the method evaluates: (i) expression of one or more target genes; and (ii) one or more reference genes whose expression is not characteristically associated with the hematological malignancy. E7. The method according to any one of E2-E6, wherein the method comprises evaluating the expression of the one or more target genes relative to the baseline expression of the one or more reference genes in the patient. E8. A method according to any one of E2-E7, wherein the method comprises evaluating the expression of the one or more target genes in the patient relative to the expression of the one or more target genes in an individual or a population of such individuals suffering from the hematological malignancy. E9. The method according to any one of E2-E7, wherein the method comprises evaluating the expression of the one or more target genes in the patient relative to the expression of the one or more target genes in an individual or a population of such individuals who did not successfully respond to treatment of the hematological malignancy with the CD123xCD3 bispecific molecule. E10. The method according to any one of E2-E7, wherein the method comprises evaluating the expression of the one or more target genes in the patient relative to the expression of the one or more target genes in an individual or a population of such individuals who successfully responded to treatment of the hematological malignancy with the CD123xCD3 bispecific molecule. E11. A method according to any one of E7-E10, wherein the relative expression level of the one or more target genes in the population is established by averaging the gene expression levels in cell samples obtained from the population of individuals. E12. The method according to any one of E2-E11, wherein the patient exhibits an expression level of at least one of the target genes: (a) greater than the first quartile of the expression level of the target gene in a population of individuals suffering from the hematological malignancy; or (b) greater than the first quartile of the expression level of the target gene in a population of individuals who have not successfully responded to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a logarithmic difference of at least about 0.4 relative to the expression level of the target gene in a population of individuals who have not successfully responded to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule. 2 or (d) within at least the first quartile of the expression level of the target gene in a population of individuals who successfully responded to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule. E13. The method according to any one of E2-E11, wherein the patient exhibits an expression level of at least one of the target genes: (a) greater than the second quartile of the expression level of the target gene in a population of individuals suffering from the hematological malignancy; or (b) greater than the second quartile of the expression level of the target gene in a population of individuals who have not successfully responded to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a logarithmic difference of at least about 0.5 relative to the expression level of the target gene in a population of individuals who have not successfully responded to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule. 2 Fold change; or (d) within at least the second quartile of the expression level of the target gene in a population of individuals who successfully responded to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule. E14. The method according to any one of E2-E11, wherein the patient exhibits an expression level of at least one of the target genes: (a) greater than the third quartile of the expression level of the target gene in a population of individuals suffering from the hematological malignancy; or (b) greater than the third quartile of the expression level of the target gene in a population of individuals who have not successfully responded to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a logarithmic difference of at least about 0.6 relative to the expression level of the target gene in a population of individuals who have not successfully responded to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule 2 Fold change. E15. A method for treating a hematological malignancy, wherein the method comprises: (a) determining whether the patient is a suitable responder to the hematological malignancy using a CD123xCD3 bispecific molecule using a method according to any one of E6-E14; (b) administering to said patient a therapeutic dose of said CD123 x CD3 bispecific molecule if said patient is determined to be a suitable responder to such treatment; wherein said administration of said CD123 x CD3 bispecific molecule stimulates killing of cells of said hematological malignancy in said patient. E16. The method according to E15, wherein the method further comprises evaluating the expression of the one or more target genes in the patient one or more times after the start of the treatment. E17. A method for treating a hematological malignancy, comprising: (a) administering a therapeutically effective dose of a CD123 x CD3 bispecific molecule; (b) determining the expression of one or more target genes in a cell sample obtained from said patient at one or more time points after administration of said CD123 x CD3 bispecific molecule relative to a corresponding baseline expression level obtained before administration of said CD123 x CD3 bispecific molecule; (c) determining whether expression of said one or more target genes is increased relative to said corresponding baseline expression levels, wherein determination of such increased gene expression identifies said patient as a suitable responder for treatment with a CD123xCD3 bispecific molecule; and (d) administering to any such suitable responder patient an adjusted or additional therapeutically effective dose of said CD123xCD3 bispecific molecule, wherein said administration of the CD123 x CD3 bispecific molecule stimulates killing of cells of said hematological malignancy in said patient. E18. The method according to any one of E2-E17, wherein the cell sample is a blood sample. E19. The method according to any one of E2-E17, wherein the cell sample is a bone marrow sample. E20. The method according to any one of E2-E17, comprising detecting the expression level of the one or more target genes and / or the one or more reference genes in a sample of the patient's bone marrow. E21. The method according to any one of E2-E20, wherein the assessing expression or the determining whether the patient is a suitable responder to the use of the CD123 x CD3 bispecific molecule for the treatment of hematological malignancies is performed by: (a) determining the gene expression level of each target gene in one or more cell samples using a gene expression platform; and (b) comparing the target gene expression level with the expression level of one or more reference genes. E22. The method according to any one of E2-E21, wherein said assessing expression or said determining whether said patient is a suitable responder to use of a CD123 x CD3 bispecific molecule for treatment of a hematological malignancy is performed by: (a) measuring the raw RNA level of each target gene in another cell sample using a gene expression platform, wherein the gene expression platform includes a reference gene set of housekeeping genes, and (b) Using the measured RNA levels of an internal reference gene, a relative expression value is assigned to each measured raw RNA level of the target gene. E23. The method according to any one of E2-E22, wherein the one or more target genes include: (a) one or more of the following: CXCL9, CXCL10, CXCL11 and STAT1; and / or (b) one or more of the following: CCL5, CD27, CD274, CD276, CD8A, CMKLR1, CXCL9, CXCR6, HLA-DQA1, HLA-DRB1, HLA-E, IDO1, LAG3, NKG7, PDCD1LG2, PSMB10, STAT1, and TIGIT; and / or (c) one or more of the following: AREG, CSF3, CXCL1, CXCL2, CXCL3, CCL20, FOSL1, IER3 (NM_003897.4), IL6, and PTGS2; and / or (d) one or more of the following: CCL2, CCL3 / L1, CCL4, CCL7 and CCL8; and / or (e) one or more of the following: MAGEA3 / A6, MAGEA1, MAGEA12, MAGEA4, MAGEB2, MAGEC1 and MAGEC2; and / or (f) one or more of the following: APOL6, DTX3L, GBP1, IFI16, IFI27, IFI35, IFI6, IFIH1, IFIT1, IFIT2, IFIT3, IFITM1, IFITM2, IRF1, IRF9, ISG15, MX1, OAS1, OAS2, PARP9, PSMB9, STAT2, TMEM140, and TRIM21; and / or (g) one or more of the following: PSMB8, PSMB9 and PSMB10; and / or (h) IL-10; and or (i) CD274; and / or (j) PDCD1LG2. E24. The method according to E23, wherein the one or more target genes further include IFNG. E25. The method according to any one of E2-E24, wherein the one or more reference genes include one or more of the following: ABCF1, G6PD, NRDE2, OAZ1, POLR2A, SDHA, STK11IP, TBC1D10B, TBP and UBB. E26. A method according to any one of E2-E25, wherein a gene signature score is determined for the one or more target genes. E27. The method according to E26, wherein the gene signature score is determined by a method comprising: (a) measuring the raw RNA level of each target gene in a cell sample using a gene expression platform that includes a reference gene set of housekeeping genes, (b) normalizing each measured raw RNA level to the geometric mean of the housekeeping gene, and optionally further normalizing each RNA value to a standard, (c) Perform log transformation on each normalized RNA value, (d) multiplying each log-transformed RNA value by the corresponding weight factor to generate a weighted RNA value, and (e) Add the weighted RNA values, and optionally an adjustment factor constant, to generate a single gene signature score. E28. The method according to E26 or E27, wherein the gene signature score is determined using the target genes, scoring weights and optional adjustment factors provided in Tables 6 and 12A-12G. E29. The method according to any one of E26-E28, wherein the gene signature score is a gene signature score determined for one or more of the following: (a) IFNγ signaling signature; (b) tumor inflammation signature; (c) myeloid inflammatory signature; (d) inflammatory chemokine signature; (e) MAGE tag; (f) IFN downstream signaling signature; (g) Immunoproteasome labeling; (h) IL-10 labeling; (i) CD274 tag; and / or (j) PDCD1LG2 labeling. E30. The method according to any one of E26-E29, wherein the patient gene signature score is: (a) greater than the first quartile of the score of the gene signature calculated from the expression levels of one or more of the target genes in a population of individuals suffering from the hematological malignancy; or (b) greater than the first quartile of the score of the gene signature calculated from the expression levels of one or more of the target genes in a population of individuals who did not successfully respond to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a logarithmic score of at least about 0.4 relative to the expression level of one or more of said target genes calculated from a population of individuals who did not successfully respond to treatment of said hematological malignancy with the CD123 x CD3 bispecific molecule 2 Fold change; or (d) within at least the first quartile of a score of said gene signature calculated from the expression levels of one or more of said target genes in a population of individuals who have successfully responded to treatment of said hematological malignancy with the CD123 x CD3 bispecific molecule, indicating a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule. E31. The method according to any one of E26-E29, wherein the patient gene signature score is: (a) greater than the second quartile of the gene signature calculated from the expression levels of one or more of the target genes in a population of individuals suffering from the hematological malignancy; or (b) greater than the second quartile of said gene signature calculated from the expression levels of one or more of said target genes in a population of individuals who did not successfully respond to treatment