T cells for use in the treatment of relapsed or refractory acute myeloid leukemia

Abstract

This disclosure provides, in particular, a method for treating relapsed or refractory acute myeloid leukemia, wherein one or more symptoms or biomarkers are improved after treatment by administering a therapeutically effective amount of an allogeneic composition containing Vδ1+ (Vδ1+) gamma delta (γδ) T cells to a subject in need. This disclosure also provides appropriate doses of a composition containing allogeneic Vδ1+ gamma delta (γδ) T cells for administration to a subject suffering from relapsed or refractory AML. In some embodiments, these Vδ1+ gamma delta (γδ) T cells are not transduced.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 457,937, filed on 7 April 2023, the contents of which are incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] Acute myeloid leukemia (AML) is a type of blood cancer that affects adults and children, characterized by the infiltration of bone marrow and other tissues by clonally proliferating immature myeloid cells. Approximately 20,050 cases of AML are diagnosed annually (NCI Surveillance Epidemiology and End Results Program, SEER). Current treatment strategies include chemotherapy, allogeneic stem cell transplantation, and certain targeted therapies, but managing AML remains a clinical challenge due to resistance to chemotherapy and the fact that a significant proportion of treated patients develop relapsed or refractory disease.

[0003] AML has a low survival rate in older patients (65 years and older), with an overall mean 5-year survival rate of approximately 30%, mainly due to resistance to standard treatment. For example, chemotherapy with a combination of cytarabine and anthracyclines is effective in inducing complete remission, but ultimately leads to the selection of chemoresistant clones that promote refractory relapses. The median cumulative incidence of relapse is 29.4% after stem cell transplantation and 46.8% after induction chemotherapy, indicating that a significant portion of patients develop refractory or relapsed AML with a poor prognosis. (Esther NO et al. (2021), Am J Blood Res, 11(4):325-360).

[0004] The presence of gamma delta T cells has been shown to be positively correlated with the prognosis of many solid tumors and hematological malignancies (Deniger, DC et al., Clin. Cancer Res. (2014), 20(22):5708-19; Gentles AJ et al., Nat. Med. (2015), 21(8):938-945). In this field, there is a need for safe and effective treatment options for AML, especially relapsed or refractory AML. [Overview of the project]

[0005] This disclosure provides, in particular, a method for treating relapsed or refractory acute myeloid leukemia, wherein one or more symptoms or biomarkers are improved after treatment by administering a therapeutically effective dose of a composition (e.g., an allogeneic composition) containing Vδ1+ gamma delta (γδ) T cells to a subject in need. This disclosure also provides appropriate doses of a composition containing allogeneic Vδ1+ gamma delta (γδ) T cells for administration to a subject suffering from relapsed or refractory AML.

[0006] Vδ1+γδT cells are an enriched subset of γδT cells, primarily consisting of Vδ1+γδT cells, which recognize malignant cells through the expression of a diverse repertoire of innate cytotoxic receptors (NCRs) that interact with stress ligands, pAg, lipid Ag, and many other non-peptide molecules specifically upregulated on affected cells. While αβT cells require an MHC-antigen axis for activation, gamma delta T lymphocytes do not require major histocompatibility complex (MHC)-mediated antigen presentation to exert cytotoxic effects, thus giving γδT cells an advantage over αβT cells in initial responsiveness. This is because γδT cells can directly recognize dysregulated molecular patterns on cancer cells and are activated by these molecular patterns. While not intending to be bound by any particular theory, the non-MHC-restricted immunomodulatory and antitumor activity of Vδ1+γδ T cells (GDX012) of this disclosure is intended to provide a novel allogeneic cell therapy for the treatment of relapsed or refractory AML, which is particularly difficult to treat. The inventors of this application have developed an "off-the-shelf" immunotherapy for relapsed or refractory AML that is not resolved by other forms of treatment. The GDX012 therapy is also described in WO2021 / 186137, the contents of which are incorporated herein by reference in their entirety.

[0007] For example, when administered systemically by intravenous infusion, GDX012 exhibits homing to the bone marrow and is detectable for at least 28 days. While we do not wish to be bound by any particular theory, in addition to its high cytotoxic activity against AML blasts, GDX012 also exhibits homing to and persists in peripheral blood and bone marrow, and is intended to exhibit long-lasting cytotoxic effects in target tissues with a single dose in the treatment of relapsed or refractory AML. Thus, the Vδ1+γδT cells (GDX012) of this disclosure provide a safe and effective treatment for relapsed or refractory AML. In some embodiments, GDX012 is administered in multiple doses and / or used in combination with other treatments.

[0008] In some embodiments, GDX012 is administered after lymphocytosis therapy. In some embodiments, cytarabine and fludarabine are administered as lymphocytosis therapy. Cytarabine and fludarabine are effective when used in combination as lymphocytosis agents. Although we do not wish to be bound by any particular theory, cytarabine is converted intracellularly to its active metabolite, ara-C 5'-triphosphate (ara-CTP). Fludarabine enhances the intracellular accumulation of ara-CTP, thereby increasing the cytotoxic effect of cytarabine.

[0009] For example, while the use of Vδ2+ T cells in the treatment of cancer, including relapsed or refractory AML, is being explored (Vydra et al., Clinical Lymphoma (2023), Myeloma and Leukemia), the clinical use of Vδ1+ T cells is difficult due to their relatively low abundance in peripheral blood lymphocytes, less than approximately 0.5%. This disclosure provides a method for treating relapsed or refractory AML by administering an allogeneic composition of Vδ1+ T cells (GDX012) obtained to subjects suffering from relapsed or refractory AML in a safe and administerable dose up to a high yield for therapeutic use. Relapsed or refractory AML is typically associated with a higher disease burden in the bone marrow and peripheral blood, and a greater number of leukemic blasts, compared, for example, to a measurable residual disease (MRD) state. This disclosure provides an effective dose of GDX012 for restoring or minimizing one or more symptoms or biomarkers of relapsed or refractory acute myeloid leukemia.

[0010] In some embodiments, the Specified provides a method for treating acute myeloid leukemia by administering a therapeutically effective amount of a composition comprising Vδ1+ gamma delta (γδ) T cells to a subject requiring treatment for acute myeloid leukemia, wherein the acute myeloid leukemia is relapsed or refractory.

[0011] In some embodiments, one or more symptoms or biomarkers improve after administration.

[0012] In some embodiments, these gamma delta T cells are not transduced.

[0013] In some embodiments, the composition contains at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 99% of the total number of viable cells, specifically gamma delta T cells.

[0014] In some embodiments, the composition contains at least 50% Vδ1+γδT cells relative to the total number of viable cells.

[0015] In some embodiments, the composition contains at least 70% Vδ1+γδT cells relative to the total number of viable cells.

[0016] In some embodiments, the composition contains at least 90% Vδ1+γδT cells relative to the total number of viable cells.

[0017] In some embodiments, the composition contains at least 99% of Vδ1+γδT cells relative to the total number of viable cells.

[0018] In some embodiments, this composition contains less than 5% residual Vδ2+ cells.

[0019] In some embodiments, this composition contains less than 0.5% residual Vδ2+ cells.

[0020] In some embodiments, this composition contains less than 0.1% residual Vδ2+ cells.

[0021] In some embodiments, this composition does not contain residual Vδ2+ T cells.

[0022] In some embodiments, the subject has previously received chemotherapy.

[0023] In some embodiments, the subject has received at least two courses of intensive induction chemotherapy. In some embodiments, the subject has received two courses of intensive induction chemotherapy. In some embodiments, the subject has received three courses of intensive induction chemotherapy. In some embodiments, the subject has received four courses of intensive induction chemotherapy. In some embodiments, the subject has received five courses of intensive induction chemotherapy.

[0024] In some embodiments, the subject has approximately 5% leukemic blasts in the bone marrow after complete remission (CR), complete remission with partial hematopoietic recovery (CRh), complete remission with incomplete hematopoietic recovery (CRi), or morphological leukemia-free state (MLFS). In some embodiments, the subject has approximately 5%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, and approximately 50% leukemic blasts in the bone marrow after complete remission (CR), complete remission with partial hematopoietic recovery (CRh), complete remission with incomplete hematopoietic recovery (CRi), or morphological leukemia-free state (MLFS). In some embodiments, the subject has 5% to 50% leukemic blasts in the bone marrow. In some embodiments, the subject has approximately 10% leukemic blasts in the bone marrow. In some embodiments, the subject has more than 15% leukemic blasts in its bone marrow. In some embodiments, the subject has more than 20% leukemic blasts in its bone marrow. In some embodiments, the subject has more than 25% leukemic blasts in its bone marrow. In some embodiments, the subject has more than 30% leukemic blasts in its bone marrow. In some embodiments, the subject has more than 35% leukemic blasts in its bone marrow. In some embodiments, the subject has more than 40% leukemic blasts in its bone marrow. In some embodiments, the subject has more than 45% leukemic blasts in its bone marrow. In some embodiments, the subject has more than 50% leukemic blasts in its bone marrow.

[0025] In some embodiments, this subject has about 5% leukemic blasts in the peripheral blood after (CR), complete remission with partial hematopoietic recovery (CRh), complete remission with incomplete hematopoietic recovery (CRi), or morphologic leukemia-free state (MLFS). In some embodiments, after complete remission (CR), complete remission with partial hematopoietic recovery (CRh), complete remission with incomplete hematopoietic recovery (CRi), or morphologic leukemia-free state (MLFS), the subject has more than about 5%, more than about 10%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50% leukemic blasts in the peripheral blood. In some embodiments, this subject has 5% - 50% leukemic blasts in the peripheral blood. In some embodiments, this subject has more than about 5% leukemic blasts in the peripheral blood. In some embodiments, this subject has more than about 10% leukemic blasts in the peripheral blood. In some embodiments, this subject has more than about 15% leukemic blasts in the peripheral blood. In some embodiments, this subject has more than about 20% leukemic blasts in the peripheral blood. In some embodiments, this subject has more than about 25% leukemic blasts in the peripheral blood. In some embodiments, this subject has more than about 30% leukemic blasts in the peripheral blood. In some embodiments, this subject has more than about 35% leukemic blasts in the peripheral blood. In some embodiments, this subject has more than about 40% leukemic blasts in the peripheral blood. In some embodiments, this subject has more than about 45% leukemic blasts in the peripheral blood. In some embodiments, this subject has more than about 50% leukemic blasts in the peripheral blood.

[0026] In some embodiments, this therapeutically effective amount is about 8×10 10 , 4×10 10 , 8×10 9 , 4×10 9 , 2.4×10 9 , 1.2×10 9 , 8×10 8 , 4×10 8 , 8×10 7 or 4×10 7 viable T cells.

[0027] In some embodiments, this therapeutically effective amount is approximately 8 × 10 9 Contains 1 surviving T cell.

[0028] In some embodiments, this therapeutically effective amount is approximately 4 × 10 9 Contains 1 surviving T cell.

[0029] In some embodiments, this therapeutically effective amount is approximately 2.4 × 10⁻⁶ 9 Contains 1 surviving T cell.

[0030] In some embodiments, this therapeutically effective amount is approximately 1.2 × 10⁻⁶ 9 Contains 1 surviving T cell.

[0031] In some embodiments, this therapeutically effective amount is approximately 8 × 10 8 Contains 1 surviving T cell.

[0032] In some embodiments, this therapeutically effective amount is approximately 4 × 10 8 Contains 1 T cell.

[0033] In some embodiments, this therapeutically effective amount is approximately 4 × 10 8 Contains fewer than 1 T cell.

[0034] In some embodiments, this therapeutically effective amount is approximately 5 × 10 4 Contains fewer than 1 alpha-beta T cells.

