Methods for treating AML by identifying and targeting myeloid / lymphocytic leukemia stem cells

Abstract

The present disclosure provides methods of treating acute myeloid leukemia (AML) and determining responsiveness to an AML treatment regimen, which methods involve identifying the presence or absence of myeloid / lymphocytic leukemia stem cells (M / L LSCs) in a sample from a subject.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 386,505, filed December 8, 2022, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Acute myeloid leukemia (AML) is a blood cancer in which a subject's bone marrow produces abnormal myeloblasts, red blood cells, or platelets. AML is one of the most common acute leukemias in adults. The accumulation of AML cells in the bone marrow and blood can rapidly lead to infection, anemia, excessive bleeding, and death. Venetoclax, a BCL-2 inhibitor, has recently emerged as an important component of acute myeloid leukemia (AML) treatment. Venetoclax, in combination with multiple primary chemotherapy regimens, can induce responses in approximately 60-70% of previously untreated elderly AML patients, the majority of whom are ineligible for conventional induction therapy. However, resistance to venetoclax-based therapy and relapse after initial response have been reported. There is a need in the art for methods to predict response to venetoclax treatment and for methods to treat AML in patients who are refractory to or predicted to relapse after venetoclax treatment. There is also a need in the art for methods of treating AML, particularly in elderly patients and patients with relapsed AML who are unsuitable for conventional induction therapy. Summary of the Invention

[0003] The present disclosure provides a method of treating acute myeloid leukemia (AML) in a subject, the method including identifying myeloid / lymphocytic leukemia stem cells (M / L LSCs) in a sample from the subject, wherein the cells are identified as M / L LSCs if they are at least CD34+ and CD7+.

[0004] The present disclosure provides methods for identifying whether a subject with AML will respond to treatment with a combination comprising at least one BCL-2 inhibitor and at least one hypomethylating agent, the method including identifying myeloid / lymphoid LSCs (M / L LSCs) in a sample from the subject, wherein the cells are identified as M / L LSCs if they are at least CD34+ and CD7+.

[0005] The present disclosure provides a method of treating acute myeloid leukemia (AML) in a subject, the method comprising: a) measuring expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one myeloid / lymphocytic leukemia stem cell (M / L LSC) based on the expression measured in step (a), wherein the cell is identified as an M / L LSC if the cell is at least CD34+ and CD7+; and c) if at least one M / L LSC is identified, administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one M / L LSC targeting agent, or if no M / L LSC is identified, administering to the subject a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

[0006] The present disclosure provides a method for identifying whether a subject with AML will respond to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent, the method comprising: a) measuring expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one myeloid / lymphocytic leukemia stem cell (M / L LSC) based on the expression measured in step (a), wherein the cell is identified as an M / L LSC if it is at least CD34+ and CD7+; and c) identifying the subject as not responding to the treatment if the presence of at least one M / L LSC is identified, or identifying the subject as responding to the treatment if no M / L LSC is identified.

[0007] The present disclosure provides a method of treating AML in a subject, the method comprising: a) measuring expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying a number and / or proportion of M / L LSCs in the plurality of cells based on the expression measured in step (a), wherein the cell is identified as an M / L LSC if the cell is at least CD34+ and CD7+; c) comparing the number and / or proportion of m M / L LSCs identified in step (b) with a predetermined cutoff value; and d) administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one M / L LSC targeting agent if the number and / or proportion of M / L LSCs is equal to or greater than the predetermined cutoff value, or administering to the subject a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent if the number and / or proportion of M / L LSCs is less than the predetermined cutoff value.

[0008] The present invention provides a method for identifying whether a subject with AML will respond to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent, the method comprising the steps of: a) measuring the expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the number and / or proportion of M / L LSCs in the plurality of cells based on the expression measured in step (a), wherein the cell is identified as an M / L LSC if it is at least CD34+ and CD7+; c) comparing the number and / or proportion of m M / L LSCs identified in step (b) with a predetermined cutoff value; and d) identifying the subject as not responding to the treatment if the number and / or proportion of M / L LSCs is equal to or greater than the predetermined cutoff value, or identifying the subject as responding to the treatment if the number and / or proportion of M / L LSCs is less than the predetermined cutoff value.

[0009] In some embodiments, the at least one M / L LSC targeting agent comprises administering at least one of: i) an anti-CD7 antibody; ii) an anti-CD7 immunotherapy, preferably wherein the immunotherapy comprises CAR-T cells and / or NK cells that specifically target CD7; iii) an agent that inhibits CD7 signaling, preferably wherein the agent that inhibits CD7 signaling is an inhibitor of PI3 kinase and related pathways; iv) an agent that inhibits CD7 activation, preferably wherein the agent that inhibits CD7 activation inhibits CD7 activation by K12 and / or any additional CD7 ligand; and v) an agent that targets an M / L LSC-specific pathway and / or surface marker.

[0010] In some embodiments, the at least one hypomethylating agent is selected from azacitidine and decitabine.

[0011] In some embodiments, the at least one BCL-2 inhibitor is selected from venetoclax and navitoclax.