of said hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a logarithmic score of at least about 0.5 relative to the expression level of one or more of said target genes calculated from a population of individuals who did not successfully respond to treatment of said hematological malignancy with the CD123 x CD3 bispecific molecule 2 Fold change; or (d) within at least the second quartile of the score of said gene signature calculated from the expression levels of one or more of said target genes in a population of individuals who have successfully responded to treatment of said hematological malignancy with the CD123 x CD3 bispecific molecule, indicating a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule. E32. The method according to any one of E26-E29, wherein the patient gene signature score is: (a) greater than the third quartile of the score of the gene signature calculated from the expression levels of one or more of the target genes in a population of individuals suffering from the hematological malignancy; or (b) greater than the third quartile of the score of the gene signature calculated from the expression levels of one or more of the target genes in a population of individuals who did not successfully respond to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a logarithmic score of at least about 0.6 relative to the expression level of one or more of said target genes calculated from a population of individuals who did not successfully respond to treatment of said hematological malignancy with the CD123 x CD3 bispecific molecule 2 Fold change, Indicating a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule. E33. The method according to any one of E28-E29, wherein: (a) the gene signature is an IFNγ signaling signature, and a patient gene signature score of at least about 2.5 indicates a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule and / or (b) the gene signature is a tumor inflammation signature, and a patient gene signature score of at least about 5.5 indicates a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule; and / or (c) the gene signature is an IFN downstream signaling signature, and a patient gene signature score of at least about 4.5 indicates a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule. E34. The method according to any one of E28-E29, wherein the gene signature is an IFNγ signaling signature, a tumor inflammation signature, or an IFN downstream signaling signature, and the patient gene signature score is: (a) greater than the first quartile of the score of the gene signature calculated from the expression levels of one or more of the target genes in a population of individuals suffering from the hematological malignancy; or (b) greater than the first quartile of the score of the gene signature calculated from the expression levels of one or more of the target genes in a population of individuals who did not successfully respond to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a logarithmic score of at least about 0.4 relative to the expression level of one or more of said target genes calculated from a population of individuals who did not successfully respond to treatment of said hematological malignancy with the CD123 x CD3 bispecific molecule 2 Fold change; or (d) within at least the first quartile of a score of said gene signature calculated from the expression levels of one or more of said target genes in a population of individuals who have successfully responded to treatment of said hematological malignancy with the CD123 x CD3 bispecific molecule, E35. The method according to any one of E28-E29, wherein the gene signature is an IFNγ signaling signature, a tumor inflammation signature, or an IFN downstream signaling signature, and the patient gene signature score is: (a) greater than the second quartile of the score of the gene signature calculated from the expression levels of one or more of the target genes in a population of individuals suffering from the hematological malignancy; or (b) greater than the second quartile of the score of the gene signature calculated from the expression levels of one or more of the target genes in a population of individuals who did not successfully respond to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a logarithmic score of at least about 0.5 relative to the expression level of one or more of said target genes calculated from a population of individuals who did not successfully respond to treatment of said hematological malignancy with the CD123 x CD3 bispecific molecule 2 Fold change; or (d) within at least the second quartile of the score of said gene signature calculated from the expression levels of one or more of said target genes in a population of individuals who have successfully responded to said hematological malignancy treatment using the CD123 x CD3 bispecific molecule, E36. The method according to any one of E28-E29, wherein the gene signature is an IFNγ signaling signature, a tumor inflammation signature, or an IFN downstream signaling signature, and the patient gene signature score is: (a) greater than the third quartile of the score of the gene signature calculated from the expression levels of one or more of the target genes in a population of individuals suffering from the hematological malignancy; or (b) greater than the third quartile of the score of the gene signature calculated from the expression levels of one or more of the target genes in a population of individuals who did not successfully respond to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a logarithmic score of at least about 0.6 relative to the expression level of one or more of said target genes calculated from a population of individuals who did not successfully respond to treatment of said hematological malignancy with the CD123 x CD3 bispecific molecule 2 Fold change, E37. The method according to E29, wherein an IFN Dominance Module score is determined and wherein a patient IFN Dominance Module score of at least about 25 indicates a more favorable patient response to treatment with said CD123 x CD3 bispecific molecule. E38. The method according to E29, wherein the IFN Dominance Module Score is determined and wherein the patient's IFN Dominance Module Score is: (a) greater than the first quartile of said IFN Dominance Module score calculated from the expression levels of one or more of said target genes in a population of individuals suffering from said hematological malignancy; or (b) greater than the first quartile of the IFN Dominance Module score calculated from the expression levels of one or more of said target genes in a population of individuals who have not successfully responded to treatment of said hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) within at least the first quartile of said IFN Dominance Module score calculated from the expression levels of one or more of said target genes in a population of individuals who have successfully responded to treatment of said hematological malignancy with the CD123 x CD3 bispecific molecule, This indicates a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule. E39. The method according to E29, wherein the IFN Dominance Module Score is determined and wherein the patient's IFN Dominance Module Score is: (a) greater than the second quartile of said IFN Dominance Module score calculated from the expression levels of one or more of said target genes in a population of individuals suffering from said hematological malignancy; or (b) greater than the second quartile of the IFN Dominance Module score calculated from the expression levels of one or more of said target genes in a population of individuals who have not successfully responded to treatment of said hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) within at least the second quartile of said IFN Dominance Module score calculated from the expression level of one or more of said target genes in a population of individuals who have successfully responded to treatment of said hematological malignancy with the CD123 x CD3 bispecific molecule, This indicates a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule. E40. The method according to any one of E26-E39, wherein patients displaying a gene expression signature characterized by an immune-enriched and IFNγ-dominant tumor microenvironment are indicative of a more favorable patient response to treatment with the CD123 x CD3 bispecific molecule. E41. The method according to any one of E1 to E40, wherein the CD123 x CD3 bispecific molecule is a bispecific antibody or a bispecific molecule comprising a scFv. E42. The method according to E41, wherein the CD123 x CD3 bispecific molecule is JNJ-63709178, XmAb14045 or APVO436. E43. The method according to any one of E1-E40, wherein the CD123 x CD3 bispecific molecule is a covalently bonded bispecific diabody having two, three or four polypeptide chains. E44. The method according to E43, wherein the CD123 x CD3 bispecific molecule is a diabody comprising: (a) a first polypeptide chain having an 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 first and the second polypeptide chains are covalently bonded to each other via disulfide bonds. E45. A method according to any one of E1-E44, wherein the patient's hematological malignancy is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), blast crisis CML, Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-lymphocytic leukemia (B-ALL), acute T-lymphocytic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), Richter syndrome, Richter transformation of CLL, hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL) including mantle cell lymphoma (MCL) and small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis and Burkitt's lymphoma. E46. The method according to E45, wherein the hematological malignancy of the patient is ML. E47. A method according to E45, wherein the hematological malignancy of the patient is MDS. E48. A method according to E45, wherein the hematological malignancy of the patient is BPDCN. E49. The method according to E45, wherein the hematological malignancy of the patient is T-ALL. E50 The method according to any one of E2-E49, wherein the hematological malignancy of the patient is refractory to chemotherapy. E51. The method according to E1 or E50, wherein the hematological malignancy of the patient is refractory to cytarabine / anthracycline-based cytotoxic chemotherapy. E52. A method according to E1 or E50, wherein the hematological malignancy of the patient is refractory to demethylating agent chemotherapy. E53. The method according to any one of E2-E52, further comprising determining the level of CD123 expressed by blasts (cancer cells) compared to the corresponding baseline level of CD123 expressed by normal PBMCs. E54. The method according to E56, wherein the expression level is measured by cell surface expression of CD123. E55. The method according to E54, wherein the surface expression of CD123 is increased by at least about 20% relative to a baseline expression level. E56. The method according to E55, wherein the increase in CD123 expression renders the patient more responsive to treatment with the CD123xCD3 bispecific molecule. E57. The method according to any one of E1-E4 or E6-E56, wherein the therapeutic dose of the CD123xCD3 bispecific molecule comprises at least one dose selected from the group consisting of 30, 100, 300 and 500 ng / kg patient weight / day. E58. The method according to E57, wherein the therapeutic dose comprises a dose of 30 ng / kg / day. E59. The method according to E57, wherein the therapeutic dose comprises a dose of 100 ng / kg patient weight / day. E60. The method according to E57, wherein the therapeutic dose comprises a dose of 300 ng / kg patient weight / day. E61. The method according to E57, wherein the therapeutic dose comprises a dose of 500 ng / kg patient weight / day. E62. The method according to any one of E1-E4 or E6-E561, wherein the therapeutic dose is administered by continuous infusion. E63. A method according to any one of E1-E62, wherein the patient is a human patient. Example

