Use of Bispecific CD123 x CD3 Diabodies for the Treatment of Hematologic Malignancies
20230220087 · 2023-07-13
Assignee
Inventors
Cpc classification
G01N2333/70596
PHYSICS
C12Q2600/106
CHEMISTRY; METALLURGY
C07K16/2866
CHEMISTRY; METALLURGY
C07K16/2809
CHEMISTRY; METALLURGY
G01N2800/52
PHYSICS
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present invention is directed to a method of treating a hematologic malignancy such as acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), including hematologic malignancies that are refractive to chemotherapeutic and/or hypomethylating agents. The method concerns administering a CD123×CD3 bispecific binding molecule to a patient in an amount effective to stimulate the killing of cells of said hematologic malignancy in said patient. The present invention is particularly directed to the embodiment of such method in which a cellular sample (Z from the patient prior to such administration evidences an expression of one or more target genes that is increased relative to a baseline level of expression of such genes, for example, a baseline level of expression of such genes in a reference population of individuals who are suffering from the hematologic malignancy, or with respect to the level of expression of a reference gene.
Claims
1. A method of determining whether a patient would be a suitable responder to the use of a CD123×CD3 bispecific molecule to treat a hematologic malignancy, wherein said method comprises: (a) evaluating the expression of one or more target genes in a cellular sample from said patient prior to the administration of said CD123×CD3 bispecific molecule, relative to the expression of one or more target and/or reference genes; and (b) identifying the patient as a suitable responder for treatment with a CD123×CD3 bispecific molecule if the expression of said one or more target genes is found to be increased relative to said expression of said one or more target and/or reference genes, wherein said one or more target genes are selected from the group consisting of: SERPHINH1, NOTCH2, FCGR3A/B, FPR1, FBP1, PDGFA, CRABP2, THBS1, ICOS and CD8B.
2. The method of claim 1, wherein said method evaluates: (i) the expression of one or more target genes; and (ii) one or more reference genes whose expression is not characteristically associated with said hematologic malignancy.
3. The method of any one of claims 1-2, wherein said method comprises evaluating the expression of said one or more target genes relative to the baseline expression of said one or more reference genes of said patient.
4. The method of any one of claims 1-3, wherein said method comprises evaluating the expression of said one or more target genes of said patient relative to the expression of said one or more target genes of: (a) an individual who is suffering from said hematologic malignancy, or of a population of such individuals; or (b) an individual who did not successfully respond to the use of a CD123×CD3 bispecific molecule to treat said hematologic malignancy, or of a population of such individuals; or (c) an individual who successfully responded to the use of a CD123× CD3 bispecific molecule to treat said hematologic malignancy, or of a population of such individuals.
5. The method of any one of claims 1-4, wherein the relative expression level of said one or more target genes in said population is established by averaging the gene expression level in cellular samples obtained from said population of individuals.
6. The method of any one of claims 1-5, wherein said patient exhibits an expression level of at least one of said target genes: (a) that is greater than the first quartile of the expression levels of said target gene in a population of individuals who are suffering from said hematologic malignancy; or (b) that is greater than the first quartile of the expression levels of said target gene in a population of individuals who did not successfully respond to a treatment for said hematologic malignancy that used a CD123×CD3 bispecific molecule; or (c) that is within at least the first quartile of the expression levels of said target gene in a population of individuals who successfully responded to a treatment for said hematologic malignancy that used a CD123×CD3 bispecific molecule.
7. The method of any one of claims 1-6, wherein said method further comprises administering a treatment dosage of said CD123×CD3 bispecific molecule to said patient if said patient is determined to be a suitable responder to such treatment, and wherein said administration of said CD123×CD3 bispecific molecule stimulates the killing of cells of said hematologic malignancy in said patient.
8. A method of treating a hematologic malignancy, wherein said method comprises: (a) employing the method of any one of claims 1-6 to determine whether a patient would be a suitable responder to the use of a CD123×CD3 bispecific molecule to treat said hematologic malignancy; (b) administering a treatment dosage of said CD123×CD3 bispecific molecule to said patient if said patient is determined to be a suitable responder to such treatment; wherein said administration of said CD123×CD3 bispecific molecule stimulates the killing of cells of said hematologic malignancy in said patient.
9. The method of any one of claims 1-8, wherein said cellular sample is a bone marrow sample.
10. The method of any one of claims 1-9, wherein said evaluation of expression or said determination of whether said patient would be a suitable responder to the use of a CD123×CD3 bispecific molecule to treat a hematologic malignancy is performed by: (a) determining the gene expression levels for each target gene in one or more cellular sample(s) using a gene expression platform; and (b) comparing said target gene expression levels to the expression levels of one or more reference genes.
11. The method of any one of claims 1-10, wherein said evaluation of expression or said determination of whether said patient would be a suitable responder to the use of a CD123×CD3 bispecific molecule to treat a hematologic malignancy is performed by: (a) measuring the raw RNA levels in for each target gene in one more cellular sample using a gene expression platform, wherein the gene expression platform comprises a reference gene set of housekeeping genes, and (b) assigning a relative expression value, for each of the measured raw RNA levels for the target genes using the measured RNA levels of the internal reference genes.
12. The method of any one of claims 1-11, wherein said one or more reference genes comprise one or more of: ABCF1, G6PD, NRDE2, OAZ1, POLR2A, SDHA, STK11IP, TBC1D10B, TBP, and UBB.
13. The method of any one of claims 1-12, wherein a gene signature score is determined for said one or more target genes.
14. The method of claim 13, wherein said gene signature score is determined by a process comprising: (a) measuring the raw RNA levels for each target gene in one more cellular sample using a gene expression platform comprising a reference gene set of housekeeping genes, (b) normalizing each of the measured raw RNA levels to the geometric mean of said housekeeping genes, and optionally further normalizing each RNA value to a standard, (c) log transforming each normalized RNA value, (d) summing the log transformed RNA values for each target gene in the signature, and (e) dividing the sum of the normalized log transformed RNA values by the number of target genes in the signature, to generate a gene signature score.
15. The method of any one of claims 13-14, wherein a patient gene signature score that: (a) is greater than the first quartile of scores for said gene signature calculated from the expression levels of one or more of said target genes in a population of individuals who are suffering from said hematologic malignancy; or (b) is greater than the first quartile of scores for 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 a treatment for said hematologic malignancy that used a CD123×CD3 bispecific molecule; or (c) is within at least the first quartile of the scores for said gene signature calculated from the expression levels of one or more of said target genes in a population of individuals who successfully responded to a treatment for said hematologic malignancy that used a CD123×CD3 bispecific molecule, is indicative of a more favorable patient response to treatment with said CD123×CD3 bispecific molecule.
16. The method of any one of claims 1-15, wherein said CD123×CD3 bispecific molecule is a bispecific antibody or bispecific molecule comprising an scFv.
17. The method of claim 16, wherein said CD123×CD3 bispecific molecule is JNJ-63709178, XmAb14045 or APVO436.
18. The method of any one of claims 1-15, wherein said CD123×CD3 bispecific molecule is a covalently bonded bispecific diabody having two, three, or four polypeptide chains.
19. The method of claim 18, wherein said diabody comprises: (a) a first polypeptide chain having the amino acid sequence of SEQ ID NO:21; and (b) a second polypeptide chain having the amino acid sequence of SEQ ID NO:23; and wherein the first and said second polypeptide chains are covalently bonded to one another by a disulfide bond.
20. The method of any one of claims 1-19, wherein said hematologic malignancy of said patient is selected from the group consisting of: acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), blastic crisis of CML, Abelson oncogene-associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B lymphoblastic leukemia (B-ALL), acute T lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), Richter's syndrome, Richter's 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.
21. The method of claim 21, wherein said hematologic malignancy of said patient is AML.
22. The method of any one of claims 1-21, wherein said hematologic malignancy of said patient is refractory to chemotherapy.
23. The method of any one of claims 1-22, wherein said treatment dosage of said CD123×CD3 bispecific molecule includes at least one dose selected from the group consisting of about 30, about 60, about 100, about 200, about 300, about 400 and about 500 ng/kg patient weight/day.
24. The method of any one of claims 1-23, wherein said treatment dosage is administered by continuous infusion.
25. The method of any one of claims 1-24, wherein said patient is a human patient.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0101]
[0102]
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DETAILED DESCRIPTION OF THE INVENTION
[0104] The present invention is directed to a method of treating a hematologic malignancy such as acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), including hematologic malignancies that are refractive to chemotherapeutic and/or hypomethylating agents. The method concerns administering a CD123×CD3 bispecific binding molecule to a patient in an amount effective to stimulate the killing of cells of said hematologic malignancy in said patient. The present invention is particularly directed to the embodiment of such method in which a cellular sample from the patient prior to such administration evidences an expression of one or more target genes that is increased relative to a baseline level of expression of such genes, for example, a baseline level of expression of such genes in a reference population of individuals who are suffering from the hematologic malignancy, or with respect to the level of expression of a reference gene.
[0105] As indicated above, chemotherapy resistance and relapse remain significant sources of mortality for children and adults with acute myeloid leukemia (AML). Receiving conventional chemotherapy, only 26.9% of patients are expected to survive beyond 5 years.
[0106] The therapeutic approach in patients with acute myeloid leukemia (AML) has not changed substantially in more than 30 years. The standard front line therapy is a two-drug regimen of cytarabine given in conjunction with daunorubicin (the so-called 7+3 induction therapy, abbreviated herein as “CTX”). The hypomethylating agents (abbreviated herein as “HMA”) decitabine and azacitidine are commonly administered to older patients or to those considered unfit for the CTX regimen. However, estimates from the literature indicate that up to 45% of patients are refractory to standard frontline chemotherapy. Further intensification of conventional cytotoxic chemotherapy has been deemed to not be feasible due to the severity of acute and long-term side effects upon normal tissues commonly induced by these drugs (Tasian, S. K. (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, M. C. et al. (2018) “The Pharmacology of T Cell Therapies,” Mol. Ther. Methods Clin. Dev. 8:210-221; Dhodapkar, M. V. 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).