[0035] In some embodiments, this therapeutically effective amount is approximately 1 × 10⁻⁶ 4 Contains fewer than 1 alpha-beta T cells.

[0036] In some embodiments, gamma delta T cells do not express chimeric antigen receptors (CARs).

[0037] In some embodiments, gamma delta T cells express chimeric antigen receptors (CARs).

[0038] In some embodiments, the composition is administered intravenously, transarterally, subcutaneously, intradermally, intratumorally, intralymphatically, intramedullarily, intramuscularly, or intraperitoneally.

[0039] In some embodiments, this composition is administered intravenously.

[0040] In some embodiments, the composition is administered intraarterially. In some embodiments, the composition is administered subcutaneously. In some embodiments, the composition is administered intradermally. In some embodiments, the composition is administered intratumorally. In some embodiments, the composition is administered intraly into lymph nodes. In some embodiments, the composition is administered intramedullarily. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered intraperitoneally.

[0041] In some embodiments, the composition is administered in one or more doses.

[0042] In some embodiments, this composition is administered in a single dose.

[0043] In some embodiments, this composition is administered once a week, once a month, or once every two months. In some embodiments, this composition is administered once a week. In some embodiments, this composition is administered once every two months. In some embodiments, this composition is administered once every two weeks.

[0044] In some embodiments, this composition is administered once a month.

[0045] In some embodiments, this composition is administered for a period of 1 month, 2 months, 4 months, 6 months, 12 months, 14 months, 18 months, or 24 months. In some embodiments, this composition is administered for a period of 1 month. In some embodiments, this composition is administered for a period of 2 months. In some embodiments, this composition is administered for a period of 4 months. In some embodiments, this composition is administered for a period of 6 months. In some embodiments, this composition is administered for a period of 12 months. In some embodiments, this composition is administered for a period of 14 months. In some embodiments, this composition is administered for a period of 18 months. In some embodiments, this composition is administered for a period of 24 months.

[0046] In some embodiments, this composition is administered for a period exceeding 24 months.

[0047] In some embodiments, this chemotherapy includes the administration of mitoxantrone, etoposide, cytarabine, or anthracycline. In some embodiments, this chemotherapy includes the administration of mitoxantrone. In some embodiments, this chemotherapy includes the administration of etoposide. In some embodiments, this chemotherapy includes the administration of cytarabine. In some embodiments, this chemotherapy includes the administration of anthracycline.

[0048] In some embodiments, this chemotherapy includes the administration of fludarabine and cyclophosphamide.

[0049] In some embodiments, this chemotherapy includes the administration of fludarabine and cytarabine.

[0050] In some embodiments, this chemotherapy includes a 7-day administration of cytarabine and a 3-day administration of anthracycline.

[0051] In some embodiments, this chemotherapy includes at least two cycles of administration of a hypomethylating agent. In some embodiments, this chemotherapy includes two cycles of administration of a hypomethylating agent. In some embodiments, this chemotherapy includes three cycles of administration of a hypomethylating agent. In some embodiments, this chemotherapy includes four cycles of administration of a hypomethylating agent.

[0052] In some embodiments, this chemotherapy includes multiple cycles of monotherapy with a hypomethylating agent. In some embodiments, this chemotherapy includes at least four cycles of monotherapy with a hypomethylating agent. In some embodiments, this chemotherapy includes five cycles of monotherapy with a hypomethylating agent. In some embodiments, this chemotherapy includes administration of more than five cycles of monotherapy with a hypomethylating agent.

[0053] In some embodiments, cytarabine is administered with venetoclax, fludarabine, or other B-cell lymphoma 2 (BCL2) inhibitors. In some embodiments, cytarabine is administered with venetoclax. In some embodiments, cytarabine is administered with fludarabine. In some embodiments, cytarabine is administered with one or more B-cell lymphoma 2 inhibitors.

[0054] In some embodiments, the subject is at least 2 years old.

[0055] In some embodiments, the subject is at least 12 years old.

[0056] In some embodiments, the subject is at least 18 years old.

[0057] In some embodiments, one or more symptoms or biomarkers that improve after treatment are the substantial absence of myeloblasts.

[0058] In some embodiments, one or more symptoms or biomarkers that improve after treatment is a myeloblast count of less than approximately 5%.

[0059] In some embodiments, one or more symptoms or biomarkers that improve after treatment are a myeloblast and / or peripheral blood blast count of 5% to 25%.

[0060] In some embodiments, one or more symptoms or biomarkers that improve after treatment is a myeloblast count of less than approximately 10%.

[0061] In some embodiments, one or more post-treatment symptoms or biomarkers that are improved is a myeloblast count of less than approximately 15%.

[0062] In some embodiments, one or more post-treatment symptoms or biomarkers that are improved is a myeloblast count of less than approximately 20%.

[0063] In some embodiments, one or more post-treatment symptoms or biomarkers that are improved is a myeloblast count of less than approximately 25%.

[0064] In some embodiments, one or more symptoms or biomarkers that improve after treatment are the substantial absence of peripheral hemoblasts.

[0065] In some embodiments, one or more symptoms or biomarkers that improve after treatment are peripheral hemoblasts of less than approximately 5%.

[0066] In some embodiments, one or more symptoms or biomarkers that improve after treatment is a peripheral hemoblast count of less than approximately 10%.

[0067] In some embodiments, one or more symptoms or biomarkers that improve after treatment is a peripheral hemoblast count of less than approximately 15%.

[0068] In some embodiments, one or more symptoms or biomarkers that improve after treatment is a peripheral hemoblast count of less than approximately 20%.

[0069] In some embodiments, one or more symptoms or biomarkers that improve after treatment is a peripheral hemoblast count of less than approximately 25%.

[0070] In some embodiments, the one or more symptoms or biomarkers that improve after treatment is at least a 50% reduction in the pre-treatment myeloblast rate based on the European Leukemia Net (ELN) 2022 response criteria for AML.

[0071] In some embodiments, one or more symptoms or biomarkers that improve after treatment is the absence of extramedullary lesions.

[0072] In some embodiments, the one or more symptoms or biomarkers that improve after treatment are 0.5 × 10 9 This refers to the absolute neutrophil count (ANC) of more than one liter.

[0073] In some embodiments, the one or more symptoms or biomarkers that improve after treatment are 1.0 × 10 9 This refers to the absolute neutrophil count (ANC) of more than one liter.

[0074] In some embodiments, one or more symptoms or biomarkers that improve after treatment are 50 × 10 9 This refers to a platelet count of more than [number] plates per liter.

[0075] In some embodiments, one or more symptoms or biomarkers that improve after treatment are 100 × 10 9 This refers to a platelet count of more than [number] plates per liter.

[0076] In some embodiments, the measurable residual disease (MRD) after treatment is less than approximately 0.1%.

[0077] In some embodiments, MRD is measured by flow cytometry of bone marrow cells and / or blood.

[0078] The numerical values ​​used in this application, whether with or without the terms "about" or "approximately," are intended to include any normal variations that would be recognized by a person skilled in the art.

[0079] Other features, purposes, and advantages of this disclosure are evident in the detailed description below. However, it should be understood that the detailed description, while illustrating embodiments of this disclosure, is for illustrative purposes only and not limiting. Various changes and modifications within the scope of this disclosure will be evident to those skilled in the art from the detailed description.

[0080] definition To facilitate understanding of this disclosure, certain terms are defined below first. Additional definitions of the following terms and other terms are provided throughout this specification.

[0081] Approximately or about: As used herein, the terms “approximately” or “about,” when applied to one or more values ​​of interest, refer to a value similar to a given reference value. In certain embodiments, unless otherwise stated or evident from the context, the terms “approximately” or “about” refer to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in any direction (greater than or less than) of a given reference value (except where such a number exceeds 100% of a possible value). In some embodiments, the term refers to a range of values ​​that fall within 10% of a given reference value. In some embodiments, the term refers to a range of values ​​that fall within 5% of a given reference value. The term “between” includes the specified boundary values, as well as all intervening values ​​and decimals.

[0082] Proliferated Vδ1+ T cell population: As used herein, “proliferated” or “proliferated population of Vδ1+ T cells” includes a population of cells that is larger or contains more cells than a non-proliferated population. Such a population may be a majority population, a minority population, or a mixed population with proliferation of a certain proportion or particular cell type within the population. The term “proliferation step” will be understood to refer to the process that results in proliferation or a proliferated population. Thus, a proliferated or proliferated population may be more numerous or contain more cells than a population that has not undergone a proliferation step or a population prior to a proliferation step. Furthermore, any numbers shown herein to indicate proliferation (e.g., growth rate or proliferation rate) will be understood to reflect the number or size of the population of cells or an increase in the number of cells and to indicate the amount of proliferation.

[0083] Vδ1+ T cell enriched population: As used herein, enriched population refers to an allogeneic composition of Vδ1+ cells in which αβ cells are depleted and less than 5%, less than 0.5%, or less than 0.1% of residual Vδ2+ cells. In some embodiments, at least 90%, at least 95%, or at least 99% of the cells are Vδ1+γδT cells. In some embodiments, 99.1% to 99.9% of the cells are Vδ1+γδT cells. In some other embodiments, this Vδ1+ T cell population comprises at least 60%, at least 70%, at least 80%, or at least 90% of the total viable cells.

[0084] Interleukin-4-like activity: As used herein, a growth factor having "interleukin-4-like activity" means any compound that has the same activity as IL-4 in terms of its ability to promote similar physiological effects on γδT cells in culture, including, but not limited to, IL-4 and IL-4 mimics, or any functional equivalent of IL-4. Physiological effects promoted by IL-4 on γδT cells have been shown to include reduced expression levels of NKG2D and NCR, inhibition of cytotoxic function, and improved selective survival. IL-4 has also been shown to significantly inhibit the secretion of inflammatory cytokines, including IFN-γ and TNF-α, from activated TCRγδ+ T cells.

[0085] Interleukin-15-like activity: As used herein, a growth factor having “interleukin-15-like activity” means any compound having the same activity as IL-15 in terms of its ability to promote similar physiological effects on γδT cells in culture, including, but not limited to, IL-15 and IL-15 mimics, or any functional equivalent of IL-15, including IL-2 and IL-7. Physiological effects promoted by IL-15, IL-2, and IL-7 on cultured γδT cells include induction of cell differentiation toward a more cytotoxic phenotype, such as upregulation of the expression levels of NKG2D and innate cytotoxic receptors (NCRs) (NKp30 and NKp44), increased antitumor cytotoxic function, and increased production of pro-inflammatory cytokines such as IFN-γ.

[0086] Measurable physiological effects: As used herein, “measurable physiological effects” means any measurable change in the physiological state of target cells according to the standard definition. For example, a change in the physiological state of cells may be detected by a change in their activation state (recognized by an upregulation or downregulation of the expression level of the early activation cell marker CD69); or by a change in their differentiation state (recognized by an upregulation or downregulation of the innate cytotoxic receptor NCR that mediates the activation of NKG2D or NK cells) several hours or days after contact with such cytokines. Measurable physiological effects may also be changes in the rate of cell proliferation, measured by staining with the amine-reactive dye CFSE (carboxyfluorescein diacetate, succinimidyl ester) or by other techniques known in the art.

[0087] Leukemic blasts: Blasts are abnormal, immature white blood cells that proliferate uncontrollably in patients with acute myeloid leukemia, filling the bone marrow and interfering with the production of other cells essential for survival, namely red blood cells and platelets. Blasts found in the bone marrow or peripheral blood in acute myeloid leukemia are called "leukemic blasts" and are a measure of the disease.