[0171] Having now generally described the invention, the invention will be more readily understood by reference to the following examples, which are provided by way of illustration and the embodiments are not intended to limit the invention unless otherwise specified. Example 1 Gene expression signatures of patient populations particularly suitable for treatment with the CD123 x CD3 bispecific binding molecules of the invention

[0172] To demonstrate the correlation between gene expression patterns in patients with hematological malignancies, particularly AML, and favorable outcomes of CD123 x CD3 bispecific binding molecule therapy, RNA was isolated from 78 bone marrow ("BM") samples (36 at baseline, 27 after the first treatment cycle, and 15 after the second treatment cycle) obtained from 40 patients with relapsed or refractory AML enrolled in a Phase 1 / 2 clinical trial of vortuzumab (NCT#02152956, an exemplary CD123 x CD3 bispecific binding molecule) with individual patient consent. TMGene expression was assessed using the NanoString PanCancer 10360 system (NanoString Technologies, Inc), which enables direct multiplexed mRNA quantification of low-abundance transcripts with high sensitivity and linearity in a single reaction (Vadakekolathu, J., et al. (2017) "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 a dose of ≥500 ng / kg / day. NanoString PanCancer 10360 TM The assay (NanoString Technologies, Inc.) compares the expression profiles of 750 genes covering key pathways at the tumor interface, tumor microenvironment, and immune response, including levels of 14 immune cell types and 32 immuno-oncology signatures. NanoString PanCancerIO 360 TM The assay also compared the expression profiles of controls and internal reference genes for data normalization, as provided below.