[0012] In some aspects, step (a) comprises performing PCR, high-throughput sequencing, next-generation sequencing, Northern blot, reverse transcription PCR (RT-PCR), real-time PCR (qPCR), quantitative PCR, qRT-PCR, flow cytometry, mass spectrometry, microarray analysis, digital droplet PCR, Western blot, Cellular Indexing of Transcriptome and Epitopes by Sequencing (CITE-SEQ), any single-cell omics technology, or any combination thereof.

[0013] In some embodiments, the subject is i) a subject with AML who has not received any treatment for AML, or ii) a subject with AML who has previously received at least one treatment for AML, preferably wherein the at least one treatment for AML comprises a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

[0014] In some embodiments, identifying the subject as responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent comprises identifying the subject as having a long-term remission after receiving treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

[0015] In some embodiments, identifying the subject as non-responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent comprises identifying the subject as refractory to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent and / or identifying the subject as relapsing after treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

[0016] In some embodiments, the biological sample comprises blood, a bone marrow biopsy, a bone marrow aspirate, a Pseudomonas aeruginosa biopsy, a tissue biopsy, cerebrospinal fluid, or any combination thereof.

[0017] Any of the above aspects or any of the aspects described herein may be combined with any other aspect.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the singular also includes the plural unless the context clearly dictates otherwise. By way of example, the terms "a," "an," and "the" are understood to be singular or plural, and the term "or" is understood to be inclusive. By way of example, "an element" means one or more elements. Throughout this specification, "comprising" or variations thereof (e.g., "comprises" or "comprising") will be understood to mean the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about." As used herein, the term "or" is inclusive and understood to include both "or" and "and" unless specifically stated or obvious from the context.

[0019] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. References cited herein are not considered prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the following detailed description and claims.

[0020] These and other features will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0021] [Figure 1A] 1 shows the survival probability after treatment with a combination of venetoclax and azacitidine for AML patient groups exhibiting one of the following: CD7- / CD34-, CD7+ / CD34-, CD7- / CD34+, and CD7+ / CD34+ cells. [Figure 1B] 1 shows the survival probability after treatment with a combination of venetoclax and azacitidine for a group of AML patients who did not undergo stem cell transplantation and who exhibit one of the following: CD7- / CD34-, CD7+ / CD34-, CD7- / CD34+, or CD7+ / CD34+ cells. [Figure 1C] Figure 1 shows the survival probability after treatment with a combination of venetoclax and azacitidine in a group of AML patients who underwent stem cell transplantation and were censored for those displaying one of the following: CD7- / CD34-, CD7+ / CD34-, CD7- / CD34+, and CD7+ / CD34+ cells. DETAILED DESCRIPTION OF THE INVENTION

[0022] Acute myeloid leukemia (AML) is a blood cancer that is the most commonly diagnosed type of leukemia in adults. It is estimated that approximately 11,000 people will die from AML in the United States in 2020, and 20,000 new cases will be diagnosed. The average age of a person diagnosed with AML is approximately 68 years, with the disease occurring most frequently after age 45. However, younger patients, including children, are also diagnosed with AML. The prognosis for patients diagnosed with AML is generally poor, with long-term survival rates of only 40–50% for younger patients and a median overall survival of less than one year for older patients. New therapies aimed at complementing standard induction therapy with infusional cytarabine with intermittent anthracycline have provided some improvement in treatment outcomes, but these improvements remain limited. Therefore, more specialized and personalized treatments are needed, especially for older patients who are not suitable candidates for induction therapy.

[0023] Recent studies have demonstrated that acute myeloid leukemia (AML) exhibits a high level of biological diversity, which may explain the difficulty in identifying effective therapeutic strategies for AML treatment. Furthermore, it has recently been recognized that leukemic stem cells (LSCs), which can give rise to identical daughter cells as well as differentiated cells, perpetuate and maintain AML.

[0024] As an alternative to standard induction therapy, the current FDA-approved standard of care for elderly patients and those unsuitable for such aggressive chemotherapy is the combination of the BCL-2 inhibitor venetoclax with a hypomethylating agent (HMA) such as azacitidine or decitabine. Specifically, venetoclax and azacitidine (hereafter referred to as "Ven / aza therapy" or "Ven / aza-based therapy") are estimated to induce complete remission (CR) of AML in approximately 60-70% of treated patients.

[0025] However, this means that approximately 30-40% of patients ultimately do not respond to Ven / aza treatment and therefore do not achieve complete remission. There is a need in the art for methods to identify this 30-40% of patients who are likely to not respond to Ven / aza treatment. The ability to identify these patients prior to treatment would allow physicians to avoid the toxicity, expense, and reduced quality of life associated with ineffective treatment. Furthermore, these patients could receive alternative therapies, improving their chances of survival. Finally, a reliable method for identifying these patients would enable the design of clinical trials to test personalized therapies for this specific AML patient population.