[0088] Measurable (or minute) residual disease (MRD): As used herein, minute, or more appropriately, measurable residual disease (MRD) refers to an objective measure of AML remission by a sensitive method that allows for the determination of a very small number of neoplastic blasts and further distinguishes neoplastic blasts from healthy blasts. For example, patients who have achieved complete remission based solely on morphological evaluation may still have some residual cancer cells in their bone marrow, with 30%–40% being MRD positive. MRD is defined as a white blood cell (WBC) level of 1:10 compared to 1:20 in morphological evaluation. 4 ~1:10 6 This indicates the presence of leukemia cells reduced to a certain level. MRD detection in AML can be used to establish deeper remission, improve outcome prediction, inform post-remission management, identify impending relapses to enable early intervention, enable more robust post-transplant monitoring, and serve as a surrogate endpoint for drug trials.

[0089] Morphological Leukemia-Free State (MLFS): As used herein, "Morphological Leukemia-Free State (MLFS)" is characterized by a myeloblast percentage of less than 5%, the absence of circulating blasts, the absence of extramedullary lesions, and the absence of hematological recovery. The bone marrow is not merely poorly regenerated, but the bone marrow pyramids are present, the aspirate contains at least 200 cells, and the cytoplasm is at least 10% in the biopsy.

[0090] Refractory acute myeloid leukemia (AML): "Refractory AML" refers to leukemia that has not responded to treatment. Because chemotherapy drugs do not sufficiently kill leukemia cells, complete remission (CR) is not achieved, and neither CRh nor CRi are reached at the landmark evaluation point, i.e., after two courses of intensive remission induction treatment or at the specified evaluation point, for example, 180 days after the start of lower-intensity therapy.

[0091] Relapsed Acute Myeloid Leukemia (AML): “Relapsed AML” or relapsed acute myeloid leukemia (AML) refers to leukemia that has returned after treatment and remission. Relapsed AML is associated with one or more of the following symptoms and biomarkers: 5% or more myeloblasts; or reappearance of blasts in the blood in at least two peripheral blood samples at least one week apart; or development of extramedullary lesions.

[0092] Remission: As used herein, the term “remission” means a reduction in the signs and symptoms of cancer. Remission may be partial or complete. Measurable (or minimal) residual disease (MRD); cancer cells not destroyed by treatment is a more sensitive measure of remission.

[0093] Complete remission (CR): In complete remission (CR), all signs and symptoms of acute myeloid leukemia have disappeared. For example, the percentage of blast cells in the bone marrow is less than 5%, and there are no circulating blasts. There are no extramedullary lesions. Blood cell counts return to the normal range (for example, neutrophil count is 1.0 × 10⁶). 9 A blood glucose level of 1,000 / μL or higher, and a platelet count of at least 100 × 10⁶ 9 ( / L [100,000 / μL]). If a person maintains complete remission for more than five years, their cancer may be considered cured.

[0094] Partial remission (PR): As used herein, partial remission (PR) is defined as the meeting of all hematological criteria for complete remission (CR), but with a reduction in myeloblast count from 5% to 25%, and a reduction of at least 50% from the pre-treatment myeloblast count.

[0095] Complete remission with partial hematopoietic recovery (CRh): The term "CR with partial hematopoietic recovery (CRh)" represents a clinical benefit to the patient, and therefore, morphological myeloblast removal and neutrophil (at least 0.5 × 10⁶) 9 / L [500 / μL]) and platelets (at least 50 × 10) 9It is used for patients with partial recovery of both CR (50,000 / μL) and other CR criteria must be met.

[0096] Complete remission with incomplete hematopoietic recovery (CRi): The term "CR with incomplete hematopoietic recovery (CRi)" is defined as 1.0 × 10⁻⁶ 9 Residual neutropenia or 100 × 10 9 It has been adopted because it meets the CR criteria except for thrombocytopenia (less than 100,000 / μL).

[0097] Duration of response: As used herein, “Duration of response (DOR)” is defined as the time from the first recorded CR, CRh, or CRi day to the day of relapse or death.

[0098] Event-free survival (EFS): As used herein, “event-free survival” is defined as the time from the date of initial dose administration to the earliest date of treatment failure, relapse, or death.

[0099] Overall survival (OS): As used herein, "overall survival" is defined as the time from the date of first dose administration to the date of death.

[0100] Subject or patient: As used herein, “subject” or “patient” means an individual having acute myeloid leukemia, which is relapsing or refractory.

[0101] Symptoms: As used herein, “symptoms” include, but are not limited to, weight loss, fatigue, fever, night sweats, loss of appetite, weakness, chills, dizziness or lightheadedness, headache, pale skin, shortness of breath, bruising, excessive bleeding, frequent or severe nosebleeds, bleeding or clotting disorders, headache, slurred speech, balance problems, blurred vision, facial numbness, enlarged lymph nodes, bone or joint pain, and any combination thereof.

[0102] Therapeutic dose: As used herein, the term “therapeutic dose” means the amount, frequency, and duration of administration that is effective in achieving the desired outcome such that one or more symptoms or biomarkers are improved after treatment. [Modes for carrying out the invention]

[0103] This disclosure provides, in particular, a method for treating relapsed or refractory acute myeloid leukemia, wherein one or more symptoms or biomarkers are improved after treatment by administering a therapeutically effective dose of a composition (e.g., an allogeneic composition) containing Vδ1+ gamma delta (γδ) T cells to a subject in need. This disclosure also provides appropriate doses of a composition containing allogeneic Vδ1+ gamma delta (γδ) T cells for administration to subjects with a high disease burden (e.g., 5%, 10%, or 20% or more of leukemic blasts or resistant leukemic blasts), such as patients with relapsed or refractory acute myeloid leukemia.

[0104] Acute myeloid leukemia Acute myeloid leukemia (AML) is a cancer that affects adults and children and is characterized by clonally proliferating immature myeloid cells and infiltration of peripheral blood, bone marrow, and other tissues. This occurs when bone marrow stem cells produce abnormal myeloblasts that do not become healthy white blood cells, or when excess bone marrow stem cells become abnormal red blood cells or platelets. As a result, leukemic blasts, or immature cell morphologies, accumulate in the bone marrow, peripheral blood, and possibly other tissues, and the production of normal red blood cells, platelets, and mature granulocytes is reduced.

[0105] Current treatment strategies include chemotherapy, allogeneic stem cell transplantation, and novel targeted therapies in some cases. However, a significant proportion of patients exhibit relapsed or refractory disease.

[0106] Relapsed or recurrent acute myeloid leukemia (AML) refers to leukemia that returns after treatment and remission. Relapses can occur, for example, due to: chemosensitivity disorders that relapse with additional mutations after partial treatment; subclones that originate from the same founder clone as the dominant clone, are initially present at low frequency, but undergo clonal proliferation during treatment due to decreased chemotherapy sensitivity; or the emergence of new AML due to treatment toxicity in late relapses, for example, occurring more than three years after achieving the initial complete remission.

[0107] Refractory AML refers to leukemia that has not responded to treatment. Complete remission has not been achieved because chemotherapy drugs do not sufficiently kill the leukemia cells.

[0108] Patients with measurable residual disease (MRD) typically have low disease-burden leukemic blasts in the bone marrow or peripheral blood (PB), ranging from 10,000 to 100,000. Both relapsed and refractory AML are typically associated with a higher disease burden of leukemic cells and are particularly difficult to manage.

[0109] Chemotherapy is often used as a treatment for relapsed or refractory diseases. Chemotherapy uses anticancer drugs to destroy cancer cells. Chemotherapy for AML can be divided into three stages: induction, consolidation after remission, and maintenance. During induction therapy, the first treatment period after the patient's diagnosis, the goal is complete remission (CR). A patient is in CR when blood cell counts return to normal, no leukemia cells are found in bone marrow samples when examined under a microscope, and there are no longer any signs or symptoms of AML. The second stage of treatment is consolidation after remission, where various different drugs are used to destroy any remaining AML cells in small amounts that are undetectable by medical tests.

[0110] Treatment may involve repeating cycles of the same or similar drugs used in induction if complete remission lasts longer than one year. Similar or higher doses of drugs may be used. Repeated courses of cytarabine for 7 days and anthracycline for 3 days, e.g., daunorubicin, doxorubicin, idarubicin, or mitoxantrone for 7 days. Other types of chemotherapy combinations for relapsed or refractory AML include fludarabine (Fludara), cytarabine, and filgrastim (Neupogen) (FLAG); mitoxantrone (Novantron), etoposide (Vepesid), and cytarabine (MEC); high-dose cytarabine and mitoxantrone; high-dose etoposide and cyclophosphamide; and cytarabine, daunorubicin, and etoposide, clofarabine, and cytarabine (with or without filgrastim). However, many patients with relapsed or refractory AML cannot tolerate intensive chemotherapy. Lower-intensity chemotherapy regimens include, for example, azacitidine (with or without venetoclax), decitabine (with or without venetoclax), and clofarabine (with or without cytarabine). In some embodiments, fludarabine and cyclophosphamide are administered. If no further treatment is given after complete remission, AML will almost certainly relapse. If acute myeloid leukemia has spread to the CNS, chemotherapy must be delivered intrathecally. In some cases, radiotherapy is also delivered, for example, in conjunction with intrathecal chemotherapy. Radiotherapy uses high-energy beams or particles to destroy cancer cells.

[0111] Targeted therapy uses drugs that target specific molecules, such as proteins on cancer cells, to inhibit the growth and spread of cancer. For example, gilteritinib is used to treat AML with FLT3 mutations, gemtuzumab ozogamicin is used to treat AML with the CD33 marker, and enasidenib is used to treat AML with a mutated IDH2 gene. However, targeted therapy is dependent on a specific target, and cancer cells can also become resistant to targeted therapy if, for example, the target molecule itself changes and the targeted drug can no longer interact with it, or if the cancer cells grow in novel, target-independent ways.

[0112] For some patients, bone marrow / stem cell transplantation is attempted as part of post-remission therapy. Stem cell transplantation replaces stem cells in patients with relapsed or refractory AML, for example, where cytogenicity testing or molecular studies predict a poor prognosis with chemotherapy or targeted therapy alone. The goal is to destroy all cancer cells in the bone marrow, blood, and other parts of the body using high-dose chemotherapy and / or radiotherapy, and then allow healthy bone marrow to be created by the replacement hematopoietic stem cells. These cells, called hematopoietic stem cells, develop into healthy bone marrow. Hematopoietic stem cells are hematopoietic cells found in both the bloodstream and bone marrow. Stem cells in autologous transplantation originate from the patient's own body. Stem cells in allogeneic transplantation originate from someone other than the patient. Allogeneic transplantation requires a matched donor. Stem cell transplantation is performed in relapsed patients immediately after the first or second remission. Subjects who relapse after stem cell transplantation may be offered other treatments, including donor lymphocyte infusion (DLI).

[0113] Even after consolidation following remission is complete, AML still carries a risk of relapse. Low-intensity medications, such as azacitidine, decitabine, and midostaurin, can be administered continuously for several years to reduce the likelihood of disease recurrence.

[0114] While various different treatment plans are being attempted individually or in combination to improve patient outcomes, there is currently no single standard of care for patients with relapsed or refractory AML.

[0115] The probability of relapse and recurrence of AML is very high with all modes of existing treatment. Furthermore, AML is resistant to various treatments through chemorespiratory resistance. Moreover, AML is an oligoclonal disease, meaning that the dominant clone at the time of initial disease onset may not be identical to the clone associated with clinical relapse. The method of providing untransduced gamma delta T cells Vδ1+ at a specific dose of the present disclosure provides a useful treatment for a disease that is extremely difficult to treat.