[0173] Expression profiles include gene signatures for the following pathways or cells: proliferation, JAKSTAT loss, endothelial cells, B7-H3, APM loss, glycolytic activity, mast cells, cytotoxicity, cytotoxic cells, CD8 T cells, lymphoid cells, T cells, Treg cells, CTLA4, TIS, Th1 cells, TIGIT, NKCD56 dim cells, NK cells, apoptosis, hypoxia, ARG1, IL-10, IFNγ, macrophages, myeloid, neutrophils, PD-L2, stroma, dendritic cells (DC), MAGEs, IDO1, B cells, PD-1, NOS2, inflammatory chemokines, PD-L1, CD45, exhausted CD8 T cells, immunoproteasome, APM, IFN downstream regulated genes, myeloid inflammatory genes, MHC2 genes, TGFβ, MMR loss.

[0174] All IO360 gene signature analyses were performed using nCounter TM System (NanoString Technologies, Inc.) and IO 360 reporting module were performed essentially as described below.

[0175] Interferon (IFN) gamma signaling signature genes (including representative, non-limiting NCBI accession numbers for each gene) and weighting factors are shown in Table 6 below.

[0176] To calculate the IFNγ signaling signature score, perform the following steps: The raw data counts for each gene were normalized to the geometric mean of 10 housekeeping (HK) genes (ABCF1, NRDE2, G6PD, OAZ1, POLR2A, SDHA, STK11IP, TBC1D10B, TBP, UBB) for each sample. The HK normalized data were then normalized to the IO 360 panel standards, in which case these data were run on the same cartridges as the cohort samples. Each normalized gene count was then log-transformed. Once normalized and log-transformed, each gene was multiplied by the weights in Table 6. Each of these weighted counts was summed to generate a single score. The adjustment factor, a constant added to the final calculated score, was 6.457026 for the IFNγ signaling signature. The adjustment factor was derived from the lowest observed score (from TCGA and cell line analysis) to make the score range greater than 0.

[0177] In general, the possible range for IFNγ signaling signature scores is 0 to 10. For this first cohort, the range is 1 to 5. A score was calculated for each baseline (screening day - 14) sample.

[0178] The genes and weighting factors for the additional tags detected are provided below.

[0179] As detailed below, several analyses were performed comparing the IFNγ signaling signature scores (and all IO 360 signature scores) for this cohort. The fold change differences between the different patient groups are presented as forest plots, where the box size represents significance and each line represents a confidence interval (see, e.g., Figures 3A-3C and Figure 5A The distribution of IFNγ signaling signature scores between patient groups is provided as a box plot (see, e.g., Figure 5B ).