[0026] Multiple studies have delineated the characteristics of malignant stem cells responsible for the pathogenesis of myeloid leukemia. Analysis of primary human tissue specimens as well as various mouse models consistently demonstrates that leukemic stem cells (LSCs) are biologically distinct from the bulk tumor population and often demonstrate distinct drug sensitivity / resistance profiles from the majority of leukemic cell types. Similar to normal hematopoietic stem cells, conventional LSCs are also thought to be largely quiescent and capable of giving rise to progeny cells that comprise the entire tumor population. As such, LSCs represent an important target for the development of novel therapies. Numerous attempts have been made to target LSC populations, focusing on specific cell surface antigens, metabolic intervention, epigenetic strategies, mutation-targeting approaches, and immunotherapy. While multiple strategies are based on solid experimental evidence, the improvement of clinical outcomes through direct ablation of LSCs remains limited.

[0027] A major challenge in targeting LSCs is the inherent heterogeneity of malignant stem cells. In particular, LSC populations from human AML patients demonstrate significant intra- and interpatient heterogeneity in developmental stage and immunophenotype, which is thought to be caused, at least in part, by basal genetic diversity. Importantly, recent studies have demonstrated that the existence of heterogeneous basal LSC populations can mediate the differential therapeutic outcomes of conventional chemotherapy and venetoclax-based therapy.

[0028] The present disclosure is based, inter alia, on the discovery of a specific subpopulation of leukemic stem cells called myeloid / lymphoid LSCs (M / L LSCs) that can be used to predict a subject's response to treatment with a combination of a BCL-2 inhibitor (e.g., venetoclax) and a hypomethylating agent (e.g., azacitidine and decitabine).

[0029] Thus, the present disclosure provides, inter alia, methods for determining whether a subject will respond to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent based on whether M / L LSCs are identified in a sample from a subject with AML, as well as methods for treating AML in a subject, comprising administering a particular treatment to the subject based on whether a sample from the subject contains M / L LSCs.

[0030] Methods for predicting response to treatment The present invention provides a method for identifying whether a subject with AML will respond to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent, the method comprising the steps of: a) measuring the expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one myeloid / lymphocytic leukemia stem cell (M / L LSC) based on the expression measured in step (a), wherein the cell is identified as an M / L LSC if it is at least CD34+ and CD7+; and c) identifying the subject as not responding to the treatment if the presence of at least one M / L LSC is identified, or identifying the subject as responding to the treatment if no M / L LSC is identified.

[0031] The present invention provides a method for identifying whether a subject with AML will respond to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent, the method comprising the steps of: a) measuring the expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the number and / or proportion of M / L LSCs in the plurality of cells based on the expression measured in step (a), wherein the cell is identified as an M / L LSC if it is at least CD34+ and CD7+; c) comparing the number and / or proportion of m M / L LSCs identified in step (b) with a predetermined cutoff value; and d) identifying the subject as not responding to the treatment if the number and / or proportion of M / L LSCs is equal to or greater than the predetermined cutoff value, or identifying the subject as responding to the treatment if the number and / or proportion of M / L LSCs is less than the predetermined cutoff value.

[0032] The above methods can further include providing a treatment recommendation to the clinician and / or the subject. Thus, if the subject is identified as a subject who will not respond to the combination of at least one BCL-2 inhibitor and at least one hypomethylating agent, the method can further include providing a treatment recommendation that includes administering an alternative treatment. In some embodiments, the alternative treatment can include administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one M / L LSC targeting agent. If the subject is identified as a subject who will respond to treatment with the combination of at least one BCL-2 inhibitor and at least one hypomethylating agent, the method can further include providing a treatment recommendation that includes administering to the subject the combination.

[0033] In some embodiments of the aforementioned methods, identifying the subject as responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent includes identifying the subject as having a long-term remission after receiving treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

[0034] In some embodiments of the foregoing methods, identifying the subject as non-responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent comprises identifying the subject as refractory to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent and / or identifying the subject as relapsing after treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

[0035] How to Treat AML The present disclosure provides a method of treating acute myeloid leukemia (AML) in a subject, the method comprising: a) measuring expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying the presence of at least one myeloid / lymphocytic leukemia stem cell (M / L LSC) based on the expression measured in step (a), wherein the cell is identified as an M / L LSC if it is at least CD34+ and CD7+; and c) administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one M / L LSC targeting agent if at least one M / L LSC is identified, or administering to the subject a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent if no M / L LSC is identified.

[0036] The present disclosure provides a method of treating acute myeloid leukemia (AML) in a subject, the method comprising: a) measuring expression of at least CD34 and CD7 in a plurality of cells in a sample from the subject; b) identifying a number and / or proportion of M / L LSCs in the plurality of cells based on the expression measured in step (a), wherein the cell is identified as an M / L LSC if it is at least CD34+ and CD7+; c) comparing the number and / or proportion of m M / L LSCs identified in step (b) with a predetermined cutoff value; and d) administering to the subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one M / L LSC targeting agent if the number and / or proportion of M / L LSCs is equal to or greater than the predetermined cutoff value; or administering to the subject a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent if the number and / or proportion of M / L LSCs is less than the predetermined cutoff value.

[0037] The present disclosure provides methods of treating a subject with AML, the methods comprising administering to the subject with AML a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one M / L LSC targeting agent.