[0116] In some embodiments, the Disclosure provides a method for treating relapsed or refractory acute myeloid leukemia by administering a therapeutically effective amount of a composition containing Vδ1+ gamma delta (γδ) T cells to a subject in need of treatment for acute myeloid leukemia, wherein one or more symptoms or biomarkers are improved after treatment.

[0117] In some embodiments, the subjects have previously received chemotherapy. In some embodiments, the subjects have received at least two courses of intensive induction chemotherapy.

[0118] In some embodiments, the subject has 5% leukemic blasts in the bone marrow. In some embodiments, the subject has more than 5%, more than 10%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, or more than 50% leukemic blasts in the bone marrow.

[0119] In some embodiments, the subject has 5% leukemic blasts in its peripheral blood. In some embodiments, the subject has about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% leukemic blasts in its peripheral blood.

[0120] In some embodiments, the Disclosure provides a method for treating an object requiring treatment, the object being a child. In some embodiments, the object is at least 2 years of age. In some embodiments, the object is at least 12 years of age.

[0121] In some embodiments, the Disclosure provides a method for treating an object requiring treatment, the object being an adult object. In some embodiments, the object is at least 18 years of age.

[0122] Gamma delta (γδ) T cells Gamma delta (γδ) T cells are lymphocytes that mature in the thymus. Double-negative thymocytes (CD4-CD8-) differentiate into T cells that express the γδ T cell receptor (TCR). These cells then migrate to the peripheral blood (PB) and mucosal tissues, where they function as major effectors in the response to infection and cancer, preceding the response of αβ T cell lines.

[0123] According to the Lefranc & Rabbits systemic nomenclature, four subtypes of human γδT cells, namely Vδ1, Vδ2, Vδ3, and Vδ5, are defined by their TCRδ chain (LeFranc MP et al. Cell (1986) 45:237-46). While Vδ1 and Vδ2 subtypes are the most dominant, Vδ3 cells include the major form of the Vδ1-Vδ2-subtype and are found in the liver rather than peripheral blood. Vδ5 cells are found in peripheral blood or tissues.

[0124] Vδ1+γδT cells recognize target cells and mediate antitumor activity by direct lysis of transformed cells. In one embodiment, the method of the present disclosure provides GDX012 as a novel allogeneic cell therapy enriched with Vδ1+γδT cells for the treatment of relapsed or refractory AML. The cryopreserved drug is produced from leukocyte apheresis of healthy donors after αβT cell depletion prior to proliferation. GDX012 is a viable (over 70% viability), pure (over 90% CD45), IV infusion suspension consisting mainly of Vδ1+γδT cells (over 60%) expressing various cellular markers that contribute to their function, recognition and targeting of malignant cells. Analysis of the in vivo distribution of GDX012 in therapeutic mouse xenograft models has shown that, when systemically administered, GDX012 exhibits homing to the bone marrow and is detectable for at least 28 days. This suggests that, in addition to its high cytotoxic activity against AML blasts, GDX012 also exhibits a directing mechanism to the bone marrow, where it persists and possesses the ability to provide long-term cytotoxic activity in target tissues.

[0125] Vδ1+ T cell enrichment allogeneic composition In some embodiments, the composition contains less than 5% residual Vδ2+ cells. In some embodiments, the composition contains less than 0.5% residual Vδ2+ cells. In some embodiments, the composition contains less than 0.1% residual Vδ2+ cells. In some embodiments, the composition does not contain any residual Vδ2+ T cells.

[0126] In some embodiments, the composition contains at least 90% Vδ1+ cells relative to the total number of viable cells. In some embodiments, the composition contains at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.9% Vδ1+ cells relative to the total number of viable cells. In some embodiments, the composition contains 100% Vδ1+ cells relative to the total number of viable cells.

[0127] In some other embodiments, this Vδ1+ T cell population comprises at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% of the total number of viable cells.

[0128] In some embodiments, the composition contains at least 50% Vδ1+γδT cells relative to the total number of viable cells.

[0129] In some embodiments, the composition contains at least 70% Vδ1+γδT cells relative to the total number of viable cells.

[0130] In some embodiments, the composition contains at least 90% Vδ1+γδT cells relative to the total number of viable cells.

[0131] In some embodiments, the composition contains at least 99% of Vδ1+γδT cells relative to the total number of viable cells.

[0132] In some embodiments, the Vδ1+ T cells comprise at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% of the total number of viable cells. In some embodiments, the Vδ1+ T cells comprise at least 99.1% to 99.9% of the total number of viable cells. In some embodiments, the Vδ1+ T cells comprise 100% of the total number of viable cells.

[0133] In some embodiments, the composition contains at least about 60% gamma delta T cells relative to the total number of viable cells. In some embodiments, the composition contains at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99% gamma delta T cells relative to the total number of viable cells.

[0134] In some embodiments, this allogeneic composition includes an ex vivo grown cell population enriched with Vδ1+ T cells compared to the starting unproliferated cell population.

[0135] In some embodiments, the Vδ1+ composition contains less than 0.1% αβT cells relative to the total number of viable cells. In some embodiments, the Vδ1+ composition contains less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or 0.01% αβT cells.

[0136] Method for preparing a Vδ2-Vδ1+ T cell enriched allogeneic composition In some embodiments, these Vδ1+ enriched allogeneic T cells can be obtained using methods known in the art. For example, these Vδ1+ T cells can be obtained using the methods described in WO2016 / 198480, WO2017 / 072367, or WO2018 / 202808, which are incorporated herein by reference. The methods used selectively grow Vδ1+ T cells (in particular Vδ2-TCRγδ+ T cells) in culture to a high yield suitable for clinical use in the treatment of relapsed or refractory AML. The methods are performed on a sample which may also be referred to as a “starting sample.” The methods use either an unfractionated sample or a sample enriched with TCRγδ+ T cells.

[0137] In some embodiments, Vδ1+ T cell compositions grown using exogenous growth factors have improved polyclonality compared to non-grown Vδ1+ T cells (i.e., ex vivo Vδ1+ T cells) obtained simply from peripheral blood through FACS sorting. In one embodiment, the allogeneic composition comprises Vδ1+ T cells obtained using a certain growth method, the growth method comprising culturing Vδ1+ T cells in the presence of exogenous growth factors.

[0138] In some embodiments, the sample is any sample containing gamma delta T cells or their precursors, including, but not limited to, blood, bone marrow, lymphoid tissue, epithelium, thymus, liver, spleen, cancerous tissue, lymph node tissue, infected tissue, fetal tissue, and fractions or concentrated portions thereof.

[0139] In some embodiments, these Vδ1+ T cells are obtained from a blood sample. This sample may be peripheral blood, umbilical cord blood, or fractions thereof, examples of which include buffy coat cells, leukocyte apheresis products, peripheral blood mononuclear cells (PBMCs), and low-density mononuclear cells (LDMCs). In some embodiments, the blood sample is peripheral blood or a fraction thereof. In some embodiments, the sample is human blood or a fraction thereof. Cells may be obtained from the blood sample using techniques known in the art, such as density gradient centrifugation. For example, whole blood may be overlaid on an equal volume of FICOLL-HYPAQUE and then centrifuged at 400 × g for 15-30 minutes at room temperature. The interface material will contain low-density mononuclear cells, which may be collected in culture medium, washed, and centrifuged at 200 × g for 10 minutes at room temperature. The sample may be fresh or frozen.

[0140] In some embodiments, Vδ1+ T cells are obtained from human subjects.

[0141] As described herein, the compositions and methods of this disclosure may be used with allogeneic Vδ1+ T cells, i.e., cells derived from a sample obtained from another donor. In some embodiments, the Vδ1+ T cells are obtained from a healthy donor.

[0142] In some embodiments, the sample or fraction (e.g., PBMCs) may be enriched with respect to a specific cell type and / or depleted with respect to other cell types before culturing the sample or fraction. In some embodiments, the sample is enriched with T cells. In some embodiments, the sample is enriched with TCRγδ+ T cells. For example, the sample may be depleted with TCRα+ T cells, non-TCRγδ+ T cells, and / or enriched with CD3+ cells. In some embodiments, the sample is first depleted with TCRα+ T cells and then enriched with CD3+ cells.

[0143] In some embodiments, the sample may be enriched or depleted of specific cell types using techniques known in the art. In some embodiments, cells of a particular phenotype may be depleted by culturing the sample or a fraction thereof with an antibody cocktail containing antibodies that bind to specific molecules on the cells to be depleted. Preferably, the antibodies in the cocktail are bound to magnetic microbeads that can be used to magnetically deplete or enrich these target cells if the target cells are forced to pass through a magnetic column. In some embodiments, αβT cells are depleted from the sample.

[0144] In some embodiments, the collection of Vδ1+ T cells may include physical collection of Vδ1+ T cells from a culture, isolation of Vδ1+ T cells from other lymphocytes (e.g., αβT cells, γδT cells, and / or NK cells), or isolation and / or separation of Vδ1+ T cells from stromal cells (e.g., fibroblasts). In some embodiments, Vδ1+ T cells are collected by mechanical means (e.g., pipetting). In some embodiments, Vδ1+ T cells are collected by magnetic separation and / or labeling. In some embodiments, Vδ1+ T cells are collected by flow cytometry techniques such as FACS. Thus, in certain embodiments, Vδ1+ T cells are collected by means of specifically labeling Vδ1+ T cells. It will be understood that such collection of Vδ1+ T cells may include physical removal from a culture, transfer to another culture vessel, or transfer to different or different culture conditions.

[0145] In some embodiments, upon isolation from a sample, Vδ1+ T cells are generally part of a larger lymphocyte population that includes, for example, αβ T cells, B cells, and natural killer (NK) cells. In some embodiments, 0.1% to 10% of the isolated lymphocyte population are Vδ1+ T cells, and for example, 1% to 10% are Vδ1+γδ T cells. In some embodiments, the proportion of Vδ1+ T cells is measured as a ratio to CD45+ cells (a common leukocyte antigen). In some embodiments, the isolated population is depleted of other cell types (e.g., αβ T cells). In some embodiments, this αβ T cell-depleted CD45+ cell isolated population contains at least 0.1% Vδ1+ T cells, for example, at least 0.5% Vδ1+ T cells. In most cases, a population of γδ T cells (e.g., a population of blood-derived γδ T cells) contains a large population of Vδ1 T cells.

[0146] In some embodiments, the cells in the sample may be fractionated and concentrated, and then, if desired, the cells may be cultured.

[0147] In certain embodiments, the disclosure features methods for growing Vδ1+ T cells. These methods may be performed in vitro. In some embodiments, Vδ1+ T cells are grown from a population of γδ T cells isolated from a sample described herein.

[0148] In some embodiments, these Vδ1+ T cells are obtained from a sample by a method comprising culturing the sample in a medium containing a T cell mitogen and a growth factor having interleukin-4-like activity, in the absence of a growth factor having interleukin-15-like activity.

[0149] In some embodiments, these Vδ1+ T cells are obtained from a sample by a method comprising culturing the sample in a medium containing a T cell mitogen and a growth factor having interleukin-15-like activity, in the absence of a growth factor having interleukin-4-like activity.

[0150] In some embodiments, the Vδ1+ T cells are obtained from a sample by a method comprising: (1) culturing the cells in the sample in a first culture medium containing a T cell mitogen and a growth factor having interleukin-4-like activity, in the absence of a growth factor having interleukin-15-like activity; and (2) culturing the cells obtained in step (1) in a second culture medium containing a T cell mitogen and a growth factor having interleukin-15-like activity, in the absence of a growth factor having interleukin-4-like activity.