[0180] The baseline expression of the genes analyzed was associated with whether the patient had a refractory response to conventional chemotherapy (i.e., refractory response to a regimen of cytarabine given in combination with daunorubicin (e.g., 7+3 induction therapy) (abbreviated as RefCTX or CTX-refractory) or refractory response to a regimen of demethylating agents (e.g., decitabine and azacitidine) (abbreviated as RefHMA or HMA-refractory) or relapsed (relapsed). Patients with secondary AML (i.e., AML that evolved from myelodysplasia or was a product of prior chemotherapy) were included in the HMA-refractory group for these analyses. These data were also associated with the patient's response to CD123 x CD3 bispecific binding molecule therapy with vortuzumab. Figure 4 Waterfall plots of 25 evaluable patients treated with target doses are provided. These responses are scored as objective responses (OR) or as non-response (NR). In addition to patients showing complete remission (CR), OR includes all patients showing molecular complete remission (mCR), complete remission but incomplete improvement in hematology (CRi), morphological leukemia-free state (MLF) and partial response (PR). In addition to non-responding patients, NR also includes all patients showing progressive disease / treatment failure (PD) and stable disease (SD).

[0181] Figure 2 Unsupervised hierarchical clustering of 46 IO 360 labels or cell types generated from the results is provided. The results show the baseline expression levels of the 36 bone marrow samples (each in a separate column) relative to the assessed gene labels (each in a separate row). Each IO 360 label score is rescaled to a range of -3 to +3 within the score of the group to facilitate comparison across labels.

[0182] Gene expression analysis of baseline BM samples stratified AML patients into 3 clusters within the immune continuum: immune-consumption, immune-exhaustion, and immune-enrichment ( Figure 2 Patients with primary refractory disease (relapse; refractory to ≥2 induction attempts, first CR <6 months, or failure after ≥4 cycles of demethylating agents, HMA) generally showed an immune-infiltrated tumor microenvironment (TME) phenotype, including higher inflammatory chemokine scores compared with relapsed patients (3.27±0.22 vs. 2.46±0.07, p=0.026). Within this group, chemotherapy-refractory and HMA-refractory patients were further divided into immune-enriched and immune-exhausted phenotypes, respectively. Gene signatures associated with immune-exhausted and immune-enriched phenotypes are listed in Table 7 below and are Figure 3A , 3B and the forest plot shown in 5A .

[0183] Among all refractory and relapsed patients ( Figure 3A ), HMA-refractory and relapsed patients ( Figure 3B ), HMA-refractory and CTX-refractory patients ( Figure 3C ) indicated that HMA-refractory patients exhibited a more aged phenotype. Specifically, HMA-refractory patients showed features of immune exhaustion and adaptive immune resistance, including upregulation of 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) as well as a trend of increased exhausted CD8 T cells (CD244, EOMES, LAG3, and PTGER4) compared to CTX-refractory patients ( Figures 3A-3C ). Figure 3D-3O Several clusters with immune enrichment (cluster 2, Figure 3D-3I ) or immune-depleted (cluster 3, Figure 3J-3O ) spectrum-related gene signature score. Figure 3D ), macrophages ( Figure 3E ), neutrophils ( Figure 3F ), B cells ( Figure 3G )、IFNγ( Figure 3H )、PD-L1( Fig. 3I )、TIGIT( Figure 3J )、CTLA-4( Figure 3K )、Th1( Figure 3L )、CTL( Figure 3M ), CD8 T cells ( Figure 3N ) and cytotoxicity ( Fig.3O ), the gene signature scores for each cluster (immunodepleted (Depl.), immune-enriched (Enriched), and immune-depleted (Exh.) are plotted and p-values ​​(Kruskal-Wallis) are reported.

[0184] A comparative analysis of IFNγ signaling signature scores was performed between OR patients (including all patients showing CR, complete remission; mCR, molecular CR; CRi, complete remission with incomplete hematologic improvement; MLF, morphologic leukemia-free status; or PR, partial response)) and NR patients (including all patients showing SD, stable disease; or PD, progressive disease / treatment failure). Figure 4The changes (relative to baseline) in the presence of blasts in bone marrow samples of 25 patients (classified as relapsed (RL) patients or chemotherapy-refractory (CTX) or HMA-refractory (HMA) patients) are shown, as measured in their response to the CD123 x CD3 bispecific binding molecule vortuzumab at a target dose of 500 ng / kg / day. The objective response (OR) rate for therapy in primary refractory patients was 50% (7 / 14). The complete remission (CR) rate in primary refractory patients was 35.7% (5 / 14).

[0185] Figure 5A A forest plot of the baseline fold change differences between OR patients and NR patients (including PD, SD, TF, NE) is presented, showing increased expression of IFNγ signaling signatures in baseline samples of OR patients (in Figure 5A In addition, TIS and IFN downstream signatures were substantially increased in OR patients. Figure 5B The distribution of IFNγ signaling signature scores in OR patients was shown to increase. In particular, responders to vortuzumab showed significantly higher IFNγ signaling signature scores at baseline compared to non-responders (3.31±0.32 vs. 2.27±0.11, p=0.0005). The sensitivity and specificity of the IFNγ signaling signature score were measured to predict the response diagnostic ability. The data range in this group was bootstrapped for all samples using different threshold cutoff values. The confidence interval (CI) of the threshold or sensitivity and specificity values ​​was calculated using a bootstrap resampling and averaging method. In all bootstrap CIs, patients were resampled and modified curves were constructed before calculating the target statistics. As in the bootstrap comparative test, resampling is performed in a stratified manner by default. Figure 5C The area under the receiver operating characteristic (ROC) curve was plotted in Figure 2, showing the IFN γ Signal transduction tags The predictive performance area under the curve (AUC) of the score is 0.819. The figure shows the true positive rate (TPR) and false positive rate (FPR) achieved using the optimal score cutoff for this cohort for calling a sample high or low for IFN γ signaling signature scoring . A signature with no predictive power would have an ROC curve along the diagonal, while a perfectly predictive signature would have a curve that reaches the upper left corner. The shaded area around the line indicates the confidence interval. These data are also consistent with the higher frequency of responders in primary refractory patients, who generally exhibit higher INFγ signaling signatures compared to relapsed patients. Accordingly, baseline IFNγ signaling signature scores showed a strong correlation with patient response to CD123 x CD3 bispecific binding molecule therapy (AUC for patients treated with vortuzumab = 0.919; Figure 5C Comparison of immune signatures at baseline and response rates between clusters are summarized in Table 8 (cluster immune-depleted (cluster 1) and immune-infiltrated (cluster 2-3)) and Table 9 (immune-depleted (cluster 2) and immune-enriched (cluster 3)). *Response data available for 35 / 38 patients *Response data available for 35 / 38 patients