[0038] Identification of M / L LSCs From the description of the methods provided herein, it will be understood that the methods of the present disclosure include identifying at least one, number and / or proportion of M / L LSCs among a plurality of cells in a sample from a subject.

[0039] It is understood that identifying at least one of the M / L LSCs in the plurality of cells can result in identification of no M / L LSCs (i.e., the user can determine that no M / L LSCs are present in the plurality of cells).

[0040] The methods of the present disclosure allow for the identification of m-LSCs based on the novel immunophenotype of CD34+ and CD7+.

[0041] Expression of the above biomarkers, or any other biomarkers described herein, can be achieved by one of skill in the art using any suitable method known in the art, including, but not limited to, PCR, high-throughput sequencing, next-generation sequencing, Northern blot, reverse transcription PCR (RT-PCR), real-time PCR (qPCR), quantitative PCR, qRT-PCR, flow cytometry, mass spectrometry, microarray analysis, digital droplet PCR, Western blot, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-SEQ), any single-cell omics technology, or any combination thereof.

[0042] hypomethylating agents As will be appreciated by those skilled in the art, a hypomethylating agent is an agent that inhibits DNA methylation. In some aspects of the methods described herein, the hypomethylating agent can be selected from azacitidine and decitabine. In some aspects of the methods described herein, the hypomethylating agent can be any hypomethylating agent known in the art.

[0043] In some embodiments, the hypomethylating agent is azacitidine [ka] or a pharmaceutically acceptable salt, analog, derivative, salt, or ester thereof. As will be understood by those skilled in the art, azacitidine may be identified by any one of the following names: 5-azacytidine, azacytidine, ladakamycin, 4-amino-1-β-D-ribofuranosyl-s-triazin-2(1H)-one, U-18496, CC-486, and 4-amino-1-β-D-ribofuranosyl-1,3,5-triazin-2(1H)-one. As will be understood by those skilled in the art, azacitidine may be identified by its CAS number 320-67-2.

[0044] In some embodiments, the hypomethylating agent is decitabine, [ka] or a pharmaceutically acceptable salt, analog, derivative, salt, or ester thereof. As will be understood by those of skill in the art, decitabine may be identified by any one of the following names: 5-aza-2'-deoxycytidine, 4-amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1,3,5-triazin-2(1H)-one, 5-aza-2'-deoxycytidine, 5-azadeoxycytidine, 2-deoxy-5-azacytidine, and 2'-deoxy-5-azacytidine. As will be understood by those of skill in the art, decitabine may be identified by CAS number 2353-33-5.

[0045] Substitution of hypomethylating agents In any of the methods described herein, the hypomethylating agent is cytarabine [ka] or a pharmaceutically acceptable salt, analog, derivative, salt, or ester thereof. As will be understood by those skilled in the art, cytarabine may be identified by any one of the names 4-amino-1-[(2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one, aracytidine, and cytosine arabinoside. As will be understood by those skilled in the art, cytarabine may be identified by CAS number 147-94-4.

[0046] BCL-2 inhibitors In some aspects of the methods provided herein, the BCL-2 inhibitor can be selected from venetoclax and navitoclax. In some aspects of the methods provided herein, the BCL-2 inhibitor can be any BCL-2 inhibitor known in the art.

[0047] In some embodiments, the BCL-2 inhibitor is venetoclax [ka] or a pharmaceutically acceptable salt, analog, derivative, salt, or ester thereof. As will be understood by one of skill in the art, venetoclax may be identified by any one of the following names: GDC-0199, ABT-199, RG-7601, 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide, Venklekta, and Venklixt. As will be understood by one of skill in the art, venetoclax may be identified by its CAS number: 1257044-40-8.

[0048] In some embodiments, the BCL-2 inhibitor is navitoclax [ka] or a pharmaceutically acceptable salt, analog, derivative, salt, or ester thereof. As will be understood by one of ordinary skill in the art, navitoclax may be identified by any one of the following names: ABT263, ABT-263, and 4-(4-{[2-(4-chlorophenyl)-5,5-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-(4-{[(2R)-4-(morpholin-4-yl)-1-(phenylsulfanyl)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzene-1-sulfonyl)benzamide. As will be understood by one of ordinary skill in the art, navitoclax may be identified by its CAS number: 923564-51-6.

[0049] In some embodiments, the BCL-2 inhibitor can be BGB-11417.

[0050] In some embodiments, the BCL-2 inhibitor can be ZN-d5.

[0051] M / L LSC targeting agent In some aspects, the M / L LSC targeting agent can be at least one of: i) an anti-CD7 antibody; ii) an anti-CD7 immunotherapy, preferably wherein the immunotherapy comprises CAR-T cells and / or NK cells that specifically target CD7; iii) an agent that inhibits CD7 signaling, preferably wherein the agent that inhibits CD7 signaling is an inhibitor of PI3 kinase and related pathways; iv) an agent that inhibits CD7 activation, preferably wherein the agent that inhibits CD7 activation inhibits CD7 activation by K12 and / or any additional CD7 ligand; and v) an agent that targets an M / L LSC-specific pathway and / or surface marker.