[0151] "Absence of interleukin-15, interleukin-2, and interleukin-7" and "Absence of interleukin-4" refer not only to the complete absence of these cytokines in the culture medium, but also to the use of cytokines at concentrations so low that they cannot produce a measurable response or physiological effect in target cells, and therefore can be considered nonexistent for practical purposes. It should be apparent to those skilled in the art that cells cultured in the first culture medium must not be functionally related to stimulation by IL-2, IL-7, and IL-15 or functionally similar growth factors. Furthermore, cells in the second culture medium must not be functionally related to stimulation by IL-4 or functionally similar growth factors. In some embodiments, these cytokines must not be present in this cell medium at a final concentration exceeding 2 ng / ml (more preferably 1 ng / ml or less, more preferably 0.1 ng / ml or less). In some embodiments, these cytokines are absent.

[0152] In some embodiments, the growth factor having interleukin-15-like activity is any of interleukin-15 (IL-15), interleukin-2 (IL-2), or interleukin-7 (IL-7), preferably IL-15. As used herein, "IL-15" refers to native or recombinant IL-15 or its variants (e.g., mutants, mutant proteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptide mimes) that function as agonists of one or more IL-15 receptor (IL-15R) subunits. IL-15, like IL-2, is a known T cell growth factor and can assist in the growth of CTLL-2, an IL-2-dependent cell line.

[0153] IL-15 can also refer to IL-15 derived from various mammalian species, including, for example, humans, monkeys, cattle, pigs, horses, and mice. As used herein, a “mutant protein” or “variant” of IL-15 is a polypeptide that is substantially homologous to the sequence of natural mammalian IL-15 but has a different amino acid sequence due to amino acid deletions, insertions, or substitutions. Variants may contain conserved substitution sequences, meaning that a given amino acid residue is replaced by a residue with similar physiological and chemical properties. Examples of conserved substitutions include substituting one aliphatic residue for another, e.g., lie, Val, Leu, or Ala with each other, or substituting one polar residue for another, e.g., between Lys and Arg, Glu and Asp, or Gln and Asn. Other such conserved substitutions are well known, such as substitutions of entire regions with similar hydrophobic properties. Naturally occurring IL-15 variants are also included in this disclosure. Examples of such variants include proteins resulting from alternative mRNA splicing events or proteolytic cleavage of the IL-15 protein, in which case the IL-15 binding properties are retained. Alternative mRNA splicing can result in a shortened but biologically active IL-15 protein. Variations resulting from proteolysis include, for example, differences in the N-terminus or C-terminus at expression in different types of host cells, resulting from the proteolytic removal of one or more terminal amino acids (generally 1 to 10 amino acids) of the IL-15 protein.

[0154] As used herein, “IL-2” refers to native or recombinant IL-2 or its variants (e.g., mutants, mutant proteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptide mimes) that act as agonists to one or more IL-2 receptor (IL-2R) subunits. Such factors may support the growth of the IL-2-dependent cell line CTLL-2 (33, American Type Culture Collection (ATCC®) TIB214).

[0155] IL-2 may also refer to IL-2 derived from various mammalian species, including, for example, humans, monkeys, cattle, pigs, horses, and mice. Variants may contain conserved substitution sequences, meaning that a given amino acid residue is replaced by a residue with similar physiological and chemical properties. Examples of conserved substitutions include substituting one aliphatic residue for another, e.g., Ile, Val, Leu, or Ala with each other, or substituting one polar residue for another polar residue, e.g., substitution between Lys and Arg, Glu and Asp, or Gln and Asn. Other such conserved substitutions are well known, e.g., substitutions of entire regions with similar hydrophobic properties. Naturally occurring IL-2 variants are also included in this disclosure. Examples of such variants include proteins resulting from alternative mRNA splicing events or proteolytic cleavage of the IL-2 protein, in which case the IL-2 binding properties are preserved. Alternative splicing of mRNA can result in a cleaved but biologically active IL-2 protein. Examples of mutations resulting from proteolysis include differences in the N-terminus or C-terminus of expression in different types of host cells, caused by the proteolytic removal of one or more terminal amino acids (generally 1 to 10 amino acids) of the IL-2 protein.

[0156] As used herein, “IL-7” refers to native or recombinant IL-7 or its variants (e.g., mutants, mutant proteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptide mimes) that act as agonists to one or more IL-7 receptor (IL-7R) subunits. Mature human IL-7 arises as a 152-amino acid sequence (subtracting a signal peptide consisting of an additional 25 N-terminal amino acids).

[0157] IL-7 may also refer to IL-7 derived from various mammalian species, including, for example, humans, monkeys, cattle, pigs, horses, and mice. Variants may contain conserved substitution sequences, meaning that a given amino acid residue is replaced by a residue with similar physiological and chemical properties. Examples of conserved substitutions include substituting one aliphatic residue for another, e.g., Ile, Val, Leu, or Ala with each other, or substituting one polar residue for another, e.g., between Lys and Arg, Glu and Asp, or Gln and Asn. Other such conserved substitutions are well known, e.g., substitutions of entire regions with similar hydrophobic properties. Naturally occurring IL-7 variants are also included in this disclosure. Examples of such variants include proteins resulting from alternative mRNA splicing events or proteolytic cleavage of IL-7 proteins, in which case the IL-7 binding properties are preserved. Alternative splicing of mRNA can result in a shortened but biologically active IL-7 protein. Examples of mutations resulting from proteolysis include differences in the N-terminus or C-terminus of expression in different types of host cells, caused by the proteolytic removal of one or more terminal amino acids (generally 1 to 10 amino acids) of the IL-7 protein.

[0158] In one embodiment, the growth factor having interleukin-4-like activity is interleukin-4 (IL-4).

[0159] As used herein, “IL-4” refers to native or recombinant IL-4 or its variants (e.g., mutants, mutant proteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptide mimes) that function as agonists of one or more IL-4 receptor (IL-4R) subunits. Such factors may support the differentiation of naive helper T cells (Th0 cells) into Th2 cells. Mature human IL-4 arises as a 129-amino acid sequence (subtracting a signal peptide consisting of an additional 24 N-terminal amino acids).

[0160] IL-4 may also refer to IL-4 derived from various mammalian species, including, for example, humans, monkeys, cattle, pigs, horses, and mice. Variants may contain conserved substitution sequences, meaning that a given amino acid residue is replaced by a residue with similar physiological and chemical properties. Examples of conserved substitutions include substituting one aliphatic residue for another, e.g., Ile, Val, Leu, or Ala with each other, or substituting one polar residue for another polar residue, e.g., substitution between Lys and Arg, Glu and Asp, or Gln and Asn. Other such conserved substitutions are well known, e.g., substitutions of entire regions with similar hydrophobic properties. Naturally occurring IL-4 variants are also included in this disclosure. Examples of such variants include proteins resulting from alternative mRNA splicing events or proteolytic cleavage of the IL-4 protein, in which case the IL-4 binding properties are preserved. Alternative splicing of mRNA can result in cleaved but biologically active IL-4 proteins. Examples of mutations resulting from proteolysis include differences in the N-terminus or C-terminus of expression in different host cells, due to the proteolytic removal of one or more terminal amino acids (generally 1-10 amino acids) of the IL-4 protein. As used herein, "symptoms" include, but are not limited to, the following: Weight loss, fatigue, fever, night sweats, loss of appetite, weakness, chills, dizziness or lightheadedness, headache, pale skin, shortness of breath, bruising, excessive bleeding, frequent or severe nosebleeds, bleeding or clotting disorders, headache, slurred speech, balance problems, blurred vision, facial numbness, enlarged lymph nodes, bone or joint pain, and any combination thereof.

[0161] In some embodiments, the Vδ1+ T cells are obtained from the sample by a method comprising: (1) culturing the cells in the sample in a first culture medium containing T cell mitogen and interleukin-4 in the absence of interleukin-15, interleukin-2, and interleukin-7; and (2) culturing the cells obtained in step (1) in a second culture medium containing T cell mitogen and interleukin-15 in the absence of interleukin-4.

[0162] Methods for obtaining Vδ1+ T cells from a sample may include further growth factors. Therefore, in some embodiments, the first or second culture medium, or both, further include one or more additional growth factors. These additional growth factors may be selected from interferon-γ (IFN-γ), interleukin-21 (IL-21), interleukin-1b (IL-1b), and combinations thereof. In some embodiments, this additional growth factor is IFN-γ. These growth factors may be added to one or both culture media to further increase the levels of proliferation and purity of the cultured Vδ1+ T cells. Examples of additional growth factors include IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, human platelet lysate (HPL), and stromal cell-derived factor-1 (SDF-1). In some embodiments, such factors are used in proliferation to selectively promote the growth of Vδ1+ T cells.

[0163] T-cell mitogens mean any agents that can stimulate T cells through TCR signaling, including, but not limited to, plant lectins such as phytohemagglutinin (PHA) and concanavalin A (ConA), as well as non-plant-derived lectins, antibodies that activate T cells, and other non-lectin / non-antibody mitogens. Preferred antibody clones include anti-CD3 antibodies such as OKT-3 and UCHT-1 clones, anti-γδ antibodies such as B1 and IMMU510, or anti-Vδ1 antibodies. In the context of this disclosure, antibodies are understood to include monoclonal antibodies (mAbs), polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab')2), single-chain antibodies, single-chain variable fragments (scFv), and recombinant binding partners. In some embodiments, the antibody is an anti-CD3 monoclonal antibody (mAb). In some embodiments, the antibody is an anti-Vδ1 antibody. Other mitogens include phorbol 12-myristate-13-acetate (TPA) and related compounds, such as mezerein, or bacterial compounds (e.g., Staphylococcus enterotoxin A (SEA) and Streptococcus protein A). The T cell mitogen may be soluble or immobilized, and two or more T cell mitogens may be used in this method.

[0164] In some embodiments, the T cell mitogen is an antibody or a fragment thereof. The antibody or fragment thereof may be an anti-CD3 antibody, e.g., OKT-3. Alternatively, or further, this antibody or fragment thereof may be an anti-TCRγδ antibody, such as a pan-γδTCR antibody or an anti-TCRVδ1 antibody. References to “culture” herein include adding cells to a culture medium containing necessary and / or preferred growth factors and / or essential nutrients for a cell and / or non-hematopoietic tissue sample. Culturing may be by selective growth, such as by selecting culture conditions in which Vδ1+ T cells are preferentially grown over other cell types present in the sample. Alternatively, the growth conditions may be non-selective, and depletion of non-target cells (e.g., cells other than Vδ1+ T cells, such as αβT cells) may be carried out after culturing. Alternatively, the growth conditions may be non-selective, and depletion of non-target cells (e.g., cells other than Vδ1+ T cells, such as αβT cells) may be carried out before culturing.

[0165] In some embodiments, the culturing is carried out in the absence of supporting cells.

[0166] In some embodiments, the culturing is carried out without substantial contact with stromal cells. In some embodiments, the culturing is carried out without substantial contact with fibroblasts.

[0167] In some embodiments, these Vδ1+ T cells are harvested after a culture of at least 11 days, e.g., at least 14 days. In some embodiments, the culture period according to the method defined herein is at least 14 days. In some embodiments, the culture period according to the method defined herein is less than 45 days, e.g., less than 30 days, e.g., less than 25 days. In some embodiments, the culture period according to the method defined herein is 14 to 35 days, such as 14 to 21 days. In some embodiments, the culture period according to the method defined herein is about 21 days.

[0168] In some embodiments, the culture is carried out for a period of time (e.g., at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days, or longer, e.g., 5 to 40 days, 7 to 35 days, 14 to 28 days, or about 21 days) in an amount effective to produce a population of proliferated Vδ1+ T cells. In some embodiments, the culture period is from a few hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, or 21 hours) to about 35 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days). In some embodiments, this culture period is from 14 to 21 days.