[0186] The gene expression signature of a set of genes associated with stimulation of cytotoxic cells or with CD8+ T cells was examined in RNA from pre-treatment bone marrow samples ("Basal") and from bone marrow samples after the first cycle of treatment with vortuzumab ("Cycle 1"). The results of this study are shown in Figure 6 In. The results showed that treatment with vortuzumab was able to stimulate immune cells in the tumor microenvironment. In addition, comparison of BM samples after cycle 1 with baseline samples showed that treatment with vortuzumab resulted in increased immune cell infiltration and immune priming scores, as reflected by higher tumor inflammation signatures (6.49 ± 0.20 vs. 5.93 ± 0.12, p = 0.015) and enhanced immunoproteasome (5.72 ± 0.07 vs. 5.23 ± 0.10, p = 0.0002) and IFNγ signaling signatures (3.38 ± 0.23 vs. 2.53 ± 0.14, p = 0.0015) scores. Therefore, vortuzumab-induced tumor microenvironment (TME) gene activation indicates immune enrichment signatures rather than immune exhaustion signatures.

[0187] like Figure 7 As shown in the literature, the vortuzumab-responsive population—particularly those patients who were previously refractory to chemotherapy—exhibited higher CD123 expression.

[0188] AML blast samples collected during screening were analyzed for PD-L1 expression by flow cytometry. Figure 8 As shown in , patients who progressed early (<15 days) during vortuzumab treatment had higher baseline levels of PD-L1 on AML cells than other patients and had evidence of response (SD, OB, PR, CR). The results of this study indicate that PD-L1 expression is associated with reduced activity in vivo and support the combined use of PD-1 / PD-L1 antagonists with CD123 x CD3 bispecific binding molecule therapy (see, e.g., WO 2017 / 214092).

[0189] Together, these data indicate that the IFNγ signaling signature at baseline is associated with the response to CD123 x CD3 bispecific binding molecule therapy. Most patients (6 / 7; 86%) who showed anti-leukemia activity to CD123 x CD3 bispecific binding molecule therapy had high immune infiltration in the bone marrow, with the most sensitive population being an immune-enriched population. In addition, patients previously treated with HMA showed an immune-enriched but exhausted tumor microenvironment (e.g., bone marrow), with increased checkpoint expression, indicating the potential benefits of combining CD123 x CD3 bispecific binding molecule therapy with immune checkpoint blockade. Without being bound by any particular theory, CD123 x CD3 bispecific binding molecule therapy can stimulate an immune-exhausted tumor microenvironment, as indicated by 25% anti-leukemia activity in this population. In particular, seeing D123 x CD3 bispecific binding molecules, ART-A treatment can enhance immune activation, antigen processing / presentation, and IFNγ signaling signature scores. Example 2 Gene expression signatures for relapsed and chemotherapy-refractory patient populations

[0190] Additional analyses were performed to further explore the correlation between higher expression of gene signatures including, but not limited to, IFNγ signaling signatures, TIS, and interferon downstream signatures in immune-infiltrated AML cases and benefit from treatment with bispecific immunotherapy agents targeting CD123 x CD3, such as vortuzumab. This analysis focused on gene signatures and signature combinations (using NanoString PanCancer IO360) from 30 chemotherapy-refractory (≥2 induction attempts, first complete remission <6 months) or relapsed AML patients enrolled in the CP-MGD006-01 clinical trial (NCT#02152956). TM The assay was obtained essentially as described below). This analysis excluded samples from HMA-refractory patients and included additional samples from relapsed and chemotherapy-refractory patients that had not been previously analyzed.

[0191] This analysis stratified relapsed and refractory AML patient BM samples into two immune subtypes at baseline, which are herein referred to as immune-infiltrated and immune-depleted by aggregating three signature modules: IFN-dominance, adaptive, and myeloid scores. Fig. 9 ). The gene signatures associated with the three signature modules are listed in Table 10 below. The module score is the sum of the scores of the individual gene signatures in each sample (the calculation of each gene signature score is as provided above). Single-gene signature

[0192] Fig. 9 Unsupervised hierarchical clustering (Euclidean distance, complete link) of immune and bioactive labels in the bone marrow (BM) microenvironment of patients with relapsed / refractory AML before receiving vortuzumab immunotherapy in the CP-MGD006-01 clinical trial (NCT#02152956) is provided. Responders are individuals who exhibit anti-leukemia responses, defined as complete remission (CR), CR with incomplete hematological recovery (CRi), CR with partial hematological recovery (CRh), partial remission (PR) or "other benefits" (OB; >30% BM blast reduction). Non-responders are individuals with treatment failure (TF), stable disease (SD) or progressive disease (PD). Chemotherapy refractory is defined as the first CR with ≥2 induction attempts or an initial CR duration of <6 months. Each IO360 label score is readjusted to a range of -3 to +3 within the score of the group for comparison across labels.