[0107] Bispecific antibodies that engage T cells stimulate the release of proinflammatory cytokines. Such cytokines can increase anti-leukemia efficacy by direct cytotoxicity and by activation and recruitment of immune cells into the tumor site (Hoseini, S. S. et al. (2107) “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 flotetuzumab, a CD123×CD3 bispecific binding molecule, is being tested in a Phase ½ study of relapsed/refractory (“R/R”) AML. Despite the great potential of immunotherapy to selectively target the cancer cells causing hematologic 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, F. S. 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 that are capable of targeting a T cell to the location of a hematologic malignancy have not been fully successful.
[0108] The discovery of new treatment strategies, including immunotherapy, thus remains a priority. It has previously been reported that AML patients with an immune-enriched and IFN gamma-dominant tumor microenvironment (“TME”) experience significantly shorter relapse-free survival, suggesting refractoriness 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). In addition, certain gene expression signatures have been reported to correlate with response to the CD123×CD3 bispecific molecule, flotetuzumab (Vadakekolathu J, et al. (2020) “Immune Landscapes Predict Chemotherapy Resistance And Immunotherapy Response In Acute Myeloid Leukemia,” Sci. Transl. Med. 12(546):eaaz0463).
[0109] As used herein, the term “gene expression signature” is intended to denote a pattern of gene expression 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, pages 1-7; Cesano, A. et al. (2018) “Bringing The Next Generation Of Immuno-Oncology Biomarkers To The Clinic,” Biomedicines 6(14) doi: 10.3390/biomedicines6010014, pages 1-11; Shrestha, G. et al. (2016) “The Value Of Genomics In Dissecting The RAS-Network And In Guiding Therapeutics For RAS-Driven Cancers,” Semin. Cell Dev. Biol. 58:108-117; Gingras, I. et al. (2015) “CCR 20th Anniversary Commentary: Gene-Expression Signature in Breast Cancer—Where Did It Start and Where Are We Now?,” Clin. Cancer Res. 21(21):4743-4746; Eberhart, C. G. (2011) “Molecular Diagnostics In Embryonal Brain Tumors,” Brain Pathol. 21(1):96-104; Baylin, S. B. (2009) “Stem Cells, Cancer, And Epigenetics,” StemBook, ed. THE STEM CELL RESEARCH COMMUNITY, StemBook, doi/10.3824/stembook.1.50.1, pages 1-14; Asakura, M. et al. (2009) “Global Gene Expression Profiling In The Failing Myocardium,” Circ. J. 73(9):1568-1576; Shaffer, A. L. et al. (2001) “Signatures Of The Immune Response,” Immunity 15(3):375-385; Staudt, L. M. et al. (2005) “The Biology Of Human Lymphoid Malignancies Revealed By Gene Expression Profiling,” Adv. Immunol. 87:163-208). An observed gene expression signature, and/or changes in that signature resulting from altered (or unaltered) biological process(es), can be used to assess the presence, nature and/or severity of a pathogenic medical condition.
[0110] A central aspect of the present invention relates to the identification of a unique “10-gene expression signature” that predicts a favorable response to therapy employing CD123×CD3 bispecific binding molecules, including therapy employing the CD123×CD3 bispecific binding molecule, flotetuzumab. The 10 genes of the “10-gene expression signature” are: SERPHINH1, NOTCH2, FCGR3A/B, FPR1, FBP1, PDGFA, CRABP2, THBS1, ICOS and CD8B. The invention derives in part from the recognition that certain sub-populations of patients having a hematologic malignancy (e.g., an acute myeloid leukemia) are particularly amenable to treatment with the CD123×CD3 bispecific binding molecules (e.g., flotetuzumab). Members of this sub-population can be readily identified by their ability to exhibit elevated expression of such 10-gene expression signature.
[0111] I. Identification of Patient Populations Particularly Suitable for Treatment with the CD123×CD3 Bispecific Binding Molecules of the Invention
[0112] A. Methods for Determining “Gene Expression Signatures”
[0113] In order to determine whether a patient exhibits elevated expression of the 10-gene expression signature, so as to be thereby identified as being particularly amenable for the treatment of a hematologic malignancy using the methods and compositions of the present invention, an RNA sample from a cellular sample obtained from a patient is evaluated to determine whether it evidences increased expression of one or more “target” genes whose expression correlates with such a signature. Such evaluation may make use of pre-existing detection and/or measurements of gene expression or may incorporate the step(s) of detecting and/or measuring such gene expression. As used herein, the term “cellular sample” refers to a sample that contains cells or an extract of cells.
[0114] Any cellular sample may be employed as a source of RNA or protein for use in determining whether a patient exhibits the 10-gene expression signature that is characteristic of response to a favorable response to therapy employing CD123×CD3 bispecific binding molecules. In certain embodiments, such gene expression comparisons are conducted using RNA obtained from a bone marrow (BM) sample or from a blood sample or a sample of blast cells (cancer cells) of the patient or of a population of donors. Where RNA is obtained from such cells of a population of donors to provide a baseline expression level, the average of the employed expression levels may be used (e.g., a geometric mean may be employed). A number of different reference populations may be used for such gene expression comparisons. In particular embodiments, the expression level of at least one target gene exhibited by a patient is compared to the expression level of such target gene exhibited in: a population of individuals who are suffering from a hematologic malignancy; a population of individuals who were suffering from such hematologic malignancy at the time such reference expression level was determined and who did not successfully respond to a treatment for a hematologic malignancy (i.e., a population of individuals who did not successfully respond to a treatment for a hematologic malignancy using a CD123×CD3 bispecific molecule); and/or a population of individuals who were suffering from such hematologic malignancy at the time such reference expression level was determined and who were thereafter successfully treated for a hematologic malignancy using the methods and compositions of the present invention (i.e., a population of individuals who successfully responded to a treatment for a hematologic malignancy using a CD123×CD3 bispecific molecule). Where the comparator population is a population of individuals who are suffering from a hematologic malignancy such population preferably includes individuals who are suffering from the same hematological malignancy as the patient. Such population may include individuals that have relapsed after prior treatment with a chemotherapeutic agent and/or that were refractory to treatment with a chemotherapeutic agent (i.e., primary refractory). Where the comparator population is a population of individuals who successfully, or unsuccessfully responded to a treatment for a hematologic malignancy CD123×CD3 bispecific molecule such population preferably includes individuals who are suffering from the same hematological malignancy as the patient.
[0115] As used herein, the expression of a gene is said to be “increased” if, relative to a baseline or other comparator (e.g., expression of such gene in a population), its expression is at least about 10% greater, at least about 20% greater, at least about 30% greater, at least about 40% greater, at least about 50% greater, at least about 60% greater, at least about 70% greater, at least about 80% greater, at least about 90% greater, at least about 1.5-fold greater, at least about 2-fold greater, at least about 2.5-fold greater, at least about 3-fold greater, at least about 3.5-fold greater, at least about 4-fold greater, at least about 4.5-fold greater, at least about 5-fold greater, at least about 5.5-fold greater, at least about 6-fold greater, at least about 6.5-fold greater, at least about 7-fold greater, at least about 7.5-fold greater, at least about 8-fold greater, at least about 8.5-fold greater, at least about 9-fold greater, at least about 10-fold greater. Such increases can be alternatively described in terms of “log 2-fold changes.” With respect to increases in expression, a log 2-fold change of 0.4 is equivalent to about 30% greater expression a log 2-fold change of 0.5 is equivalent to about 40% greater expression; a log 2-fold change of 0.6 is equivalent to about 50% greater expression; a log 2-fold change of 0.7 is equivalent to about 60% greater expression; a log 2-fold change of 0.8 is equivalent to about 70% greater expression; a log 2-fold change of 0.9 is equivalent to about 90% greater expression; a log 2-fold change of 1 is equivalent to a 2-fold increase; a log 2-fold change of 1.5 is equivalent to a 2.8-fold increase; a log 2-fold change of 2 is equivalent to a 4-fold increase; a log 2-fold change of 2.5 is equivalent to a 5.7-fold increase; a log 2-fold change of 3 is equivalent to an 8-fold increase; a log.sub.2-fold change of 3.5 is equivalent to an 11.3-fold increase; a log 2-fold change of 4 is equivalent to a 16-fold increase, etc. Log.sub.2 fold changes are commonly used when comparing counts to array data and are also appropriate for t-tests.
[0116] Alternatively, such increases are described in terms of a “gene signature score” wherein the expression of each of a cluster of target genes is measured, normalized to one or more housekeeping genes and/or internal standards, and summed to generate a single gene signature score. Optionally, after normalization and prior to summing, the expression of each target gene may be log transformed, and/or weighted. Methods for calculating such scores are known in the art and specific methods are provided herein (see, Example 1 below).
[0117] The 10-gene signature score of a patient is also said to be “increased” if it is greater than the first quartile of gene signature scores (i.e., greater than the bottom 25%), greater than the second quartile of gene signature scores (i.e., greater than the lower 50%), greater than the third quartile of gene signature scores (i.e., greater than the lower 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 are suffering from a hematologic malignancy.
[0118] The 10-gene signature score of a patient is also said to be “increased” if it is greater than the first quartile of gene signature scores (i.e., greater than the bottom 25%), greater than the second quartile of gene signature scores (i.e., greater than the lower 50%), greater than the third quartile of gene signature scores (i.e., greater than the lower 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 did not successfully respond to a treatment for a hematologic malignancy (e.g., a population of individuals who did not successfully respond to a treatment for a hematologic malignancy CD123×CD3 bispecific molecule).
[0119] The 10-gene signature score of a patient is also said to be “increased” if it is within at least the first quartile of gene signature scores (i.e., within the bottom 25%), 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 a hematologic malignancy using the methods and compositions of the present invention (e.g., a population of individuals who successfully responded to a treatment for a hematologic malignancy using a CD123×CD3 bispecific molecule).
[0120] A finding of an increased 10-gene signature score is indicative of a more favorable patient response to treatment for hematologic malignancy with the CD123×CD3 bispecific molecules of the present invention.