[0169] When using two culture media, it will be understood that the culturing in each medium may take place for different lengths of time. For example, cells may be cultured in the first culture medium for a period ranging from approximately 2 to approximately 21 days. In some embodiments, cells may be cultured for approximately 3 to approximately 14 days. In some embodiments, cells may be cultured for approximately 4 to approximately 8 days. Cells may be cultured in the second culture medium for a period ranging from approximately 2 to approximately 30 days. In some embodiments, cells may be cultured for approximately 5 to approximately 21 days. In some embodiments, cells may be cultured for approximately 10 to approximately 15 days.

[0170] In some embodiments, culture is carried out in a container containing a gas-permeable material. Such a material is permeable to gases such as oxygen, carbon dioxide, and / or nitrogen, allowing gas exchange between the contents of the container and the surrounding air. References to “container” herein include culture dishes, culture plates, single-well dishes, multi-well dishes, multi-well plates, flasks, multi-wall flasks, bottles (e.g., roller bottles), bioreactors, bags, tubes, etc. Such containers are known in the art for use in methods involving the proliferation of non-adherent cells and other lymphocytes. Containers containing gas-permeable material have been found to increase the yield of isolated Vδ1+ T cells. Such containers have also been found to preferentially support Vδ1+ T cells and other lymphocytes over fibroblasts and other stromal cells (e.g., epithelial cells), including adherent cell types. In some embodiments, fibroblasts and / or other stromal cells (e.g., epithelial cells) are not present in cultures carried out in containers containing gas-permeable material.

[0171] Such a container comprising a gas-permeable material may further comprise a non-porous gas-permeable material. Therefore, in some embodiments, this gas-permeable material is non-porous. In some embodiments, this gas-permeable material is a membrane film such as silicone, fluoroethylene polypropylene, polyolefin, or ethylene vinyl acetate copolymer. Furthermore, such a container may comprise only a portion of the gas-permeable material, a gas-permeable membrane film, or a non-porous gas-permeable material. Therefore, in some embodiments, the container comprises a top, a bottom, and at least one side wall, wherein at least a portion of the bottom of the container comprises a gas-permeable material that is substantially horizontal when the top is above the bottom. In some embodiments, the container comprises a top, a bottom, and at least one side wall, wherein at least a portion of the bottom comprises a gas-permeable material that is horizontal when the top is above the bottom. In some embodiments, the container comprises a top, a bottom, and at least one side wall, the at least one side wall comprising a gas-permeable material which may be in a vertical plane if the top is above the bottom, or in a horizontal plane if the top is not above the bottom. In such embodiments, it will be understood that only the bottom or a portion of the side wall may comprise the gas-permeable material. Alternatively, the entire bottom or the entire side wall may comprise the gas-permeable material. In some embodiments, the top of the container comprising the gas-permeable material may be sealed using, for example, an O-ring. It will be understood that such embodiments prevent the contents of the container from spilling out or reduce their evaporation. In some embodiments, the container comprises a liquid-sealed container comprising a gas-permeable material that allows gas exchange. In some embodiments, the top of the container comprising the gas-permeable material is in a horizontal plane and above the bottom, and is not sealed. In some embodiments, the top is configured to allow gas exchange from the top of the container. In some embodiments, the bottom of the gas-permeable container is configured to allow gas exchange from the bottom of the container.In some embodiments, the container containing the gas-permeable material may be a liquid-sealed container and may further include inlet and outlet ports or tubes. In some embodiments, this container containing the gas-permeable material comprises a top, a bottom, and optionally at least one side wall, wherein at least portions of the top and the bottom contain the gas-permeable material, and, if present, at least portions of this at least one side wall contain the gas-permeable material. Exemplary containers are described in WO2005 / 035728 and US9255243, which are incorporated herein by reference. These containers, e.g., G-REX® cell culture devices provided by Wilson Wolf Manufacturing, e.g., G-REX 6-well plates, G-REX 24-well plates, and G-REX 10 containers, are also commercially available.

[0172] In some embodiments, the sample is cultured in a substantially serum-free medium (e.g., serum-free medium or medium containing a serum substitute (SR)). Thus, in some embodiments, the sample is cultured in serum-free medium. Such serum-free medium may also contain a serum substitute medium, which is based on chemically defined components to avoid the use of human or animal-derived serum. In some embodiments, the sample is cultured in a medium containing serum (e.g., human AB serum or fetal bovine serum (FBS)). In some embodiments, the sample is cultured in a medium containing a serum substitute. In some embodiments, the sample is cultured in a medium that does not contain animal-derived products. Culture the sample in serum-free medium has the advantage of avoiding problems related to filtration, precipitation, contamination, and serum supply. Furthermore, animal-derived products are not suitable for use in the manufacture of clinical-grade human therapeutics.

[0173] Numerous basal media suitable for use in the proliferation of γδ T cells are available in specific media such as AIM-V, Iscoves medium, and RPMI-1640 (Life Technologies). Other media factors as defined herein, such as serum, serum proteins, and selective agents, such as antibiotics, may be supplemented to the media. For example, in some embodiments, RPMI-1640 medium contains 2 mM glutamine, 10% FBS, 10 mM HEPES, pH 7.2, 1% penicillin-streptomycin, sodium pyruvate (1 mM; Life Technologies), non-essential amino acids (e.g., 100 mM Gly, Ala, Asn, Asp, Glu, Pro, and Ser; 1 × MEM non-essential amino acids (Life Technologies)), and 10 pl / L β-mercaptoethanol. In another embodiment, AIM-V medium may be supplemented with CTS immunoserum substitute and amphotericin B. Conveniently, during isolation and / or growth, cells are cultured at 37°C in a suitable culture medium in a humidified atmosphere containing 5% CO2. Examples of other components that may be added to the culture medium include, but are not limited to, plasma or serum, purified proteins such as albumin, lipid sources such as low-density lipoprotein (LDL), vitamins, amino acids, steroids, and any other supplements that support or promote cell growth and / or survival.

[0174] In some embodiments, Vδ1+ T cells obtained according to the methods described herein may be isolated from other cells that may be present in the final culture using techniques known in the art, including fluorescence-activated cell sorting, immunomagnetic separation, affinity column chromatography, density gradient centrifugation, and cell panning.

[0175] In some embodiments, the resulting Vδ1+ T cells (GDX012) may be used immediately for therapeutic, experimental, or commercial applications as described herein, or the cells may be cryopreserved for later use.

[0176] Treatment methods for relapsed or refractory AML In some aspects of this disclosure, what is provided herein is a method for treating relapsed or refractory acute myeloid leukemia by administering a therapeutically effective dose of a composition containing Vδ1+ gamma delta (γδ) T cells to a subject requiring treatment for acute myeloid leukemia, such that one or more symptoms or biomarkers are improved after treatment. The allogeneic composition may comprise a dose suitable for administration to a patient. In some embodiments, what is provided herein is a dose of an allogeneic composition containing γδ1+ T cells for use in the treatment of a patient with relapsed or refractory AML.

[0177] In some embodiments, these Vδ1+ gamma delta (γδ) T cells are not transduced. In some embodiments, Vδ1+ gamma delta (γδ) T cells do not express chimeric antigen receptors (CARs). In some embodiments, Vδ1+ gamma delta (γδ) T cells express chimeric antigen receptors (CARs).

[0178] In some embodiments, the therapeutically effective amount of Vδ1+ gamma delta (γδ) T cells is approximately 8 × 10⁻⁶. 10 , 4×10 10 , 8×10 9 , 4×10 9 , 2.4×10 9 , 1.2 × 10 9 , 8×10 8 , 4×10 8 , 8×10 7 or 4 x 10 7 Contains 1 surviving T cell.

[0179] In some embodiments, the therapeutically effective amount of Vδ1+ gamma delta (γδ) T cells is approximately 8 × 10⁻⁶. 10 Contains 10⁴ viable T cells. In some embodiments, the therapeutically effective amount of Vδ1+ gamma delta (γδ) T cells is approximately 4 × 10⁴. 10 Contains 10⁴ viable T cells. In some embodiments, the therapeutically effective amount of Vδ1+ gamma delta (γδ) T cells is approximately 8 × 10⁴. 9 Contains 10⁴ viable T cells. In some embodiments, the therapeutically effective amount of Vδ1+ gamma delta (γδ) T cells is approximately 4 × 10⁴.9 Contains 10⁴ viable T cells. In some embodiments, the therapeutically effective amount of Vδ1+ gamma delta (γδ) T cells is approximately 2.4 × 10⁴. 9 Contains 10⁴ viable T cells. In some embodiments, the therapeutically effective amount of Vδ1+ gamma delta (γδ) T cells is approximately 1.2 × 10⁴. 9 Contains 10⁴ viable T cells. In some embodiments, the therapeutically effective amount of Vδ1+ gamma delta (γδ) T cells is approximately 8 × 10⁴. 8 Contains 10⁴ viable T cells. In some embodiments, the therapeutically effective amount of Vδ1+ gamma delta (γδ) T cells is approximately 4 × 10⁴. 8 Contains 10⁴ viable T cells. In some embodiments, the therapeutically effective amount of Vδ1+ gamma delta (γδ) T cells is approximately 8 × 10⁴. 7 Contains 10⁴ viable T cells. In some embodiments, the therapeutically effective amount of Vδ1+ gamma delta (γδ) T cells is approximately 4 × 10⁴. 7 Contains 10⁴ viable T cells. In some embodiments, the therapeutically effective amount of Vδ1+ gamma delta (γδ) T cells is approximately 4 × 10⁴. 7 Contains fewer than 100 surviving T cells.

[0180] In some embodiments, this therapeutically effective amount is approximately 5 × 10 4 It contains fewer than 10 alpha-beta T cells. In some embodiments, this therapeutically effective amount is about 1 × 10⁶ 4 Contains fewer than 1 alpha-beta T cells.

[0181] In some embodiments, the composition may comprise a certain dose for administration to a patient (such as a therapeutically effective dose). In some embodiments, the patient is administered a certain dose of Vδ1+ T cells calculated per kg of the patient's body weight. In some embodiments, the certain dose of Vδ1+ T cells described herein is approximately 1 × 10⁻⁶ 5 , 5×10 5 , 1 x 10 6 , 1.5×10 6 , 2×10 6 , 3 x 10 6 , 5×10 6 , 1 x 10 7 , 1.5×107 、 2×10 7 、 3×10 7 、 5×10 7 、 1×10 8 、 2×10 8 、 or 5×10 8 cells / kg. In some embodiments, a dose of Vδ1+ T cells is at least about 1×10 5 、 5×10 5 、 1×10 6 、 1.5×10 6 、 2×10 6 、 3×10 6 、 5×10 6 、 1×10 7 、 1.5×10 7 、 2×10 7 、 3×10 7 、 5×10 7 、 1×10 s 、 2×10 s 、 or 5×10 8 cells / kg. In some embodiments, a dose of Vδ1+ T cells is at most about 1×10 6 、 1.5×10 6 、 2×10 6 、 3×10 6 、 5×10 6 、 1×10 7 、 1.5×10 7 、 2×10 7 、 3×10 7 、 5×10 7 、 1×10 s 、 2×10 s 、 or 5×10 8 cells / kg. In some embodiments, a dose of Vδ1+ T cells is about 1×10 6 ~ 1×10 8 cells / kg.

[0182] In some embodiments, the dose of the allogeneic composition is 5×10 4 abT cells / kg or less, for example, about 10 4 、 10 3 、 or 10 2 αβT cells / kg or less. Thus, in some embodiments, this dose is about 5×10 per kg4 Contains fewer than 1 abT cells. In some embodiments, this dose is approximately 1 × 10⁶ per kg. 4 Contains fewer than 1 αβT cells.