[0052] In some aspects of the methods of the present disclosure, the immunotherapy can include administering a therapeutically effective amount of at least one antibody, at least one checkpoint inhibitor, at least one chimeric antigen receptor modified T cell (CAR-T cell), or any combination thereof. The immunotherapy can include adoptive cell transfer therapy.

[0053] In some embodiments, the immunotherapy can be an immunotherapy that specifically targets at least one M / L LSC antigen, including, but not limited to, CD7. Thus, a non-limiting example of an immunotherapy that specifically targets at least one monocyte antigen can be a CAR-T cell comprising a chimeric antigen receptor that includes an antigen-binding domain that binds to CD7.

[0054] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. An antibody that binds to a target refers to an antibody that can bind to a target with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeted to the target. In one embodiment, the extent to which an anti-target antibody binds to an unrelated non-target protein is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA) or Biacore assay. In certain embodiments, an antibody that binds to a target has a dissociation constant (Kd) of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 8 M or less, e.g., 10 8 M~10 13 M, e.g., 10 9 M~10 13 M). In certain embodiments, the anti-target antibody binds to an epitope of the target that is conserved among different species.

[0055] A "blocking antibody" or "antagonist antibody" is an antibody that partially or completely blocks, inhibits, interferes with, or neutralizes the normal biological activity of an antigen to which it binds. For example, an antagonistic antibody may block signaling through an immune cell receptor (e.g., a T cell receptor) to restore a dysfunctional functional response by T cells to antigenic stimulation (e.g., proliferation, cytokine production, target cell killing).

[0056] An "agonist antibody" or "activating antibody" is an antibody that mimics, promotes, stimulates, or enhances the normal biological activity of an antigen to which it binds. Agonist antibodies can also enhance or initiate signaling by the antigen to which they bind. In some embodiments, agonist antibodies cause or activate signaling without a natural ligand. For example, agonist antibodies may increase memory T cell proliferation, increase cytokine production by memory T cells, inhibit regulatory T cell function, and / or inhibit regulatory T cell suppression of effector T cell function, such as effector T cell proliferation and / or cytokine production.

[0057] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0058] CAR-T cells are T cells genetically engineered to stably express at least one chimeric antigen receptor (CAR). The CAR can comprise an extracellular domain, a transmembrane domain, and a cytoplasmic domain. The CAR can comprise an antigen-binding domain. The antigen-binding domain can be located in the extracellular domain. In some embodiments of the methods of the present disclosure, the antigen-binding domain binds to at least one AML cell surface protein. In some embodiments of the methods of the present disclosure, the antigen-binding domain binds to CD64 and / or LILRB4. The CAR can also comprise an extracellular spacer (hinge) domain. The extracellular spacer can be located in the extracellular domain. The CAR can comprise a signaling domain. The signaling domain can be a T cell activation domain. The signaling domain can be located in the cytoplasmic domain. The CAR can comprise at least one costimulatory domain. The CAR can comprise at least two costimulatory domains. The CAR can comprise at least three costimulatory domains. The costimulatory domain can be located in the cytoplasmic domain.

[0059] In some embodiments of the disclosed methods, the CAR-T cells can be autologous to the subject. In some embodiments, the CAR-T cells can be allogeneic to the subject.

[0060] In some forms of the methods of the present invention, CAR-T cells can be administered alone or as a pharmaceutical composition in combination with other components, such as diluents and / or IL-2 or other cytokines or cell populations. Briefly, a pharmaceutical composition can include a plurality of CAR-T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. CAR-T cells and related compositions can be administered intravenously to a subject.

[0061] The CAR-T cell can comprise a chimeric antigen receptor. The chimeric antigen receptor can comprise an antigen-binding domain. The antigen-binding domain can bind to CD64 and / or LILRB4.

[0062] subject As used herein, the term "subject" includes human and non-human animals, as well as cell lines, cell cultures, tissues, and organs. In some aspects, the subject is a mammal. The mammal can be, for example, a human or a suitable non-human mammal, such as a primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep, or pig. The subject can also be a bird or poultry. In some aspects, the subject is a human.

[0063] As used herein, the term "subject in need thereof" refers to a subject having a disease or a subject at high risk of developing a disease. "Subject" includes mammals. A mammal can be, for example, a human or a suitable non-human mammal, such as a primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep, or pig. A subject can also be a bird or poultry. In some embodiments, a mammal is a human. A subject in need thereof can be a subject previously diagnosed or identified as having a disease or disorder disclosed herein. A subject in need thereof can also be a subject suffering from a disease or disorder disclosed herein. Alternatively, a subject in need thereof can be a subject at high risk of developing such a disease or disorder compared to the general population (i.e., a subject who is more susceptible to developing such a disorder compared to the general population). A subject in need thereof can have a refractory or resistant disease or disorder disclosed herein (i.e., a disease or disorder disclosed herein that does not respond, or has not yet responded, to treatment). The subject may be resistant to treatment at the start of the treatment or may become resistant during treatment. In some embodiments, the subject in need thereof has undergone all known effective therapies for the disease or disorder disclosed herein, none of which have been effective. In some embodiments, the subject in need thereof has undergone at least one previous therapy.

[0064] In some aspects of the methods of the present disclosure, the subject is a human.