[0121] In one embodiment, a patient is identified as exhibiting an elevated 10-gene expression signature and to thus be particularly amenable to the treatment of hematologic malignancy using the methods and compositions of the present invention by determining whether the expression of a target gene is “increased” relative to the baseline level of its expression in the patient being evaluated when such patient was healthy, or before such patient had received a diagnosis of hematologic malignancy, or relative to the expression of that gene at a time during such patient's course of a chemotherapy treatment regimen or during such patient's course of a treatment regimen involving a CD123×CD3 bispecific binding molecule.
[0122] In a second embodiment, a patient is identified as exhibiting an elevated 10-gene expression signature and as thus being particularly amenable to the treatment of hematologic malignancy using the methods and compositions of the present invention by comparing the level of expression of one or more target gene(s) to the averaged or weighted baseline level of expression of such target gene(s) in a population of individuals who are suffering from a hematologic malignancy. A target gene whose expression is greater than such an averaged or weighted baseline level is said to exhibit an “increased” level of expression, and the methods and compositions of the present invention are particularly suitable for use in treating hematologic malignancy in such patients. For example, the methods and compositions of the present invention are particularly suitable for use in patients who exhibit an “increased” level of target gene(s) expression that is greater than the first quartile (i.e., greater than the bottom 25%) of the expression levels of such target gene(s) in a population of individuals who are suffering from a hematologic malignancy. The methods and compositions of the present invention are particularly suitable for use in patients who exhibit an “increased” level of target gene(s) expression that is greater than the second quartile (i.e., greater than the bottom 50%) of the expression levels of such target gene(s) in a population of individuals who are suffering from a hematologic malignancy. The methods and compositions of the present invention are particularly suitable for use in patients who exhibit an “increased” level of target gene(s) expression that is greater than the third quartile (i.e., greater than the bottom 75%) of the expression levels of such target gene(s) in a population of individuals who are suffering from a hematologic malignancy. The methods and compositions of the present invention are particularly suitable for use in patients who exhibit an “increased” level of target gene(s) expression that is greater than 85%, greater than 90%, or greater than 95% of the expression levels of such target gene(s) in a population of individuals who are suffering from a hematologic malignancy.
[0123] In a third embodiment, a patient is identified as exhibiting an elevated 10-gene expression signature and as thus being particularly amenable to the treatment of hematologic malignancy using the methods and compositions of the present invention by comparing the level of expression of one or more target gene(s) to the averaged or weighted baseline level of expression of such target gene(s) in a population of individuals who have previously been unsuccessfully treated for a hematologic malignancy using the methods and compositions of the present invention (e.g., a population of individuals who did not successfully respond to a treatment for a hematologic malignancy using a CD123×CD3 bispecific molecule). A target gene whose expression is equal or greater than such an averaged or weighted baseline level is said to exhibit an “increased” level of expression, and the methods and compositions of the present invention are particularly suitable for use in treating hematologic malignancy in such patients. The methods and compositions of the present invention are particularly suitable for use in patients who exhibit an “increased” level of target gene(s) expression that is greater than the first quartile (i.e., greater than the bottom 25%) of the expression levels of such target gene(s) in such population of unsuccessfully-treated individuals. The methods and compositions of the present invention are particularly suitable for use in patients who exhibit an “increased” level of target gene(s) expression that is greater than the second quartile (i.e., greater than the bottom 50%) of the expression levels of such target gene(s) in such population of unsuccessfully-treated individuals. The methods and compositions of the present invention are particularly suitable for use in patients who exhibit an “increased” level of target gene(s) expression that is greater than the third quartile (i.e., greater than the bottom 75%) of the expression levels of such target gene(s) in such population of unsuccessfully-treated individuals. The methods and compositions of the present invention are particularly suitable for use in patients who exhibit an “increased” level of target gene(s) expression that is greater than 85%, greater than 90%, or greater than 95% of the expression levels of such target gene(s) in such population of unsuccessfully-treated individuals.
[0124] In a fourth embodiment, a patient is identified as exhibiting an elevated 10-gene expression signature and as thus being particularly amenable to the treatment of hematologic malignancy using the methods and compositions of the present invention by comparing the level of expression of one or more target gene(s) to the averaged or weighted baseline level of expression of such target gene(s) in a population of individuals who have previously been successfully treated for a hematologic malignancy using the methods and compositions of the present invention (e.g., a population of individuals who successfully responded to a treatment for a hematologic malignancy using a CD123×CD3 bispecific molecule). A target gene whose expression is equal or greater than such an averaged or weighted baseline level is said to exhibit an “increased” level of expression, and the methods and compositions of the present invention are particularly suitable for use in treating hematologic malignancy in such patients. The methods and compositions of the present invention are particularly suitable for use in patients who exhibit an “increased” level of target gene(s) expression that is within at least the first quartile (i.e., within the bottom 25%) of the expression levels of such target gene(s) in such population of successfully-treated individuals. The methods and compositions of the present invention are particularly suitable for use in patients who exhibit an “increased” level of target gene(s) expression that is within at least the second quartile (i.e., between the bottom 25% and 50%) of the expression levels of such target gene(s) in such population of successfully-treated individuals. The methods and compositions of the present invention are particularly suitable for use in patients who exhibit an “increased” level of target gene(s) expression that is within at least the third quartile (i.e., between the bottom 50% and 75%) of the expression levels of such target gene(s) in such population of successfully-treated individuals. The methods and compositions of the present invention are even more particularly suitable for use in patients who exhibit an “increased” level of target gene(s) expression that is within at least the fourth quartile (i.e., above the bottom 75%) of the expression levels of such target gene(s) in such population of previously-treated individuals.
[0125] In certain embodiments, whether a target gene's expression is “increased” is determined by comparing the level of its expression to the level of expression of one or more genes that are not associated with disease or that do not exhibit increased expression as a consequence of a disease state (“reference” genes). Because reference genes are often expressed at different levels, the geometric mean of the reference genes' expression can be utilized to calculate scaling factors. A geometric mean is obtained by multiplying each gene per sample value in a data set and then taking the n.sup.th root (where n is the count of numbers in the set) of the resulting product. A geometric mean is similar to an arithmetic mean, in that it indicates the central tendency of a set of numbers. However, unlike an arithmetic mean, the geometric mean is less sensitive to variation in the magnitude of count levels between probes. To compare biological signatures across a cohort of samples the geometric mean from a set of “reference” gene(s) may be used to normalize individual samples across a data set in order for comparisons between biological genes to be made independent of differences due to technical variation such as sample mass input and sample quality.
[0126] Preferred “reference” genes are 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, A. J. 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 the maintenance of basic cellular functions are a preferred class of reference genes.
[0127] In a further embodiment, the CD123×CD3 binding molecule therapy of the present invention may additionally comprise the administration of 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 may be used in accordance with this embodiment include atezolizumab, avelumab, and durvalumab (see, e.g., U.S. Pat. Nos. 9,873,740; 8,779,108). The amino acid sequence 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.
[0128] In an alternative further embodiment, the CD123×CD3 binding molecule therapy of the present invention may additionally comprise 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 may be used in accordance with this embodiment include: nivolumab (also known as 5C4, BMS-936558, ONO-4538, MDX-1106, and marketed as OPDIVO® by Bristol-Myers Squibb), pembrolizumab (formerly known as lambrolizumab, also known as MK-3475, SCH-900475, and marketed as KEYTRUDA® by Merck), EH12.2H7 (commercially available from BioLegend), pidilizumab (CAS Reg. No.: 1036730-42-3 also known as CT-011, CureTech), retifanlimab (CAS Reg. No.: 2226345-85-1 also know as MGA012), and DART-I (disclosed in WO 2017/019846), (also see, 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; WO 2012/135408; WO 2012/145493; WO 2013/014668; WO 2014/179664; WO 2014/194302; WO 2015/112800; WO 2017/019846, and WO 2017/214092).
[0129] B. Exemplary “Target” Genes
[0130] Table 1 discloses the target genes and a representative, non-limiting GenBank® Accession Number for each gene identified in the 10-gene signature.
TABLE-US-00001 TABLE 1 Gene GenBank Description Accession No. † CD8B NM_004931.5 CRABP2 NM_001878.4 FCGR3A/B NM_000569.8/ NM_001244753.2 FBP1 NM_000507.4 FPR1 NM_002029.4 ICOS NM_012092.4 NOTCH2 NM_024408.4 PDGFA NM_002607.5 SERPINH1 NM_001235.5 THBS1 NM_003246.4 † non-limiting representative example, alleles and/or splice variants are also encompassed.