[0193] BM samples from 92% of patients (11 of 12) with evidence of an anti-leukemic response to CD123 x CD3 bispecific binding molecule therapy with vortuzumab had immune-infiltrated TME relative to nonresponders ( Fig. 9 ).

[0194] Fig.10 A forest plot of the baseline fold change difference between responders (CR, CRi, CRh, PR, and OB) and non-responders (PD, SD, TF) from an analysis of 30 chemotherapy-refractory or relapsed AML patients is presented. Consistent with the analysis provided in Example 1 above, the IFNγ signaling signature, IFN downstream signature, and tumor inflammation signature ( Fig.10 In addition, most of the gene signatures that comprise the IFN advantage module were increased in the baseline samples of responders versus nonresponders (in Fig.10 (with a star in the middle).

[0195] IFNγ signaling signature between refractory and relapsed patients ( Fig.11A ), IFN downstream label ( Fig. 11B ), tumor inflammation signature (TIS, Fig. 11C ) and IFN advantage module ( Fig.11D ) The distribution of scores is plotted on Figures 11A-11D The distribution of scores in refractory patients was increased. The distribution of scores for the nine gene signatures constituting the IFN dominance module and tumor inflammation signature in nonresponders (NR) and responders (OR) is plotted on Figures 12A-12J Middle: IFNγ signaling signature ( Fig. 12A ); IFN downstream label ( Fig. 12B); myeloid inflammation signature ( Fig. 12C ); Immunoproteasome labeling ( Fig.12D ); Inflammatory chemokine signature ( Fig.12E ); MAGE tag ( Fig.12F );PD-L1 tag( Figure 12G );PD-L2 label ( Fig.12H );IL10 tag( Fig.12I ); Tumor Inflammation Signature (TIS, Fig.12J ). The score distribution of these gene signatures increased in the patients who responded. In particular, as shown in Table 11, responders showed significantly higher IFNγ signaling signature, IFN downstream signature, TIS and IFN advantage module scores at baseline compared to non-responders. Unpaired comparisons were performed using the Mann Whitney U test.

[0196] The sensitivity (true positive rate) and specificity (false positive rate) of the scores of the nine gene signatures constituting the IFN advantage module, TIS, and IFN advantage module for predicting response diagnostic ability (ROC AUC) in this group of patients were measured essentially as described above. The ROC curves showing the predictive performance are presented in Figures 13A-13K Middle: IFNγ signaling signature ( Fig.13A , AUC = 0.750); IFN downstream signature ( Fig. 13B , AUC = 0.755); myeloid inflammation signature ( Fig. 13C , AUC=0.69); immunoproteasome labeling ( Fig.13D , AUC=0.505); Inflammatory chemokine signature ( Fig.13E , AUC=0.764); MAGE tag ( Fig.13F , AUC=0.736); PD-L1 signature ( Figure 13G , AUC=0.699); PD-L2 signature ( Fig.13H , AUC = 0.727); IL10 signature ( Fig.13I , AUC=0.745); TIS( Fig.13J , AUC=0.852) and IFN advantage module ( Figure 13K , AUC = 0.806).

[0197] On-treatment BM samples (available in 19 patients at the end of cycle 1) showed increased antigen presentation and immune activation relative to baseline samples (compared using the Mann Whitney U test for unpaired determinations), as evidenced by higher TIS scores (6.47 ± 0.22 vs. 5.93 ± 0.15, p = 0.0006, Fig.14A ), antigen processing mechanism (APM) signature score (5.67±0.16 vs. 5.31±0.12, p=0.002, Fig. 14C ), IFN-γ signaling signature score (3.58±0.27 vs. 2.81±0.24, p=0.0004, Fig. 14B ) and PD-L1 signature score (3.43±0.28 vs. 2.73±0.21, p=0.0062; Fig.14D The results confirm the clinical benefit in AML patients with immune-infiltrated TME and support the local immunomodulatory effect of CD123xCD3 bispecific binding molecule therapy.

[0198] As described above, AML patients with immune-enriched and IFNγ-dominant tumor microenvironments ("TME") have been reported to experience significantly shorter relapse-free survival, indicating refractory to standard induction chemotherapy (Vadakekolathu, J. et al. (2017) "T Immune Gene Expression Profiling in Children and Adults with AcuteMyeloid Leukemia Identifies Distinct Phenotypic Patterns," Blood 130: 3942A). These data indicate that baseline IFNγ signaling signatures, IFN downstream signatures, and IFN advantage module scores are closely associated with refractory to standard chemotherapy and response to CD123 x CD3 bispecific binding molecule therapy. In addition, in the highly pre-treated individuals evaluated herein (average 4 previous lines of therapy), most of the gene signatures constituting the IFN advantage module and tumor inflammation signature (TIS) were shown to be associated with response to CD123 x CD3 bispecific binding molecule therapy. Each of these scores was significantly higher in patients with chemotherapy-refractory AML compared with AML that relapsed on treatment and in individuals with evidence of anti-leukemic activity compared with nonresponders. The ROC curves and AUC values ​​reflected the strong correlation. Gene Tag

[0199] IO 360 gene counting is used System (NanoString Technologies, Inc.), essentially as follows: RNA (~100 ng per sample) was purified from bone marrow aspirates and incubated with a mixture of reporter and capture probes for hybridization. Transcript counts were analyzed on the nCounter FLEX analysis system using a high-resolution setting. Reporter gene count (RCC) output files were used to calculate gene signature scores using predefined linear combinations (weighted averages) of biologically relevant gene sets, essentially as previously described, as detailed herein.