[0065] In some aspects of the methods of the present disclosure, the subject is a subject with AML who has not received any treatment for the AML.

[0066] In some embodiments of the methods of the present disclosure, the subject has AML and has previously received at least one AML treatment, in some embodiments, the at least one treatment comprises a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

[0067] In some embodiments of the method of the present disclosure, the subject is a subject in which at least one M / L LSC is present in the AML cell population.In some embodiments of the method of the present disclosure, the subject is a subject in which the AML cell population comprises a proportion of M / L LSC that is greater than a predetermined cutoff percentage.In some embodiments of the method of the present disclosure, the subject is a subject in which the AML cell population comprises a number of M / L LSC that is greater than a predetermined cutoff number.

[0068] sample In some aspects of the disclosed methods, the sample can include blood, a bone marrow biopsy, a bone marrow aspirate, a chloroma biopsy, a tissue biopsy, cerebrospinal fluid, or any combination thereof.

[0069] In some aspects, the sample can be a bone marrow biopsy.

[0070] In some embodiments, the sample can be a bone marrow aspirate.

[0071] In some aspects, the sample can be a chloroma biopsy.

[0072] General definition As used herein, phrases such as "one or more of A, B, or C," "one or more of A, B, or C," "one or more of A, B, and C," "one or more of A, B, and C," "selected from the group consisting of A, B, and C," "selected from A, B, and C," and the like are used interchangeably and, unless otherwise specified, all refer to a selection from the group consisting of A, B, and / or C, i.e., one or more A, one or more B, one or more C, or any combination thereof.

[0073] Unless otherwise specified, any reference to a method of treatment should be understood to include the use of an agent to provide treatment as described herein. Furthermore, unless otherwise specified, any reference to a method of treatment should be understood to include the use of an agent to prepare a medicament for treating such a condition. Treatment includes treatment of humans or non-human animals, including rodents and other disease models used herein.

[0074] As used herein, the term "treating" or "treat" refers to the management and care of a patient for the purpose of combating a disease, condition, or disorder, and includes administering an agent described in the present disclosure, or a pharmaceutically acceptable salt, polymorph, or solvate thereof, to alleviate the symptoms or complications of the disease, condition, or disorder, or to eliminate the disease, condition, or disorder. The term "treat" may also include treatment of a cell or animal model in vitro. It should be understood that reference to "treating" or "treat" includes the alleviation of established symptoms of the condition. "Treating" or "treating" the condition, disorder, or condition includes (1) preventing the onset of clinical symptoms of the condition or delaying the onset of clinical symptoms of the condition, disorder, or condition in a person who may be affected by or predisposed to the condition, disorder, or condition, but who has not yet experienced or exhibited clinical or subclinical symptoms of the condition, disorder, or condition; (2) inhibiting the condition, disorder, or condition, i.e., arresting, alleviating, or delaying the onset of the disease or its recurrence (in the case of maintenance therapy) or the progression of at least one clinical or subclinical symptom thereof; or (3) palliating or alleviating the disease, i.e., causing regression of the condition, disorder, or condition or at least one clinical or subclinical symptom thereof.

[0075] It is understood that the agents described in this disclosure or pharmaceutically acceptable salts, polymorphs or solvates thereof can also be or may be used to prevent associated diseases, conditions or disorders or to identify suitable candidates for such purposes.

[0076] As used herein, the terms "prevent," "prevent," or "protect from" mean reducing or eliminating the onset of symptoms or complications of such disease, condition, or disorder.

[0077] As used herein, the phrase "pharmaceutically acceptable" refers to compounds, anions, cations, materials, compositions, carriers, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0078] It is understood that the agents described herein can be administered to a subject in at least one therapeutically effective amount. As used herein, the term "therapeutically effective amount" refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent a specified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. This effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend on the subject's weight, height, and health, the nature and extent of the condition, and the therapeutic agent or combination of therapeutic agents selected for administration. The therapeutically effective amount for a given situation can be determined by routine experimentation, which is within the skill and judgment of the clinician. Clinicians can also determine therapeutically effective amounts of the agents described herein using established dosages and administration protocols for the agents described herein.

[0079] It is understood that for any agent, the therapeutically effective amount can be estimated initially either in cell culture assays, for example, of tumor cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine effective doses and routes of administration in humans. For example, ED 50 (the dose that is therapeutically effective in 50% of the population) and LD 50 Therapeutic / prophylactic efficacy (the dose lethal to 50% of the population) and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the ratio LD 50 / ED 50 Pharmaceutical compositions that exhibit high therapeutic indices are preferred. The dosage can vary within this range depending on the dosage form used, sensitivity of the patient, and the route of administration.

[0080] Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect. Factors that may be considered include the severity of the disease state, the subject's general health, the subject's age, weight, and sex, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerability / response to therapy. Long-acting pharmaceutical compositions may be administered every 3-4 days, every week, or once every two weeks, depending on the half-life and clearance rate of the particular formulation.