[0131] C. Exemplary “Reference” Genes
[0132] Housekeeping genes that are constitutively expressed at the same level in normal and malignant cells comprise a exemplary class 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 enzymes of: carbohydrate metabolism, the citric acid cycle, lipid metabolism, amino acid metabolism, NADH dehydrogenases, cytochrome C oxidase, ATPases, lysosomal enzymes, proteasome proteins, ribonucleases, thioreductases, etc.), cellular structural integrity (such as genes encoding cytoskeletal proteins, proteins involved in organelle synthesis, mitochondrial proteins, etc.), and cell-surface proteins (such as genes encoding cellular 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 that are suitable for this purpose include genes that encode: [0133] sterol regulatory element binding proteins (e.g., ATF1, ATF2, ATF4, ATF6, ATF7, ATF7, BTF3, E2F4, ERH, HMGB1, ILF2, IER2, JUND, TCEB2, etc.); [0134] repressors (e.g., PUF60, etc.); [0135] RNA splicing proteins (e.g., BAT1, HNRPD, HNRPK, PABPN1, SRSF3, etc.); [0136] translation factors (e.g., EIF1, EIF1AD, EIFIB, 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.); [0137] tRNA synthetases (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.); [0138] RNA binding proteins (e.g., ELAVL1, etc.); [0139] 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.); [0140] 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, 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.); [0141] RNA polymerases (e.g., POLR1C, POLR1D, POLR1E, POLR2A, POLR2B, POLR2C, POLR2D, POLR2E, POLR2F, POLR2G, POLR2H, POLR2I, POLR2J, POLR2K, POLR2L, POLR3C, POLR3E, POLR3GL, POLR3K, etc.); [0142] 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.); [0143] heat shock proteins (e.g., HSPA4, HSPA5, HSPA8, HSPA9, HSPA14, HSBP1, etc.); [0144] histones (e.g., HIST1H2BC, H1FX, H2AFV, H2AFX, H2AFY, H2AFZ, etc.); [0145] 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, RABEPI, RABEPK, RABGEF1, RABGGTA, RABGGTB, CENPB, CTBP1, CCNBIIP1, CCNDBP1, CCNG1, CCNH, CCNK402, CCNL 1, 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.); [0146] apoptosis proteins (e.g., DAD1, DAP3, DAXX, etc.); [0147] oncogene proteins (e.g., ARAF, MAZ, MYC, etc.); [0148] DNA repair/replication proteins (e.g., MCM3AP, XRCC5, XRCC6, etc.); [0149] metabolism proteins (e.g., PRKAG1, PRKAA1, PRKAB1, PRKACA, PRKAG1, PRKAR1A, PRKRIP1, etc.); [0150] carbohydrate metabolism proteins (e.g., ALDOA, B3GALT6, B4GALT3, B4GALT5, B4GALT7, GSK3A, GSK3B, TPIl, PGK1, PGAM5, ENOPHI, LDHA, TALDO1, TSTA3); [0151] citric acid cycle proteins (e.g., SDHA, SDHAF2, SDHB, SDHC, SDHD, etc.); [0152] lipid metabolism proteins (e.g., HADHA, etc.); [0153] amino acid metabolism proteins (e.g., COMT, etc.); [0154] NADH dehydrogenases (e.g., NDUFA2, NDUFA3, NDUFA4, NDUFA5, NDUFA6, NDUFA7, NDUFA8, NDUFA9, NDUFA10, NDUFA11, NDUFA12, NDUFA13, NDUFAF2, NDUFAF3, NDUFAF4, NDUFB2, NDUFB3, NDUFB4, NDUFB5, NDUFB6, NDUFB7, NDUFB10, NDUFB11, NDUFB8, NDUFB9, NDUFC1, NDUFC2, NDUFC2, NDUFS5, NDUFV2, NDUFS2, NDUFS3, NDUFS4, NDUFS5, NDUFS6, NDUFS7, NDUFS8, NDUFV1, NDUFV2, etc.); [0155] cytochrome C oxidases (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, ATP5C1, ATP5D, ATP5F1, ATP5G2, ATP5G3, ATP5H, ATP5J, ATP5J2, ATP5J2, ATP5L, ATP50, ATP5S, ATP5SL, ATP6AP1, ATP6VOA2, ATP6VOB, ATP6VOC, ATP6VOD1, ATP6VOE1, ATP6V1C1, ATP6V1D, ATP6V1E1, ATP6V1F, ATP6V1G1, ATP6V1H, ATPAF2, ATPIF1, etc.); [0156] lysosomal proteins (e.g., CTSD, CSTB, LAMP1, LAMP2, M6PR, etc.); [0157] proteasomal 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, UBALDI, UBAP1, UBAP2L, UBB, UBC, UBE2A, UBE2B, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2F, UBE2G2, UBE2H, UBE2I, UBE2J1, UBE2J2, UBE2K, UBE2L3, UBE2M, UBE2N, UBE2NL989, UBE2Q1, UBE2R2, UBE2V1, 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.); [0158] ribonucleases (e.g., RNH, etc.); [0159] thioreductases (e.g., TXN2, TXNDC11, TXNDC12, TXNDC15, TXNDC17, TXNDC9, TXNL1, TXNL4A, TXNL4B, TXNRD1, Cytoskeletal, ANXA6, ANXA7, ARPC1A, ARPC2, ARPC5L, CAPZA2, CAPZB, RHOB, RHOT1, RHOT2, TUBB, WDR1, etc.); [0160] 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.); [0161] mitochondrial proteins (e.g., MTX2, etc.); [0162] cell surface proteins (e.g., AP2S1, CD81, GPAA1, LGALS9, MGAT2, MGAT4B, VAMP3, etc.); [0163] cell adhesion proteins (e.g., CTNNA1, CTNNB1, CTNNBIP1, CTNNBL1, CTNND1458, etc.); [0164] ion channels 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, SLC25A46, SLC25A5, SLC27A4, SLC30A1, SLC30A5, SLC30A9, SLC35A2, SLC35A4, SLC35B1, SLC35B2, SLC35C2, SLC35E1, SLC35E3, SLC35F5, SLC38A2, SLC39A1, SLC39A3, SLC39A7, SLC41A3, SLC46A3, SLC48A1, Receptors, ACVR1, ACVR1B, CD23, etc.); [0165] HLA/immunoglobulin/cell recognition proteins (e.g., BAT1, BSG, MIF, TAPBP, etc.); [0166] kinases/signaling proteins (e.g., ADRBK1, AGPAT1, ARF1, ARF3, ARF4, ARF5, ARL2, CSF1, CSK, DCT, EFNA3, FKBPlA, GDI1, GNAS1, GNAI2, HAX1, ILK, MAPKAPK2, MAP2K2, MAP3K11, PITPNM, RAC1, RAP1B, RAGA, STK19, STK24, STK25, YWHAB, YWHAH, YWHAQ, YWHAZ, etc.); [0167] growth factors (e.g., AIF1, HDGF, HGS, LTBP4, VEGFB, ZFP36L1, tissue necrosis factor, CD40, casein kinase, CSNK1E, CSNK2B, etc.); and [0168] miscellaneous proteins (e.g., ALAS1, ARHGEF2, ARMET, AES, BECN1, BUD31, CKB, CPNE1, ENSA, FTH1, GDI2, GUK1, HPRT, IFITMI, JTB, MMPL2, NME2, NONO, P4HB, PRDX1, PTMA, RPA2, SULT1A3, SYNGR2, TTC1, C11Orfl3, C14orf2, C21orf33, SPAG7, SRM, TEGT, DAZAP2, MEA1, etc.).
[0169] Exemplary housekeeping genes include those listed in Table 2. Table 2 also provides a representative, non-limiting NCBI Accession Number for each gene. Any combination or sub-combination of such genes (and/or splice variants of the same) may be employed.
TABLE-US-00002 TABLE 2 NCBI Official Accession Gene Full Name Symbol No. ID ABCF1 NM_ 23 Homo sapiens ATP-binding cassette, 001090.2 sub-family F (GCN20), member 1 (ABCF1), transcript variant 2, mRNA ACTB NM_ 60 Homo sapiens actin, beta (ACTB), 001101.2 mRNA ALAS1 NM_ 211 Homo sapiens aminolevulinate, 000688.4 delta-, synthase 1 (ALAS1), transcript variant 1, mRNA B2M NM_ 567 Homo sapiens beta-2-microglobulin 004048.2 (B2M), mRNA CLTC NM_ 1213 Homo sapiens clathrin, heavy 004859.2 polypeptide (Hc) (CLTC), mRNA EEF1G NM_ 1937 Homo sapiens eukaryotic translation 001404.4 elongation factor 1 gamma (EEF1G), mRNA G6PD NM_ 2539 Homo sapiens glucose-6-phosphate 000402.2 dehydrogenase (G6PD), nuclear gene encoding mitochondrial protein, mRNA GAPDH NM_ 2597 Homo sapiens glyceraldehyde-3- 002046.3 phosphate dehydrogenase (GAPDH), mRNA GUSB NM_ 2990 Homo sapiens glucuronidase, beta 000181.1 (GUSB), mRNA HPRT1 NM_ 3251 Homo sapiens hypoxanthine 000194.1 phosphoribosyltransferase 1 (Lesch- Nyhan syndrome) (HPRT1), mRNA LDHA NM_ 3939 Homo sapiens lactate 005566.1 dehydrogenase A (LDHA), mRNA NRDE2 NM_ 55051 Necessary For RNA Interference, 017970.3 Domain Containing OAZ1 NM_ 4946 Homo sapiens ornithine 004152.3 decarboxylase antizyme 1 (OAZ1), transcript variant 1, mRNA PGK1 NM_ 5230 Homo sapiens phosphoglycerate 000291.2 kinase 1 (PGK1), mRNA POLR1B NM_ 84172 Homo sapiens polymerase (RNA) I 019014.3 polypeptide B, 128 kDa (POLR1B), mRNA POLR2A NM_ 5430 Homo sapiens polymerase (RNA) II 000937.2 (DNA directed) polypeptide A, 220 kDa (POLR2A), mRNA PPIA NM_ 5478 Homo sapiens peptidylprolyl 021130.4 isomerase A (PPIA), transcript variant 1, mRNA RPL19 NM_ 6143 Homo sapiens ribosomal protein 000981.3 L19 (RPL19), mRNA RPLP0 NM_ 6175 Homo sapiens ribosomal protein, 001002.3 large, P0 (RPLP0), transcript variant 1, mRNA SDHA NM_ 6389 Homo sapiens succinate 004168.1 dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA), nuclear gene encoding mitochondrial protein, mRNA STK11IP NM_ 114790 Serine/Threonine Kinase 11 052902.3 Interacting Protein TBC1D10B NM_ 26000 TBC1 domain family, member 10B 015527.3 TBP NM_ 6908 Homo sapiens TATA-box binding 003194.3 protein (TBP), transcript variant 1, mRNA TBP NM_ 6908 Homo sapiens TATA-box binding 001172085.1 protein (TBP), transcript variant 2, mRNA TUBB NM_ 203068 Homo sapiens tubulin, beta 178014.2 (TUBB), mRNA UBB NM_ 7314 Ubiquitin B 018955.3
[0170] In certain embodiments, the following reference genes are utilized ABCF1, G6PD, NRDE2, OAZ1, POLR2A, SDHA, STK11IP, TBC1D10B, TBP, and UBB).