[0183] In some embodiments, the allogeneic composition is frozen before administration and then thawed. In some embodiments, the dose of the allogeneic composition is calculated before freezing. In some embodiments, the dose is calculated after thawing. In some embodiments, the allogeneic composition is not frozen. In some embodiments, the method further includes the administration of chemotherapy. In some embodiments, the patient is treated with chemotherapy at least 3 days before administration of the allogeneic composition. In some embodiments, the therapeutic agents are fludarabine and cyclophosphamide.

[0184] In some embodiments, the subject receives an initial dose of Vδ1+ T cells (e.g., 10 per kg of the subject's body weight). 6 ~10 8 For example, 10 Vδ1+ T cells per kg of body weight of the subject. 6 ~10 7 The patient receives an initial dose of Vδ1+ T cells, followed by one or more subsequent doses of Vδ1+ T cells (e.g., two, three, four, or five doses). In some embodiments, the one or more subsequent doses are administered 30 days after the previous dose. In some embodiments, the one or more subsequent doses are administered 15 days after the previous dose. In some embodiments, the one or more subsequent doses are administered less than 15 days after the previous dose, for example, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous dose, for example, less than 4, 3, or 2 days after the previous dose.

[0185] In some embodiments, one or more additional therapeutic agents are administered to the subject. The additional therapeutic agents may be selected from the group consisting of immunotherapeutic agents, cytotoxic agents, proliferation inhibitors, radiotherapeutic agents, anti-angiogenic agents, or combinations of two or more such agents. The additional therapeutic agent is administered concurrently with, before, or after the administration of proliferated Vδ1+ T cells. The additional therapeutic agent is an immunotherapeutic agent that can act on targets within the subject's body (e.g., the subject's own immune system) and / or on the transplanted Vδ1+ T cells.

[0186] In some embodiments, the composition is administered by any convenient route; for example, the composition described herein may be administered to a patient intravenously, transarterially, subcutaneously, intradermally, intratumorally, intralymphatically, intramedullarily, intramuscularly, intravenously, or intraperitoneally, for example, by intradermal or subcutaneous injection. In some embodiments, the composition is administered as an intravenous infusion.

[0187] In some embodiments, the composition is administered in one or more doses. In some embodiments, the composition is administered in a single dose. In some embodiments, the composition is administered once a week, once every two weeks, once a month, or once every two months. In some embodiments, the composition is administered once a month. In some embodiments, the composition is administered for a period of one month, two months, four months, six months, twelve months, fourteen months, eighteen months, or twenty-four months. In some embodiments, the composition is administered for a period longer than twenty-four months.

[0188] Lymphocyte apheresis

[0189] According to this disclosure, in some embodiments, the administration of Vδ1+γδT cells is performed after low-intensity lymphocyte apheresis chemotherapy.

[0190] In some embodiments, this lymphocyte apheresis therapy includes the administration of fludarabine and cytarabine.

[0191] In some embodiments, this lymphocyte apheresis therapy comprises at least one dose of cytarabine and at least one dose of fludarabine.

[0192] In some embodiments, the subject is treated with lymphocyte depletion at least once before administration of GDX012. In some embodiments, lymphocyte depletion is performed before one or more additional doses of GDX012 are administered. In some embodiments, lymphocyte depletion is followed by at least two doses of GDX012 therapy, separated by a time interval.

[0193] In some embodiments, if multiple treatment cycles are required (for example, if the subject requires further treatment), lymphocyte removal is performed before the start of each treatment cycle of GDX012. For example, in some embodiments, the subject undergoes lymphocyte removal, receives multiple doses of GDX012 cells according to the treatment plan, and, if necessary, receives a second treatment plan followed by a second lymphocyte removal.

[0194] In some embodiments, this lymphocyte apheresis chemotherapy includes the administration of mitoxantrone, etoposide, cytarabine, or anthracycline. In some embodiments, this lymphocyte apheresis chemotherapy includes a 7-day administration of cytarabine and a 3-day administration of anthracycline. In some embodiments, cytarabine is administered together with venetoclax or other B-cell lymphoma 2 (BCL2) inhibitors. In some embodiments, the lymphocyte apheresis chemotherapy includes at least two cycles of administration of a hypomethylating agent. In some embodiments, the lymphocyte apheresis chemotherapy includes at least three cycles of administration of a hypomethylating agent. In some embodiments, the lymphocyte apheresis chemotherapy includes at least four cycles of monotherapy with a hypomethylating agent.

[0195] In some embodiments, treatment by the method of the present disclosure leads to improvement of one or more symptoms or biomarkers of relapsing or refractory acute myeloid leukemia, for example, myeloblasts are substantially absent or reduced to less than 5%. In some embodiments, myeloblasts are less than 5%. In some embodiments, myeloblasts are between 5% and 25%. In some embodiments, myeloblasts are less than 10%. In some embodiments, myeloblasts are less than 15%. In some embodiments, myeloblasts are less than 20%. In some embodiments, myeloblasts are less than 25%.

[0196] In some embodiments, the treatment leads to improvement of one or more symptoms or biomarkers of relapsing or refractory acute myeloid leukemia, for example, substantially no peripheral hemoblasts. In some embodiments, peripheral hemoblasts are less than 5%. In some embodiments, peripheral hemoblasts are between 5% and 25%. In some embodiments, peripheral hemoblasts are less than 10%. In some embodiments, peripheral hemoblasts are less than 15%. In some embodiments, peripheral hemoblasts are less than 20%. In some embodiments, peripheral hemoblasts are less than 25%.

[0197] In some embodiments, the one or more symptoms or biomarkers that improve after treatment is at least a 50% reduction in the pre-treatment myeloblast rate based on the European Leukemia Net (ELN) 2022 response criteria for AML.

[0198] In some embodiments, further symptoms or biomarkers improved during treatment include the absence of extramedullary lesions. Hematological parameters are improved, for example, the absolute neutrophil count (ANC) is 0.5 × 10⁻⁶. 9 / liter or more, 1 x 10 9 / liter or more; platelet count 50 x 10 9 / liter or more, 100 x 10 9 It is more than one liter.

[0199] In some embodiments, the measurable residual lesion after treatment is less than 0.1%. In some embodiments, MRD is measured by flow cytometry of bone marrow cells and / or blood.

[0200] In some embodiments, γδ T cells express CD27. For example, Vδ1+ T cells may have a CD27+ cell frequency greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. Alternatively, γδ T cells may have a CD27+ cell frequency of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In certain embodiments, Vδ1+ T cells have a CD27+ cell frequency greater than 10%. Thus, in one embodiment, Vδ1+ T cells have a CD27+ cell frequency of about 20%. In further embodiments, Vδ1+ T cells have a CD27+ cell frequency greater than 20%. In one embodiment, Vδ1+ T cells have a CD27+ cell frequency of about 20%.

[0201] In some embodiments, these Vδ1+ T cells have a low proportion of cytotoxic cells, memory cells, and Treg cells, as well as cells expressing TIGIT, an immune checkpoint receptor on NK cells. For example, Vδ1+ T cells may have a frequency of TIGIT+ cells of less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%. Alternatively, Vδ1+ T cells may have a frequency of TIGIT+ cells of about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%. In some embodiments, these Vδ1+ T cells substantially do not express TIGIT. In some embodiments, these Vδ1+ T cells express CD27 and / or substantially do not express TIGIT.

[0202] A pharmaceutical composition may comprise the proliferated Vδ1+ T cell composition described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers, e.g., neutral buffered saline or phosphate-buffered saline; carbohydrates, e.g., glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, e.g., glycine; antioxidants; chelating agents, e.g., EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. A cryopreservation solution that may be used with the pharmaceutical compositions of this disclosure includes, for example, DMSO. The compositions may be formulated for any suitable administration, for example, intravenous administration.

[0203] In some embodiments, the pharmaceutical composition is substantially free of contaminants, such as endotoxins or mycoplasmas (e.g., not present at detectable levels).

[0204] All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. Furthermore, substances, methods, and examples are illustrative and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Similar or equivalent methods and substances may be used in the practice or testing of the present invention, but appropriate methods and substances are described herein. [Examples]

[0205] Various aspects of the present invention will be described in further detail in the following examples. The following examples illustrate some preferred ways of creating and carrying out the present invention. However, it should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0206] Example 1. Clinical study to evaluate the safety and efficacy of GDX012 in patients with relapsed or refractory acute myeloid leukemia. This example describes a Phase 1 / 2a, open-label, dose-escalation, and dose-expansion study conducted to evaluate the safety and efficacy of GDX012 in patients with relapsed or refractory acute myeloid leukemia.

[0207] This multicenter trial will enroll approximately 53 patients in two phases: dose escalation and dose expansion. In Phase 1 (sequential dose escalation), participants will be assigned to one of the following treatment groups, each consisting of approximately 3 to 6 participants, and will receive GDX012 at one of three dose levels for 4 to 6 months.

[0208] One group of participants received approximately 4 × 10⁶ cells on day 1 of phase 1 after lymphocyte apheresis chemotherapy. 8 ~Approx. 8×10 8 The GDX012 dose 1 (weight-based) of cells is administered by intravenous (IV) infusion. Some patients may be eligible for a second dose. For example, participants who achieve a response based on the European Leukemia Net (ELN) 2022 response criteria 30 days after the initial GDX012 infusion, who do not experience dose-limiting toxicity (DLT), and who are healthy, may, at their discretion, receive approximately 4 × 10⁶ doses after low-intensity lymphocyte apheresis chemotherapy. 8 ~Approx. 8×10 8 A second dose of GDX012 dose 1 of cells is administered by intravenous infusion. Exemplary lymphocyte depletion includes treatment with fludarabine and cytarabine. The chemotherapeutic agents fludarabine and cyclophosphamide may be administered according to standard treatment.

[0209] Another group of participants, after lymphocyte apheresis chemotherapy, showed approximately 1.2 × 10⁶ on day 1 of phase 1. 9 ~Approx. 2.4×10 9The GDX012 dose 2 (weight-based) of cells is administered by IV infusion. Some patients may be eligible for the second dose. For example, participants who achieve a response based on the ELN2022 response criteria on day 30 after the initial GDX012 infusion, who do not experience DLT, and who are healthy, may, at their discretion, receive approximately 1.2 × 10⁶ doses after low-intensity lymphocyte apheresis chemotherapy. 9 ~Approx. 2.4×10 9 The cells are administered a second dose of GDX012 dose 2. Exemplary lymphocyte depletion is achieved by treatment with fludarabine and cytarabine. The chemotherapeutic agents fludarabine and cyclophosphamide may be administered according to standard treatment.

[0210] The third group of participants received approximately 4 × 10⁶ cells on day 1 of phase 1, following lymphocyte apheresis chemotherapy. 9 ~Approx. 8×10 9 The GDX012 dose 3 (weight-based) of cells is administered by IV infusion. Some patients may be eligible for a second dose. For example, participants who achieve a response based on the ELN2022 response criteria on day 30 after the initial GDX012 infusion, who do not experience DLT, and who are healthy, may, at their discretion, receive approximately 4 × 10⁶ doses after low-intensity lymphocyte apheresis chemotherapy. 9 ~Approx. 8×10 9 A third dose of GDX012 dose 2 is administered to the cells. Exemplary lymphocyte depletion is achieved by treatment with fludarabine and cytarabine. The chemotherapeutic agents fludarabine and cyclophosphamide may be administered according to standard treatment.