[0081] As used herein, the term "pharmaceutically acceptable salts" refers to derivatives of the agents described herein, wherein the agent is modified by making its acid or base salt. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include conventional non-toxic salts as well as, for example, quaternary ammonium salts of the parent agent formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, 1,2-ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, glycolic acid arsanilic acid, hexylresorcylic acid, hydrabamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxymaleic acid, hydroxynaphthoic acid, isethionic acid, lactic acid, and lactobionic acid. , laurylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, napsylic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfuric acid, tannic acid, tartaric acid, toluenesulfonic acid, and commonly occurring amino acids such as glycine, alanine, phenylalanine, arginine, and the like.

[0082] In some embodiments, the pharmaceutically acceptable salt is a sodium salt, a potassium salt, a calcium salt, a magnesium salt, a diethylamine salt, a choline salt, a meglumine salt, a benzathine salt, a tromethamine salt, an ammonia salt, an arginine salt, or a lysine salt.

[0083] Other examples of pharmaceutically acceptable salts include hexanoic acid, cyclopentanepropionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, muconic acid, etc. The present disclosure also relates to salts formed when an acidic proton present in the parent drug is either replaced by a metal ion, e.g., an alkali metal ion, alkaline earth ion, or aluminum ion, or coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc. In the salt form, it is understood that the ratio of drug to salt cation or anion can be 1:1 or any ratio other than 1:1, for example, 3:1, 2:1, 1:2, or 1:3.

[0084] It should be understood that all references to pharmaceutically acceptable salts include the solvent addition forms (solvates) or crystal forms (polymorphs) defined herein of the same salt.

[0085] As used herein, the term "refractory" is used in its broadest sense to refer to when the disease present in a subject is unresponsive to a particular therapy, i.e., when the therapy provides no or diminishing clinical benefit to that particular subject.

[0086] As used herein, the terms "combination therapy" or "concurrent therapy" include the administration of an agent disclosed herein or a pharmaceutically acceptable salt, polymorph, or solvate thereof, and at least a second agent as part of a specific treatment regimen intended to provide a beneficial effect due to the interaction of these therapeutic agents. The beneficial effect of the combination includes, but is not limited to, a pharmacokinetic or pharmacodynamic interaction resulting from the combination of therapeutic agents.

[0087] As used herein, the term "proximity in time" refers to the administration of one therapeutic agent occurring within a period of time before or after the administration of another therapeutic agent, such that the therapeutic effects of one therapeutic agent overlap with the therapeutic effects of the other therapeutic agent. In some embodiments, the therapeutic effects of one therapeutic agent completely overlap with the therapeutic effects of the other therapeutic agent. In some embodiments, "proximity in time" means that the administration of one therapeutic agent occurs within a period of time before or after the administration of another therapeutic agent, such that there is a synergistic effect between the one therapeutic agent and the other therapeutic agent. "Proximity in time" may vary depending on various factors, including, but not limited to, the age, sex, weight, genetic background, health status, medical history, and treatment history of the subject to whom the therapeutic agent is administered, the disease or condition being treated or ameliorated, the therapeutic outcome being achieved, the dosage, frequency, and duration of administration of the therapeutic agent, the pharmacokinetics and pharmacodynamics of the therapeutic agent, and the route(s) by which the therapeutic agent is administered. In some embodiments, "proximity in time" means within 15 minutes, within 30 minutes, within 1 hour, within 2 hours, within 4 hours, within 6 hours, within 8 hours, within 12 hours, within 18 hours, within 24 hours, within 36 hours, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within 1 week, within 2 weeks, within 3 weeks, within 4 weeks, within 6 weeks, or within 8 weeks. In some embodiments, multiple administrations of one therapeutic agent can be administered in close temporal proximity to a single administration of another therapeutic agent. In some embodiments, the temporal proximity can vary during a treatment cycle or dosing regimen. [Example]

[0088] Example 1 - Co-expression of CD7 and CD34 separates subpopulations with different overall survival rates The following is a non-limiting example demonstrating that subjects with AML that co-express CD7 and CD34 respond poorly to treatment with a combination of venetoclax and azacitidine.

[0089] Serial samples from subjects with AML treated with a combination of venetoclax and azacitidine were analyzed by flow cytometry to determine CD7 and CD34 expression on AML cells, and patient survival probability was then plotted as a function of time (days) after treatment.

[0090] Figure 1A shows an analysis of all AML samples, regardless of whether the subjects also underwent stem cell transplantation (SCT). As shown in Figure 1A, subjects with AML exhibiting cells that co-expressed CD7+ and CD34+ (i.e., M / L LSCs) had poorer outcomes after venetoclax / azacitidine treatment compared with subjects exhibiting cells that were CD7- / CD34- or CD7- / CD34+. Without being bound by theory, these results demonstrate that the presence of CD7+ / CD34+ cells in biological samples from subjects with AML can be used to determine poor cellular response to treatment with venetoclax and azacitidine in combination.

[0091] The data were further analyzed to exclude subjects who underwent SCT (FIG. 1B) or to censor data from subjects who underwent SCT (FIG. 1C). Similar to the results shown in FIG. 1A, FIGS. 1B and 1C show that subjects with AML who exhibited cells that coexpressed CD7+ and CD34+ (i.e., M / L LSCs) had poorer outcomes after venetoclax / azacytidine treatment compared with subjects who exhibited cells that were CD7- / CD34- or CD7- / CD34+.