[0171] D. Exemplary Methods for Evaluating Expression of Target and Reference Genes
[0172] In order to reveal the level of expression of the target gene(s) relative to the baseline or reference gene(s), the amount of mRNA in a cellular sample corresponding to each assessed target gene is determined and normalized to the expression of mRNA corresponding to the baseline or reference gene(s). Any suitable method may be employed to accomplish such an analysis. A exemplary method employs the nCOUNTER® Analysis System (NanoString Technologies, Inc.). In the nCOUNTER® Analysis System, RNA of a sample is incubated in the presence of sets of gene-specific Reporter Probes and Capture Probes under conditions sufficient to permit the sample RNA to hybridize to the probes. Each Reporter Probe carries a fluorescent barcode and each Capture Probe contains a biotin moiety capable of immobilizing the hybridized complex to a solid support for data collection. After hybridization, excess probe is removed, and the support is scanned by an automated fluorescence microscope. Barcodes are counted for each target molecule. Data analysis is may be conducted using nSolver® 4.0 Analysis Software (NanoString Technologies, Inc.), or similar. The data presented in Example 1 was obtained using PanCancer IO 360™ Gene Expression Panel kits (NanoString Technologies, Inc.) which contain a set of probes for 770 different genes (750 genes cover the key pathways at the interface of the tumor, tumor microenvironment, and immune response, and 20 internal reference genes that may be used for data normalization (Table 5). The 10-gene signature score is calculated as follows: [0173] Raw data counts for each gene are normalized to the geometric mean of the selected housekeeping (HK) genes (e.g., ABCF1, NRDE2, G6PD, OAZ1, POLR2A, SDHA, STK11IP, TBC1D10B, TBP, UBB) for each sample. [0174] HK normalized data is then normalized to IO 360 panel standards, preferably to those run on the same cartridges as the test samples. [0175] Each normalized gene count is then log transformed. [0176] Each of the normalized and log transformed RNA values is summed together. [0177] The sum of the normalized log transformed RNA values is divided by the number of target genes in the signature (i.e., 10) to generate a single score.
[0178] II. Exemplary CD123×CD3 Bispecific Binding Molecules
[0179] A. JNJ-63709178
[0180] JNJ-63709178 is a humanized IgG4 bispecific antibody with silenced Fc function. The antibody was produced using Genmab DuoBody® technology and is able to bind both CD123 on tumor cells and CD3 on T cells. JNJ-63709178 is able to recruit T cells to CD123-expressing tumor cells and induce the killing of these tumor cells in vitro (MOLM-13, OCI-AML5 and KG-1; EC50=0.51−0.91 nM). JNJ-63709178 is disclosed in WO 2016/036937, Gaudet, F. et al. (2016) “Development of a CD123×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 CD123×CD3 Duobody® Antibody JNJ-63709178 Against Primary Acute Myeloid Leukemia Bone Marrow Samples,” Blood 128:2875, which documents are herein incorporated 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.
[0181] B. XmAb14045
[0182] XmAb14045 (also known as vibecotamab) is a tumor-targeted antibody that contains both a CD123 binding domain and a cytotoxic T-cell binding domain (CD3). An XmAb Bispecific Fc domain serves as the scaffold for these two antigen binding domains and confers long circulating half-life, stability and ease of manufacture on XmAb14045. Engagement of CD3 by XmAb14045 activates T cells for highly potent and targeted killing of CD123-expressing tumor cells (US Patent Publication 2017/0349660; Chu, S. Y. et al. (2014) “Immunotherapy with Long-Lived Anti-CD123× CD3 Bispecific Antibodies Stimulates Potent T Cell-Mediated Killing of Human AML Cell Lines and of CD123+ Cells in Monkeys: A Potential Therapy for Acute Myelogenous Leukemia,” Blood 124(21):2316, which documents are herein incorporated by reference). The amino acid sequences of the heavy and light chains of XmAb14045 and similar CD123×CD3 bispecific binding molecules are disclosed in US Patent Publication 2017/0349660 and in WHO Drug Information, Proposed INN: List 120, 2018, 32(4):658-660.
[0183] C. APVO436
[0184] APVO436 is an ADAPTIR™ CD123×CD3 bispecific binding molecule that possesses an anti-CD123 scFv portion and an anti-CD3 scFv portion. Each of the scFv portions are bound to an Fc Domain that has been modified to abolish ADCC/CDC effector function. APVO436 is disclosed to bind human CD123 and CD3-expressing cells with EC.sub.50 values in the low nM range and to demonstrate potent target-specific activity against CD123-expressing tumor cell lines at low effector to target ratios. APVO436 is disclosed to be capable of potently inducing endogenous T-cell activation and proliferation accompanied by depletion of CD123 expressing cells in experiments with primary AML subject samples and normal donor samples. APVO436 (see, Comeau, M. R. et al. (2018) “APVO436, a Bispecific anti-CD123×anti-CD3 ADAPTIR™ Molecule for Redirected T-cell Cytotoxicity, Induces Potent T-cell Activation, Proliferation and Cytotoxicity with Limited Cytokine Release,” AACR Annual Meeting April 2018, Abstract 1786; Godwin, C. D. et al. (2017) “Bispecific Anti-CD123×Anti-CD3 ADAPTIR™ Molecules APVO436 andAPVO437Have Broad Activity Against Primary Human AML Cells In Vitro,” American Society of Hematology Annual Meeting, December 2017, Blood 130:2639; Comeau, M. R. et al. (2017) “Bispecific anti-CD123×anti-CD3 ADAPTIR™ 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 APVO436 CD123×CD3 bispecific binding molecules are disclosed in WO 2018/057802A1.
[0185] D. DART-A
[0186] DART-A (also known as flotetuzumab, CAS number: 1664355-28-5) is an exemplary CD123×CD3 bispecific binding molecule of the present invention. DART-A is a sequence-optimized bispecific diabody capable of simultaneously and specifically binding to an epitope of CD123 and to an epitope of CD3 (a “CD123×CD3” bispecific diabody) (US Patent Publn. No. US 2016-0200827, in PCT Publn. WO 2015/026892, in Al-Hussaini, M. et al. (2016) “Targeting CD123 In Acute Myeloid Leukemia Using A T-Cell-Directed Dual-Affinity Retargeting Platform,” Blood 127:122-131, in Vey, N. et al. (2017) “A Phase I, First-in-Human Study of MGDO06 S80880 (CD123×CD3) in AML MDS,” 2017 ASCO Annual Meeting, Jun. 2-6, 2017, Chicago, TL: Abstract TPS7070, each of which documents is herein incorporated by reference in its entirety). DART-A was found to exhibit enhanced functional activity relative to other non-sequence-optimized CD123×CD3 bispecific diabodies of similar composition, and is thus termed a “sequence-optimized” CD123×CD3 bispecific diabody. PCT Application PCT/US2017/050471 describes particular dosing regimens for administering DART-A to patients, and is herein incorporated by reference in its entirety.
[0187] DART-A comprises a first polypeptide chain and a second polypeptide chain (
[0188] An exemplary sequence for such a VL.sub.CD3 Domain is
TABLE-US-00003 SEQ ID NO:1: QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG
[0189] The Antigen Binding Domain of VL.sub.CD3 comprises:
TABLE-US-00004 CDR.sub.L1 (SEQ ID NO: 2): RSSTGAVTTSNYAN CDR.sub.L2 (SEQ ID NO: 3): GTNKRAP CDR.sub.L3 (SEQ ID NO: 4): ALWYSNLWV
[0190] An exemplary sequence for such Linker 1 is
TABLE-US-00005 SEQ ID NO: 5: GGGSGGGG.
An exemplary sequence for such a VH.sub.CD123 Domain is
TABLE-US-00006 SEQ ID NO: 6: EVQLVQSGAE LKKPGASVKV SCKASGYTFT DYYMKWVRQA PGQGLEWIGD IIPSNGATFY NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCARSH LLRASWFAYW GQGTLVTVSS
[0191] The Antigen Binding Domain of VH.sub.CD123 comprises:
TABLE-US-00007 CDR.sub.H1 (SEQ ID NO: 7): DYYMK CDR.sub.H2 (SEQ ID NO: 8): DIIPSNGATFYNQKFKG CDR.sub.H3 (SEQ ID NO: 9): SHLLRASWFAY
[0192] The second polypeptide chain will comprise, in the N-terminal to C-terminal direction, an N-terminus, a VL domain of a monoclonal antibody capable of binding to CD123 (VL.sub.CD123), an intervening linker peptide (e.g., Linker 1), a VH domain of a monoclonal antibody capable of binding to CD3 (VH.sub.CD3), and a C-terminus. An exemplary sequence for such a VL.sub.CD123 Domain is
TABLE-US-00008 SEQ ID NO: 10: DEVMTQSPDS LAVSLGERVT MSCKSSQSLL NSGNQKNYLT WYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIK
[0193] The Antigen Binding Domain of VL.sub.CD123 comprises:
TABLE-US-00009 CDR.sub.L1 (SEQ ID NO: 11): KSSQSLLNSGNQKNYLT CDR.sub.L2 (SEQ ID NO: 12): WASTRES CDR.sub.L3 (SEQ ID NO: 13): QNDYSYPYT
[0194] An exemplary sequence for such a VH.sub.CD3 Domain is
TABLE-US-00010 SEQ ID NO: 14: EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSS
[0195] The Antigen Binding Domain of VH.sub.CD3 comprises:
TABLE-US-00011 CDR.sub.H1 (SEQ ID NO: 15): TYAMN CDR.sub.H2 (SEQ ID NO: 16): RIRSKYNNYATYYADSVKD CDR.sub.H3 (SEQ ID NO: 17): HGNFGNSYVSWFAY
[0196] The sequence-optimized CD123×CD3 bispecific diabodies of the present invention are engineered so that such first and second polypeptides covalently bond to one another via cysteine residues along their length. Such cysteine residues may be introduced into the intervening linker (e.g., Linker 1) that separates the VL and VH domains of the polypeptides. Alternatively, a second peptide (Linker 2) is introduced into each polypeptide chain, for example, at a position N-terminal to the VL domain or C-terminal to the VH domain of such polypeptide chain. An exemplary sequence for such Linker 2 is SEQ ID NO:18: GGCGGG.