[0200] The IFNγ signaling signature is described in detail above. Immune cell type abundance signatures are defined in Danaher, P., et al., 2017, “Gene Expression Markers of Tumor Infiltrating Leukocytes,” J Immunother Cancer 5, 18); tumor inflammation signatures are described in Danaher, P., et al., 2018 (“Pan-cancer Adaptive Immune Resistance as Defined by the Tumor Inflammation Signature (TIS): Results From The Cancer Genome Atlas (TCGA),” J Immunother Cancer. 6(1): 63) (see also Ayers. M., et al., 2017, “IFN-γ–Related mRNA Profile Predicts Clinical Response to PD-1 blockade” J Clin Invest. 127(8): 2930-2940 and T cell-inflamed GEPs described in WO 2016 / 094377), and other signatures are described in Danaher, P., et al., (2018, “Development of Gene Expression Signatures Characterizing The Tumor-Immune Interaction,” J Clin Oncol 36, 205-205). For ease of reference, the genes and weighting factors of the selected gene signatures used in these studies are provided below.

[0201] The tumor inflammation signature (TIS) genes (including representative, non-limiting NCBI accession numbers for each gene) and weighting factors (see, e.g., WO 2016 / 094377) are shown below in Table 12A.

[0202] Interferon (IFN) downstream signature genes (including representative, non-restrictive NCBI accession numbers for each gene) and weighting factors are shown below in Table 12B. The adjustment factor for this signature is: 5.342598.

[0203] The inflammatory chemokine (Inflammatory Chemokines) signature genes (including representative, non-restrictive NCBI accession numbers for each gene) and weighting factors are shown below in Table 12C. The adjustment factor for this signature is: 6.0968.

[0204] The MAGE signature genes (including a representative, non-restrictive NCBI accession number for each gene) and weighting factors are shown below in Table 12D. The adjustment factor for this signature is: 3.965625.

[0205] Myeloid Inflammation (Myeloid Inflam) signature genes (including representative, non-restrictive NCBI accession numbers for each gene) and weighting factors are shown below in Table 12E. The adjustment factor for this signature is: 5.41931.

[0206] The immunoproteasome signature genes (including representative, non-restrictive NCBI accession numbers for each gene) and weighting factors are shown below in Table 12F. The adjustment factor for this signature is: 6.096812.

[0207] The individual gene signature genes (including a representative, non-restrictive NCBI accession number for each gene) and adjustment factors are shown below in Table 12G.

[0208] The signature scores were calculated essentially as described above, except that once normalized and log transformed, each gene was multiplied by the weights provided in Tables 12A-12F, and the specified adjustment factors were added. For single gene signatures (e.g., PDL1), no weights were used, and the log2 normalized gene expression values ​​were added to the adjustment factors provided in Table 12G.

[0209] All publications and patents mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Although the invention has been described in conjunction with specific embodiments thereof, it will be appreciated that it is capable of further modification, and this application is intended to cover any variations, uses or adaptations of the invention that generally follow the principles of the invention and include such deviations from the essential features set forth herein that fall within known or customary practices in the art to which the invention pertains and that are applicable.

Claims

1. A method of treating a chemotherapy-refractory hematological malignancy in a patient, wherein the method comprises administering to the patient a therapeutic dose of a CD123 x CD3 bispecific molecule effective to stimulate killing of cells of the hematological malignancy in the patient and thereby treat the malignancy.

2. The method of claim 1, wherein said method further comprises assessing the expression of one or more target genes and / or reference genes in a cell sample from said patient prior to and / or subsequent to said administering of said CD123 x CD3 bispecific molecule.

3. A method of determining whether a patient is a suitable responder to a CD123 x CD3 bispecific molecule for treatment of a hematological malignancy, wherein the method comprises: (a) assessing the expression of one or more target genes in a cell sample from the patient prior to administration of the CD123 x CD3 bispecific molecule relative to the expression of one or more target genes and / or reference genes; and (b) identifying the patient as a suitable responder for treatment with the CD123 x CD3 bispecific molecule if expression of said one or more target genes is found to be increased relative to said expression of said one or more target genes and / or a reference gene.

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

5. The method according to any one of claims 2-3, wherein the method comprises assessing the expression of the one or more target genes relative to the baseline expression of the one or more reference genes in the patient.

6. The method according to any one of claims 2-5, wherein the method comprises assessing the expression of the one or more target genes in the patient relative to the expression of the one or more target genes of: (a) an individual or a group of individuals suffering from such a haematological malignancy; or (b) an individual or a population of individuals who have not successfully responded to treatment of said hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) an individual or a population of such individuals who successfully responds to treatment of said hematological malignancy with the CD123 x CD3 bispecific molecule.

7. The method according to any one of claims 5-6, wherein the relative expression level of the one or more target genes in the population is established by averaging the gene expression levels in cell samples obtained from the population of individuals.

8. The method according to any one of claims 2-7, wherein the patient exhibits an expression level of at least one of the target genes: (a) greater than the first quartile of the expression level of the target gene in a population of individuals suffering from the hematological malignancy; or (b) greater than the first quartile of expression levels of the target gene in a population of individuals who have not successfully responded to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule; or (c) having a log2 fold change of at least about 0.4 relative to the expression level of the target gene in a population of individuals who did not successfully respond to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule; or (d) within at least the first quartile of the expression level of the target gene in a population of individuals who successfully responded to treatment of the hematological malignancy with the CD123 x CD3 bispecific molecule.

9. A method for treating a hematological malignancy, wherein the method comprises: (a) using the method according to any one of claims 3 to 8 to determine whether the patient is a suitable responder to treatment of the hematological malignancy with a CD123 x CD3 bispecific molecule; (b) administering to said patient a therapeutic dose of said CD123 x CD3 bispecific molecule if said patient is determined to be a suitable responder to such treatment; wherein said administration of said CD123 x CD3 bispecific molecule stimulates killing of cells of said hematological malignancy in said patient.

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

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