[0092] Without being bound by theory, the results presented in this example demonstrate that the methods of the present disclosure based on the identification of CD7+ / CD34+M / L LSCs can be used to predict the response that subjects with AML should receive treatment with a combination of venetoclax and azacitidine.

Claims

1. 1. A method of treating acute myeloid leukemia (AML) in a subject, the method comprising: a) measuring the expression of at least CD34 and CD7 on a plurality of cells in a sample from the subject; b) identifying the presence of at least one myeloid / lymphocytic leukemia stem cell (M / L LSC) based on the expression measured in step (a), wherein a cell is identified as a M / L LSC if it is at least CD34+ and CD7+; c) if at least one M / L LSC is identified, administering to said subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one M / L LSC targeting agent; or If no M / L LSC is identified, administering to said subject a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

2. 1. A method for identifying whether a subject having AML will respond to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent, the method comprising: a) measuring the expression of at least CD34 and CD7 on a plurality of cells in a sample from the subject; b) identifying the presence of at least one myeloid / lymphocytic leukemia stem cell (M / L LSC) based on the expression measured in step (a), wherein a cell is identified as a M / L LSC if it is at least CD34+ and CD7+; c) identifying the subject as not responding to the treatment if the presence of at least one M / L LSC is identified; or and if no M / L LSC is identified, identifying the subject as responsive to the treatment.

3. 1. A method of treating AML in a subject, the method comprising: a) measuring the expression of at least CD34 and CD7 on a plurality of cells in a sample from the subject; b) identifying the number and / or proportion of M / L LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as an M / L LSC if it is at least CD34+ and CD7+; c) comparing the number and / or proportion of the m M / L LSCs identified in step (b) with a predetermined cutoff value; d) if the number and / or proportion of M / L LSCs is equal to or greater than the predetermined cutoff value, administering to said subject a combination of at least one BCL-2 inhibitor, at least one hypomethylating agent, and at least one M / L LSC targeting agent; or and if the number and / or proportion of M / L LSCs is below the predetermined cutoff value, administering to the subject a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

4. 1. A method for identifying whether a subject having AML will respond to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent, the method comprising: a) measuring the expression of at least CD34 and CD7 on a plurality of cells in a sample from the subject; b) identifying the number and / or proportion of M / L LSCs in the plurality of cells based on the expression measured in step (a), wherein a cell is identified as an M / L LSC if it is at least CD34+ and CD7+; c) comparing the number and / or proportion of the m M / L LSCs identified in step (b) with a predetermined cutoff value; d) identifying said subject as not responding to said treatment if the number and / or proportion of M / L LSCs is equal to or greater than said predetermined cut-off value; or and identifying the subject as responding to the treatment if the number and / or proportion of M / L LSCs is below the predetermined cutoff value.

5. The at least one M / L LSC targeting agent comprises: i) an anti-CD7 antibody; and ii) anti-CD7 immunotherapy, preferably wherein said immunotherapy comprises CAR-T cells and / or NK cells that specifically target CD7; and iii) an agent that inhibits CD7 signaling, preferably, the agent that inhibits CD7 signaling is an inhibitor of PI3 kinase and related pathways; and iv) an agent that inhibits CD7 activation, preferably wherein said agent that inhibits CD7 activation inhibits CD7 activation by K12 and / or any additional CD7 ligand; and v) an agent targeting an M / L LSC-specific pathway and / or surface marker.

6. 10. The method of any one of the preceding claims, wherein the at least one hypomethylating agent is selected from azacitidine and decitabine.

7. 10. The method of any one of the preceding claims, wherein the at least one BCL-2 inhibitor is selected from venetoclax and navitoclax.

8. 10. The method of any one of the preceding claims, wherein step (a) comprises performing PCR, high-throughput sequencing, next-generation sequencing, Northern blot, reverse transcription PCR (RT-PCR), real-time PCR (qPCR), quantitative PCR, qRT-PCR, flow cytometry, mass spectrometry, microarray analysis, digital droplet PCR, Western blot, Cellular Indexing of Transcriptome and Epitopes by Sequencing (CITE-SEQ), any single-cell omics technique, or any combination thereof.

9. The subject is i) a subject with AML who has not received any treatment for AML, or ii) The method of any one of the preceding claims, wherein the subject has AML and has previously received at least one AML treatment, preferably wherein said at least one AML treatment comprises a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

10. 10. The method of any one of the preceding claims, wherein identifying the subject as responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent comprises identifying the subject as having a long-term remission after receiving said treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

11. 10. The method of any one of the preceding claims, wherein identifying the subject as non-responsive to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent comprises identifying the subject as refractory to treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent and / or relapse in the subject after treatment with a combination of at least one BCL-2 inhibitor and at least one hypomethylating agent.

12. 10. The method of any one of the preceding claims, wherein the biological sample comprises blood, a bone marrow biopsy, a bone marrow aspirate, a Pseudomonas aeruginosa biopsy, a tissue biopsy, cerebrospinal fluid, or any combination thereof.

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