[0197] The formation of heterodimers can be driven by further engineering such polypeptide chains to contain polypeptide coils of opposing charge. Thus, in a particular embodiment, one of the polypeptide chains will be engineered to contain an “E-coil” domain (SEQ ID NO:19: EVAALEKEVAALEKEVAALEKEVAALEK) whose residues will form a negative charge at pH 7, while the other of the two polypeptide chains will be engineered to contain an “K-coil” domain (SEQ ID NO:20: KVAALKEKVAALKEKVAALKEKVAALKE) whose residues will form a positive charge at pH 7. The presence of such charged domains promotes association between the first and second polypeptides, and thus fosters heterodimerization.
[0198] It is immaterial which coil is provided to the first or second polypeptide chains. However, an exemplary sequence-optimized CD123×CD3 bispecific diabody of the present invention (“DART-A”) has a first polypeptide chain having the sequence
TABLE-US-00012 (SEQ ID NO: 21): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGEV QLVQSGAELK KPGASVKVSC KASGYTETDY YMKWVRQAPG QGLEWIGDII PSNGATFYNQ KFKGRVTITV DKSTSTAYME LSSLRSEDTA VYYCARSHLL RASWFAYWGQ GTLVTVSSGG CGGGEVAALE KEVAALEKEV AALEKEVAAL EK
[0199] DART-A Chain 1 is composed of: SEQ ID NO:1-SEQ ID NO:5-SEQ ID NO:6-SEQ ID NO:18-SEQ ID NO:19. A polynucleotide that encodes the first polypeptide chain of DART-A is
TABLE-US-00013 SEQ ID NO: 22: caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg acatgcagat ccagcacagg cgcagtgacc acatctaact acgccaattg ggtgcagcag aagccaggac aggcaccaag gggcctgatc gggggtacaa acaaaagggc tccctggacc cctgcacggt tttctggaag tctgctgggc ggaaaggccg ctctgactat taccggggca caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc gggggtggca caaaactgac tgtgctggga gggggtggat ccggcggcgg aggcgaggtg cagctggtgc agtccggggc tgagctgaag aaacccggag cttccgtgaa ggtgtcttgc aaagccagtg gctacacctt cacagactac tatatgaagt gggtcaggca ggctccagga cagggactgg aatggatcgg cgatatcatt ccttccaacg gggccacttt ctacaatcag aagtttaaag gcagggtgac tattaccgtg gacaaatcaa caagcactgc ttatatggag ctgagctccc tgcgctctga agatacagcc gtgtactatt gtgctcggtc acacctgctg agagccagct ggtttgctta ttggggacag ggcaccctgg tgacagtgtc ttccggagga tgtggcggtg gagaagtggc cgcactggag aaagaggttg ctgctttgga gaaggaggtc gctgcacttg aaaaggaggt cgcagccctg gagaaa
[0200] The second polypeptide chain of DART-A has the sequence
TABLE-US-00014 (SEQ ID NO: 23): DFVMTQSPDS LAVSLGERVT MSCKSSQSLL NSGNQKNYLT WYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIKGGGSGGG GEVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKDR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSSGGCG GGKVAALKEK VAALKEKVAA LKEKVAALKE
[0201] DART-A Chain 2 is composed of: SEQ ID NO:10-SEQ ID NO:5-SEQ ID NO:14-SEQ ID NO:18-SEQ ID NO:20. A polynucleotide that encodes the second polypeptide chain of DART-A is
TABLE-US-00015 SEQ ID NO: 24 gacttcgtga tgacacagtc tcctgatagt ctggccgtga gtctggggga gcgggtgact atgtcttgca agagctccca gtcactgctg aacagcggaa atcagaaaaa ctatctgacc tggtaccagc agaagccagg ccagccccct aaactgctga tctattgggc ttccaccagg gaatctggcg tgcccgacag attcagcggc agcggcagcg gcacagattt taccctgaca atttctagtc tgcaggccga ggacgtggct gtgtactatt gtcagaatga ttacagctat ccctacactt tcggccaggg gaccaagctg gaaattaaag gaggcggatc cggcggcgga ggcgaggtgc agctggtgga gtctggggga ggcttggtcc agcctggagg gtccctgaga ctctcctgtg cagcctctgg attcaccttc agcacatacg ctatgaattg ggtccgccag gctccaggga aggggctgga gtgggttgga aggatcaggt ccaagtacaa caattatgca acctactatg ccgactctgt gaaggataga ttcaccatct caagagatga ttcaaagaac tcactgtatc tgcaaatgaa cagcctgaaa accgaggaca cggccgtgta ttactgtgtg agacacggta acttcggcaa ttcttacgtg tcttggtttg cttattgggg acaggggaca ctggtgactg tgtcttccgg aggatgtggc ggtggaaaag tggccgcact gaaggagaaa gttgctgctt tgaaagagaa ggtcgccgca cttaaggaaa aggtcgcagc cctgaaagag
[0202] DART-A has the ability to simultaneously bind CD123 and CD3 as arrayed by human and cynomolgus monkey cells. Provision of DART-A was found to cause T cell activation, to mediate blast reduction, to drive T cell expansion, to induce T cell activation and to cause the redirected killing of target cancer cells (Table 3).
TABLE-US-00016 TABLE 3 Equilibrium Dissociation Constants (K.sub.D) for the Binding of DART-A to Human and Cynomolgus Monkey CD3 and CD123 k.sub.a (±SD) k.sub.d (±SD) K.sub.D (±SD) Antigens (M.sup.−1s.sup.−1) (s.sup.−1) (nM) Human CD3ε/δ 5.7 (±0.6) × 10.sup.5 5.0 (±0.9) × 10.sup.−3 9.0 ± 2.3 Cynomolgus CD3ε/δ 5.5 (±0.5) × 10.sup.5 5.0 (±0.9) × 10.sup.−3 9.2 ± 2.3 Human CD123-His 1.6 (±0.4) × 10.sup.6 1.9 (±0.4) × 10.sup.−4 0.13 ± 0.01 Cynomolgus CD123-His 1.5 (±0.3) × 10.sup.6 4.0 (±0.7) × 10.sup.−4 0.27 ± 0.02
[0203] More particularly, DART-A exhibits a potent redirected killing ability with concentrations required to achieve 50% of maximal activity (EC50s) in sub-ng/mL range, regardless of CD3 epitope binding specificity in target cell lines with high CD123 expression (Kasumi-3 (EC.sub.50=0.01 ng/mL)) medium CD123-expression (Molm13 (EC.sub.50=0.18 ng/mL) and THP-1 (EC.sub.50=0.24 ng/mL)) and medium low or low CD123 expression (TF-1 (EC.sub.50=0.46 ng/mL) and RS4-11 (EC.sub.50=0.5 ng/mL)). Similarly, DART-A-redirected killing was also observed with multiple target cell lines with T cells from different donors and no redirected killing activity was observed in cell lines that do not express CD123. Results are summarized in Table 4.
TABLE-US-00017 TABLE 4 EC.sub.50 of Sequence- CD123 Surface Optimized CD 123 × Expression CD3 Bispecific Target Cell (Antibody Diabodies (ng/mL) Line Binding Sites) E:T = 10:1 Max % Killing Kasumi-3 118620 0.01 94 Molm13 27311 0.18 43 THP-1 58316 0.24 40 TF-1 14163 0.46 46 RS4-11 957 0.5 60 A498 Negative No activity No activity HT29 Negative No activity No activity
[0204] Additionally, when human T cells and tumor cells (Molm13 or RS4-11) were combined and injected subcutaneously into NOD/SCID gamma (NSG) knockout mice, the MOLM13 tumors was significantly inhibited at the 0.16, 0.5, 0.2, 0.1, 0.02, and 0.004 mg/kg dose levels. A dose of 0.004 mg/kg and higher was active in the MOLM13 model. The lower DART-A doses associated with the inhibition of tumor growth in the MOLM13 model compared with the RS4-11 model are consistent with the in vitro data demonstrating that MOLM13 cells have a higher level of CD123 expression than RS4-11 cells, which correlated with increased sensitivity to DART-A-mediated cytotoxicity in vitro in MOLM13 cells.
[0205] DART-A is active against primary AML specimens (bone marrow mononucleocytes (BMNC) and peripheral blood mononucleocytes (PBMC)) from AML patients. Incubation of primary AML bone marrow samples with DART-A resulted in depletion of the leukemic cell population over time, accompanied by a concomitant expansion of the residual T cells (both CD4 and CD8) and the induction of T cell activation markers (CD25 and Ki-67). Upregulation of granzyme B and perforin levels in both CD8 and CD4 T cells was observed. Incubation of primary AML bone marrow samples with DART-A resulted in depletion of the leukemic cell population over time compared to untreated control or Control DART. When the T cells were counted (CD8 and CD4 staining) and activation (CD25 staining) were assayed, the T cells expanded and were activated in the DART-A sample compared to untreated or Control DART samples. DART-A was also found to be capable of mediating the depletion of pDCs cells in both human and cynomolgus monkey PBMCs, with cynomolgus monkey pDCs being depleted as early as 4 days post infusion with as little as 10 ng/kg DART-A. No elevation in the levels of cytokines interferon gamma, TNF alpha, IL6, IL5, IL4 and IL2 were observed in DART-A-treated animals. These data indicate that DART-A-mediated target cell killing was mediated through a granzyme B and perforin pathway.
[0206] No activity was observed against CD123-negative targets (U937 cells) or with Control DART, indicating that the observed T cell activation was strictly dependent upon target cell engagement and that monovalent engagement of CD3 by DART-A was insufficient to trigger T cell activation.
[0207] In sum, DART-A is an antibody-based molecule engaging the CD3F subunit of the TCR to redirect T lymphocytes against cells expressing CD123, an antigen up-regulated in several hematologic malignancies. DART-A binds to both human and cynomolgus monkey's antigens with similar affinities and redirects T cells from both species to kill CD123+ cells. Monkeys infused 4 or 7 days a week with weekly escalating doses of DART-A showed depletion of circulating CD123+ cells 72 h after treatment initiation that persisted throughout the 4 weeks of treatment, irrespective of dosing schedules. A decrease in circulating T cells also occurred, but recovered to baseline before the subsequent infusion in monkeys on the 4-day dose schedule, consistent with DART-A-mediated mobilization. DART-A administration increased circulating PD1+, but not TIM-3+, T cells; furthermore, ex vivo analysis of T cells from treated monkeys exhibited unaltered redirected target cell lysis, indicating no exhaustion. Toxicity was limited to a minimal transient release of cytokines following the DART-A first infusion, but not after subsequent administrations even when the dose was escalated, and a minimal reversible decrease in red cell mass with concomitant reduction in CD123+bone marrow progenitors.
[0208] E. Additional Bispecific Diabody Molecules
[0209] An alternative version of DART-A comprising an Fe Region and having the general structure shown in
TABLE-US-00018 (SEQ ID NO: 25) (“Knob-Bearing” Fc Domain) APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGX
wherein X is K or is absent and the sequence
TABLE-US-00019 (SEQ ID NO: 26) (“Hole-Bearing” Fc Domain) APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPGX
wherein X is K or is absent
[0210] The first polypeptide of an exemplary DART-A w/Fc construct comprises, in the N-terminal to C-terminal direction, an N-terminus, a VL domain of a monoclonal antibody capable of binding to CD123 (VL.sub.CD123), an intervening linker peptide (Linker 1), a VH domain of a monoclonal antibody capable of binding to CD3 (VH.sub.CD3), a Linker 2, an E-coil Domain, a Linker 5, Peptide 1, a polypeptide that contains the CH2 and CH3 Domains of an Fc Domain and a C-terminus. An exemplary Linker 5 has the sequence: GGG. An exemplary Peptide 1 has the sequence: DKTHTCPPCP (SEQ ID NO:29). Thus, the first polypeptide of such a DART-A w/Fc version 1 construct is composed of: 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).
[0211] An exemplary sequence of the first polypeptide of such a DART-A w/Fc version 1 construct has the sequence
TABLE-US-00020 (SEQ ID NO: 27): DEVMTQSPDS LAVSLGERVT MSCKSSQSLL NSGNQKNYLT WYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIKGGGSGGG GEVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKDR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSSGGCG GGEVAALEKE VAALEKEVAA LEKEVAALEK GGGDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLWC LVKGEYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK
[0212] The second chain of such a DART-A w/Fc version 1 construct will comprise, in the N-terminal to C-terminal direction, an N-terminus, a VL domain of a monoclonal antibody capable of binding to CD3 (VL.sub.CD3), an intervening linker peptide (Linker 1), a VH domain of a monoclonal antibody capable of binding to CD123 (VH.sub.CD123), a Linker 2, a K-coil Domain, and a C-terminus. Thus, the second polypeptide of such a DART-A w/Fc version 1 construct is composed of: SEQ ID NO:1-SEQ ID NO:5-SEQ ID NO:6-SEQ ID NO:18-SEQ ID NO:20. Such a polypeptide has the sequence
TABLE-US-00021 (SEQ ID NO: 28): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGEV QLVQSGAELK KPGASVKVSC KASGYTETDY YMKWVRQAPG QGLEWIGDII PSNGATFYNQ KFKGRVTITV DKSTSTAYME LSSLRSEDTA VYYCARSHLL RASWFAYWGQ GTLVTVSSGG CGGGKVAALK EKVAALKEKV AALKEKVAAL KE
[0213] The third polypeptide chain of such a DART-A w/Fc version 1 will comprise the CH2 and CH3 Domains of an IgG Fc Domain. An exemplary polypeptide that is composed of Peptide 1 (DKTHTCPPCP; SEQ ID NO:29) and the CH2 and CH3 Domains of an Fc Domain (SEQ ID NO:26, wherein X is K) and has the sequence of
TABLE-US-00022 SEQ ID NO: 30: DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVK GEYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPGK
[0214] Additional CD123×CD3 bispecific diabodies comprising alternative optimized anti-CD3 binding domains are provided in WO 2019/160904. In particular, such diabodies comprise the VH.sub.CD123 Domain of SEQ ID NO:6 and the VL.sub.CD123 Domain is SEQ ID NO:10 and further comprise an Fe Region.
[0215] III. Pharmaceutical Formulations
[0216] The compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient) which can be used in the preparation of unit dosage forms. Such compositions comprise a prophylactically or therapeutically effective amount of a CD123×CD3 bispecific binding molecule and a pharmaceutically acceptable carrier.
[0217] Exemplary pharmaceutical formulations comprise a CD123×CD3 bispecific binding molecule and an aqueous stabilizer and, optionally, a pharmaceutically acceptable carrier.
[0218] As used herein, the term “pharmaceutically acceptable carrier” is intended to refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle that is approved by a regulatory agency or listed in the U.S. Pharmacopeia or in another generally recognized pharmacopeia as being suitable for delivery into animals, and more particularly, 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 and the like. Water may be used as carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
[0219] Generally, the ingredients of compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a liquid formulation, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as a vial, an ampoule or sachette indicating the quantity of active agent. Where the composition is to be 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 for injection or saline can be provided so that the ingredients may be mixed prior to administration.
[0220] The invention also provides a pharmaceutical pack or kit comprising one or more containers containing a CD123×CD3 bispecific binding molecule alone or with a stabilizer and/or a pharmaceutically acceptable carrier. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease can also be included in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
[0221] IV. Kits
[0222] The present invention provides kits that comprise a CD123×CD3 bispecific binding molecule, instructional material (for example, relating to storage, dosage, indications, side effects, counter-indications, etc.), and optionally a stabilizer and/or carrier that can be used in the above methods. In such kits, the CD123×CD3 bispecific binding molecule may be packaged in a hermetically sealed container such as an ampoule, a vial, a sachette, etc. that may indicate the quantity of the molecule contained therein. The container may be formed of any pharmaceutically acceptable material, such as glass, resin, plastic, etc. The CD123×CD3 bispecific binding molecule of such kit may be supplied as a liquid solution, a dry sterilized lyophilized powder or a water-free concentrate in a hermetically sealed container that can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject. Such liquid or lyophilized material should be stored at between 2 and 8° C. in its original container and the material should be administered within about 24 hours, with about 12 hours, within about 6 hours, within about 5 hours, within about 3 hours, or within about 1 hour after being reconstituted. The kit can further comprise one or more other prophylactic and/or therapeutic agents useful for the treatment of cancer, in one or more containers; and/or the kit can further comprise one or more cytotoxic antibodies that bind one or more cancer antigens associated with cancer. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic. The kit can further comprise instructions for use, or other printed information.
[0223] Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease can also be included in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
[0224] V. Methods of Administration
[0225] The CD123×CD3 bispecific binding molecule pharmaceutical formulations of the present invention may be provided for the treatment, prophylaxis, and amelioration of one or more symptoms associated with a disease, disorder or infection by administering to a subject an effective amount of a molecule of the invention, or a pharmaceutical composition comprising a fusion protein or a conjugated molecule of the invention. In a one aspect, such compositions are substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side effects). In a specific embodiment, the subject is an animal, preferably a mammal such as non-primate (e.g., bovine, equine, feline, canine, rodent, etc.) or a primate (e.g., monkey such as, a cynomolgus monkey, human, etc.). In a particular embodiment, the subject or patient is a human.
[0226] Methods of administering a CD123×CD3 bispecific binding molecule pharmaceutical formulation 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×CD3 bispecific binding molecules are administered intravenously. The compositions may be administered by any convenient route, for example, by infusion, and may be administered together with other biologically active agents.
[0227] Administration by infusion is maybe accomplished using an infusion pump. “Infusion pumps” are medical device that deliver fluids into a patient's body in a controlled manner, especially at a defined rate and for a prolonged period of time. Infusion pumps may be powered mechanically, but are more typically electrically powered. Some infusion pumps are “stationary” infusion pumps, and are designed to be used at a patient's bedside. Others, called “ambulatory” infusion pumps, are designed to be portable or wearable. A “syringe” pump is an infusion pump in which the fluid to be delivered is held in the reservoir of a chamber (e.g., a syringe), and a moveable piston is used to control the chamber's volume and thus the delivery of the fluid. In an “elastomeric” infusion pump, fluid is held in a stretchable balloon reservoir, and pressure from the elastic walls of the balloon drives fluid delivery. In a “peristaltic” infusion pump, a set of rollers pinches down on a length of flexible tubing, pushing fluid forward. In a “multi-channel” infusion pump, fluids can be delivered from multiple reservoirs at multiple rates. A “smart pump” is an infusion pump that is equipped a computer-controlled fluid delivery system so as to be capable of alerting in response to a risk of an adverse drug interaction, or when the pump's parameters have been set beyond specified limits. Examples of infusion pumps are well-known, and are provided in, for example, [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.
[0228] In certain embodiments, the CD123×CD3 bispecific binding molecule pharmaceutical formulations of the invention be administered by infusion facilitated by one or more ambulatory pumps, so that the patient will be ambulatory during the therapeutic regimen. In certain embodiments, the CD123×CD3 bispecific binding molecule pharmaceutical formulations of the invention be administered by continuous infusion. In a specific embodiment, a 7-day continuous infusion regimen comprises a treatment dosage of about 30 ng/kg patient weight/day for 3 days followed by a treatment dosage of about 100 ng/kg/day for 4 days (for example, a treatment dosage of 30 ng/kg patient weight/day for 3 days followed by a treatment dosage of 100 ng/kg/day for 4 days; etc.). In another specific embodiment, a 7-day continuous infusion regimen comprises a treatment dosage of about 30 ng/kg patient weight/day for 1 day, followed by a treatment dosage of about 60 ng/kg patient weight/day for 1 day, followed by a treatment dosage of about 100 ng/kg/day for 1 day, followed by a treatment dosage of about 200 ng/kg/day for 1 day, followed by a treatment dosage of about 300 ng/kg/day for 1 day, followed by a treatment dosage of about 400 ng/kg/day for 1 day, followed by a treatment dosage of about 500 ng/kg/day for 1 day. In certain embodiments, such 7-day continuous infusion regiment is followed by a 21-day continuous infusion regiment in which a treatment dosage of 500 ng/kg/day is administered every day for 21 days. In certain embodiments, such 21-day continuous infusion regiment is followed by a 21-day continuous infusion regiment in which a treatment dosage of about 500 ng/kg/day is administered during days 1-4 of each week of such 21-day regiment and during days 5-7 of each week no treatment dosage is administered.
[0229] In any of the above-described courses of treatment, the proportion of CD8+T-lymphocytes in the tumor microenvironment may additionally be monitored. Such monitoring may occur prior to the administration of the CD123×CD3 bispecific binding molecule, during the course of CD123×CD3 binding molecule therapy, and/or after the conclusion of a cycle of CD123×CD3 binding molecule therapy.
[0230] VI. Uses of the Compositions of the Invention
[0231] The CD123×CD3 bispecific binding molecules of the invention may be used to treat any disease or condition associated with or characterized by the expression of CD123. In particular, the CD123×CD3 bispecific binding molecules of the invention may be used to treat hematologic malignancies. The CD123×CD3 bispecific binding molecules of the invention are particularly suitable for use in the treatment of hematologic malignancies, including chemo-refractory hematologic malignancies. As used herein, a chemo-refractory hematologic malignancy is a hematologic 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).
[0232] Thus, without limitation, such molecules may be employed in the diagnosis or treatment of acute myeloid leukemia (AML) (including primary chemo-refractory AML), chronic myelogenous leukemia (CML), including blastic crisis of CML and Abelson oncogene-associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B lymphoblastic leukemia (B-ALL), acute T lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), including Richter's syndrome or Richter's transformation of call, 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. The CD123×CD3 bispecific binding molecules of the invention may additionally be used in the manufacture of medicaments for the treatment of the above-described conditions.
[0233] The CD123×CD3 bispecific binding molecules of the invention are particularly suitable for use in the treatment of acute myeloid leukemia (AML, including primary chemo-refractory acute myeloid leukemia), hematologic myelodysplastic syndrome (MDS), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL), or acute T lymphoblastic leukemia (T-ALL).
EXAMPLES
[0234] Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention unless specified.
Example 1
Gene Expression Signatures of Patient Populations Particularly Suitable for Treatment with a CD123×CD3 Bispecific Binding Molecule of the Invention
[0235] In order to demonstrate a correlation between the expression patterns of the genes of patients having a hematologic malignancy, particularly AML, and the favorable outcome of CD123×CD3 bispecific binding molecule therapy, RNA was isolated from bone marrow (“BM”) samples obtained from patients with individual patient consent from patients with relapsed or refractory AML enrolled in a phase ½ clinical trial of flotetuzumab (NCT #02152956, an exemplary CD123×CD3 bispecific binding molecule).
[0236] The expression of 770 immune-related genes, including predefined immune gene signature scores examined in baseline bone marrow samples of a subgroup of patients treated with the exemplary CD123×CD3 bispecific molecule flotetuzumab. Briefly, The NanoString PanCancer IO360™ assay was used to interrogate the expression of 770 genes, including the abundance of 14 immune cell types and 32 immuno-oncology signatures in bone marrow samples of a subgroup of patients (n=38) with relapsed/refractory AML treated with flotetuzumab at the recommended phase 2 dose (RP2D; 38 bone marrow samples collected at baseline and 34 bone marrow samples collected on treatment (post-cycles 1 [n=25] and 2 [n=9]). IO 360 gene counts were generated using the nCounter® system (NanoString Technologies, Inc.) essentially as follows: RNA (˜100 ng per sample) was extracted from unfractionated bone marrow aspirates, and was incubated with reporter and capture probe mix for hybridization. Transcript counts were analyzed on the nCounter FLEX analysis system using the high-resolution setting. Reporter code count (RCC) output files are used to calculate gene signature scores. Signature scores for predefined signatures described by NanoString were calculated as pre-defined linear combinations (weighted averages) of biologically relevant gene sets essentially as described in WO 2020/092404 and Vadakekolathu J, et al. (2020) “Immune Landscapes Predict Chemotherapy Resistance And Immunotherapy Response In Acute Myeloid Leukemia,” Sci. Transl. Med. 12(546).eaaz0463. In addition, a ranked gene list (χ.sup.2 values) was generated using Orange3 software package (Version 3.25.0). Unsupervised hierarchical clustering (Euclidean distance, complete linkage).
[0237] As previously reported patients with primary induction failure (PIF)/early relapse (ER) showed higher immune infiltration relative to those exhibiting late relapse (LR). PD-L1 and inflammatory chemokine scores were higher in PIF/ER and HMA-treated patients compared with LR. The tumor inflammation signature score (TIS) correlated with the antigen processing machinery and inflammatory chemokine scores (P<0.0001), suggesting the occurrence of antigen presentation and T cell chemoattraction in highly T cell-inflamed samples (Vadakekolathu J, et al. (2020) “Immune Landscapes Predict Chemotherapy Resistance And Immunotherapy Response In Acute Myeloid Leukemia.” Sci. Transl. Med. 12(546):eaaz0463).
[0238] Further analysis was performed by ranking the 770 immune-related genes in the gene expression panel. This ranking identified a parsimonious expression signature encompassing the top 10 genes associated with complete response to the exemplary CD123×CD3 bispecific molecule flotetuzumab, defined as either complete remission (CR), complete remission with incomplete hematopoietic recovery (CRi) or complete remission with partial hematopoietic recovery (CRh). The 10 identified genes: CD8B, CRABP2, FCGR3A/B, FBP1, FPR1, ICOS, NOTCH2, PDGFA, SERPINH1, and THBS1 are listed in Table 1 above. Table 1 also provides a representative, non-limiting NCBI Accession Number for each gene. A heatmap showing the expression of these 10 genes associated with complete response to the exemplary CD123×CD3 bispecific molecule flotetuzumab is provided in
[0239] Analysis of functional protein association networks was performed using STRING (string-db.org). These data indicated that the 10 genes associated with complete response were enriched in ontologies and pathways related to antigen binding and processing, VEGF-activated receptor activity, Notch signaling, micro-RNA regulation in cancer and T helper type 1 (Th1) and Th2 differentiation (Table 5). These data support the potential role of enhanced and sustained antigen presentation in the TME in promoting anti-leukemia responses from CD123×CD3 bispecific binding molecules such as flotetuzumab.
TABLE-US-00023 TABLE 5 Gene Ontologies (GO) and KEGG Pathways Captured by the Top 10 Genes Associated with Complete Response to Flotetuzumab Count in GO term Description Gene Set FDR GO: 0005102 Signaling receptor binding 25 of 1513 1.63 × 10.sup.−12 GO: 0042605 Peptide antigen binding 7 of 22 1.00 × 10.sup.−10 GO: 0003823 Antigen binding 8 of 56 3.98 × 10.sup.−10 GO: 0019838 Growth factor binding 9 of 126 4.05 × 10.sup.−9 GO: 0005518 Collagen binding 7 of 61 2.31 × 10.sup.−8 GO: 0005161 Platelet-derived growth factor 5 of 15 6.26 × 10.sup.−8 receptor binding GO: 0005515 Protein binding 38 of 1.25 × 10.sup.−7 326605 GO: 0005021 Vascular endothelial growth 4 of 7 4.24 × 10.sup.−7 factor-activated receptor activity GO: 0046977 TAP binding 4 of 7 4.24 × 10.sup.−7 GO: 0044877 Protein-containing complex 15 of 968 4.26 × 10.sup.−7 binding GO: 0048407 Platelet-derived growth factor 4 of 11 1.26 × 10.sup.−6 binding hsa05165 Human papillomavirus infection 20 of 317 1.56 × 10.sup.−20 hsa04330 Notch signaling pathway 10 of 48 1.49 × 10.sup.−14 hsa05166 HTLV-I infection 14 of 250 1.43 × 10.sup.−13 hsa05206 MicroRNAs in cancer 12 of 149 2.12 × 10.sup.−13 hsa04658 Th1 and Th2 cell differentiation 10 of 888 1.49 × 10.sup.−12 hsa04612 Antigen processing and 9 of 66 5.40 × 10.sup.−12 presentation hsa04145 Phagosome 10 of 145 1.12 × 10.sup.−10 hsa05200 Pathways in cancer 151 of 515 6.68 × 10.sup.−10 hsa04015 Rapl signaling pathway 10 of 203 1.82 × 10.sup.−9 hsa04514 Cell adhesion molecules 9 of 139 1.82 × 10.sup.−9 (CAMs) hsa05330 Allograft rejection 6 of 35 9.90 × 10.sup.−9 FDR = false discovery rate
[0240] AUROC curves measuring the predictive ability of European Leukemia-Net (ELN) risk disease status (Dohner H, et al., 2017, “Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel.” Blood 129(4): 424-47), at study entry and the 10-gene signature score for anti-leukemic activity of the exemplary CD123×CD3 bispecific binding molecule flotetuzumab, either individually or in combination, are shown in
TABLE-US-00024 TABLE 6 AUROC Curve Analysis 95% CI Lower Upper Variable Area S.E. P value bound bound ELN risk 0.672 0.10 0.122 0.476 0.869 10-gene signature score 0.854 0.067 0.001 0.724 0.985 ELN risk + 10-gene 0.904 0.055 0.000 0.797 1.000 signature score SE = standard error; CI = confidence interval. AUROC = 1.0 would denote perfect prediction and AUROC = 0.5 would denote no predictive ability. AUROC curves were estimated using the SPSS software package, as previously published (Vadakekolathu J. et al. (2020) “Immune Landscapes Predict Chemotherapy Resistance And Immunotherapy Response In Acute Myeloid Leukemia,” Sci. Transl. Med. 12(546):eaaz0463; Wagner S, et al. (2019) “A Parsimonious 3-Gene Signature Predicts Clinical Outcomes In An Acute Myeloid Leukemia Multicohort Study,” Blood Adv. 3(8): 1330-1346)
[0241] All publications and patents mentioned in this specification are herein incorporated 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. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.