[0211] Based on the results of the Phase 1 study, one or two dose levels will be selected for Phase 2a. The recommended Phase 2 dose (RP2D) will be determined, and evaluable participants will be enrolled in Phase 2a (dose escalation) of this study, based on whether a single dose level or a two-dose level is selected from Phase 1, for example, depending on safety, tolerability, efficacy, and cytokinetics (CK) from the dose escalation stage. Thus, participants will be enrolled in Phase 2a and will receive GDX012 as an IV infusion at the RP2D (body weight basis) on day 1 of Phase 2.

[0212] The following are the primary outcome measures that will be recorded: (a) Number of participants with dose-limiting toxicity (DLT); (b) Maximum withstand power (MTD) of GDX012; (c) Number of participants experiencing adverse events, where an adverse event (AE) may be defined as any undesirable medical event in a clinically investigated subject who has been administered the drug; where an adverse event must be causally related to the treatment. Thus, an AE may be an undesirable, unintended sign, symptom, or illness that is time-related to the use of the drug, whether or not it is considered drug-related.

[0213] As a secondary endpoint, disease response includes participants achieving the following: (a) For example, less than 5% myeloblasts; no circulating blasts; no extramedullary lesions; at least 1.0 × 10⁻⁶ 9 Absolute neutrophil count (ANC) per liter; at least 100 × 10⁻¹⁶ 9 Complete response [CR] characterized by platelet count per liter, (b) 1.0 × 10 9 Residual neutropenia of less than 100 × 10 9 Complete response [CRi] with incomplete hematopoietic recovery, characterized by all CR criteria except thrombocytopenia of less than 1 liter / liter. (c) at least 0.5 × 10 9 / liter ANC and at least 50 x 10 9 Complete response [CRh] characterized by a platelet count of 1 / liter, with partial hematopoietic recovery (all other CR criteria met). (d) Morphological leukemia-free status [MLFS] characterized by less than 5% myeloblasts; absence of blasts; absence of extramedullary lesions; and no need for hematological recovery, or (e) Partial response characterized by all hematological criteria for CR; a reduction in myeloblast percentage from 5% to 25%; and a reduction of at least 50% in the pre-treatment myeloblast percentage based on the 2022 ELN response criteria for AML [PR]. The results of this study demonstrate, for example, the safety and tolerability of GDX012 in subjects with relapsed or refractory acute myeloid leukemia. These results also indicate how well-tolerated GDX012 treatment is and determine the optimal dose of GDX012 for treatment. This study also demonstrates disease response; specifically, an additional objective of this study is to determine whether AML is reduced or absent after treatment with GDX012.

[0214] Example 2. Lymphocyte apheresis in patients with relapsed or refractory acute myeloid leukemia prior to GDX012 therapy. The subjects receive lymphocyte apheresis before treatment with GDX012. Lymphocyte apheresis is a conditioning pre-treatment performed to reduce the circulating lymphocyte population before GDX012 therapy. In this example, cytarabine and fludarabine are administered. The lymphocyte apheresis treatment includes administering one or more doses of cytarabine and one or more doses of fludarabine to the subjects before GDX012 therapy.

[0215] A drug regimen is designed to evaluate three doses of Vδ1+ cells to be administered to subjects with relapsed or refractory acute myeloid leukemia. A conditioning phase is performed prior to the drug cycle. During the conditioning phase, subjects undergo lymphocyte depletion using cytarabine and fludarabine. On day 1, following lymphocyte depletion chemotherapy as described in Example 1, subjects receive the first of three different doses (weight-based) of GDX012 by IV infusion on day 1 of phase 1: approximately 4 × 10⁶ 8 ~Approx. 8×10 8 Cell dose 1, approximately 1.2 × 10⁻⁶ 9 ~Approx. 2.4×10 9 Cell dose 2, and approximately 4 × 10 9 ~Approx. 8×10 9 Cell dose 3.

[0216] Some subjects receive a second treatment at the same dose as the first treatment after the second round of lymphocyte apheresis. For example, among healthy patients who achieve a response based on the ELN2022 response criteria on day 30 after the initial GDX012 infusion, do not experience DLT, are treated with a second round of cytarabine and fludarabine, and then receive a second dose of one of three different doses (weight-based) of GDX012 by IV infusion on day 1 of phase 1, after lymphocyte apheresis chemotherapy as described in Example 1: approximately 4 × 10⁻¹⁴ 8 ~Approx. 8×10 8 Cell dose 1, approximately 1.2 × 10⁻⁶ 9 ~Approx. 2.4×10 9 Cell dose 2, and approximately 4 × 10 9 ~Approx. 8×10 9 Cell dose 3.

[0217] The primary endpoints include: (1) the incidence, nature, and severity of treatment-related adverse events, which are any undesirable or unintended signs, including clinically significant abnormal laboratory findings, symptoms, or diseases. These are measured for 30 days from the last dose of Vδ1+ cells; and (2) the proportion of subjects who experienced dose-limiting toxicity (DLT) of Vδ1+ cells, where DLT is defined as a treatment-related adverse event occurring during cycle 1 and meeting the criteria specified in the protocol. This is measured for 28 days from the first dose of Vδ1+ cells.

[0218] Secondary endpoints include: (1) Assessment of the half-life of Vδ1+ cells, measured as the time required for a 50% reduction in the maximum volume of circulating Vδ1+ cells. This is measured for 28 days from the first administration of Vδ1+ cells; (2) Duration of Vδ1+ cells, measured by the volume of Vδ1+ cells in peripheral blood every 3 months after administration to determine persistence. This is measured up to 2 years after the last administration of Vδ1+ cells; (3) Assessment of the host immune response to Vδ1+ cells through serum samples measured for antibodies against Vδ1+ cells. This is measured up to 2 years after the last administration of Vδ1+ cells; (4) Objective response rate to Vδ1+ cells, measured by the percentage of subjects with complete or partial response. AML subjects are evaluated for the antitumor activity of Vδ1+ cells based on the updated ELN criteria.

[0219] The results of this study demonstrate that lymphocyte depletion with cytarabine and fludarabine reduces circulating lymphocytes and conditions subjects for subsequent treatment with GDX012.

[0220] The results of this study demonstrate that administration of GDX012 at one of three doses (either once or twice at each dose) after lymphocyte apheresis with cytarabine and fludarabine is well-tolerated, has limited adverse events, and has limited dose-limiting time (DLT). The results of this study demonstrate that Vδ1+ cells exhibit extended half-life and improved duration, induce a limited host immune response, and have clinically meaningful objective response rates (e.g., reduced tumor burden).

[0221] While several embodiments of the present invention are described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures to perform the functions described herein and / or to obtain the results and / or one or more advantages, and each such variation and / or modification will be considered to fall within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials and configurations described herein are illustrative, and that actual parameters, dimensions, materials and / or configurations will depend on the specific application(s) in which the teachings of the present invention are used. Those skilled in the art will recognize many equivalents of the specific embodiments of the present invention described herein or can verify them using only conventional experimental methods. Therefore, it should be understood that the embodiments described herein are presented only as examples, and that embodiments of the present invention can be carried out in ways other than those specifically described and claimed, within the scope of the appended claims and their equivalents. Embodiments of the invention in this disclosure apply to each of the individual features, systems, articles, materials, kits and / or methods described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included in the scope of the invention of this disclosure, provided that they do not conflict with each other.

Claims

1. A method for treating acute myeloid leukemia, comprising administering a therapeutically effective amount of an allogeneic composition containing Vδ1+ gamma delta (γδ) T cells to a subject requiring treatment for acute myeloid leukemia, wherein the acute myeloid leukemia is relapsed or refractory.

2. The method according to claim 1, wherein one or more symptoms or biomarkers are improved after the administration.

3. The method according to claim 1 or 2, wherein the gamma delta T cells are not transduced.

4. The method according to any one of the prior claims, wherein the composition comprises at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99% gamma delta T cells relative to the total number of viable cells.

5. The method according to any one of claims 1 to 3, wherein the composition comprises at least 50% (e.g., at least 60%) Vδ1+γδT cells relative to the total number of viable cells.

6. The method according to claim 4, wherein the composition comprises at least 70% Vδ1+γδT cells relative to the total number of viable cells.

7. The method according to claim 5, wherein the composition comprises at least 90% Vδ1+γδT cells relative to the total number of viable cells.

8. The method according to claim 7, wherein the composition comprises at least 99% of Vδ1+γδT cells relative to the total number of viable cells.

9. The method according to claim 1 or 2, wherein the composition contains less than 5% residual Vδ2+ cells.

10. The method according to claim 9, wherein the composition contains less than 0.5% residual Vδ2+ cells.

11. The method according to claim 10, wherein the composition contains less than 0.1% residual Vδ2+ cells.

12. The method according to any one of the prior claims, wherein the subject has been previously treated with chemotherapy.

13. The method according to any one of the prior claims, wherein the subject is receiving at least two courses of intensive remission induction chemotherapy.

14. The method according to any one of the prior claims, wherein the subject has 5% or more than approximately 5% leukemic blasts in the bone marrow.

15. The method according to any one of the prior claims, wherein the subject has 5% or more than approximately 5% leukemic blasts in its peripheral blood.

16. The therapeutically effective amount is about 8×10 １０ , 4×10 １０ , 8×10 ９ , 4×10 ９ , 2.4×10 ９ , 1.2×10 ９ , 8×10 ８ , 4×10 ８ , 8×10 ７ or 4×10 ７ viable T cells, according to any one of the preceding claims.

17. The therapeutically effective amount is approximately 8 × 10 ９ The method according to claim 16, comprising 1 surviving T cell.

18. The therapeutically effective amount is approximately 4 × 10 ９ The method according to claim 16, comprising 1 surviving T cell.

19. The therapeutically effective amount is approximately 2.4 × 10 ９ The method according to claim 16, comprising 1 surviving T cell.

20. The therapeutically effective amount is approximately 1.2 × 10 ９ The method according to claim 16, comprising 1 surviving T cell.

21. The therapeutically effective amount is approximately 8 × 10 ８ The method according to claim 16, comprising 1 surviving T cell.

22. The therapeutically effective amount is approximately 4 × 10 ８ The method according to claim 16, comprising 1 surviving T cell.

23. The therapeutically effective amount is approximately 4 × 10 ８ The method according to claim 16, comprising fewer than 100 surviving T cells.

24. The therapeutically effective amount is approximately 5 × 10 per kg. ４ The method according to claim 16, comprising less than 1 alpha-beta T cells.

25. The aforementioned therapeutically effective amount is approximately 1 × 10⁻⁶ per kg. ４ The method according to claim 16, comprising less than 1 alpha-beta T cells.

26. The method according to any one of the prior claims, wherein the gamma delta T cells do not express a chimeric antigen receptor (CAR).

27. The method according to any one of the prior claims, wherein the gamma delta T cells express a chimeric antigen receptor (CAR).

28. The method according to any one of the prior claims, wherein the composition is administered intravenously, transarterially, subcutaneously, intradermally, intratumorally, intralymphatically, intramuscularly, or intraperitoneally.

29. The method according to any one of the prior claims, wherein the composition is administered intravenously.

30. The method according to any one of the prior claims, wherein the composition is administered in one or more doses.

31. The method according to claim 30, wherein the composition is administered in one dose.

32. The method according to any one of the prior claims, wherein the composition is administered once a week, once every two weeks, once a month, or once every two months.

33. The method according to any one of the prior claims, wherein the composition is administered once a month.

34. The method according to any one of the prior claims, wherein the composition is administered for a period of one month, two months, four months, six months, twelve months, fourteen months, eighteen months, or twenty-four months.

35. The method according to any one of the prior claims, wherein the composition is administered for a period exceeding 24 months.

36. The method according to any one of the prior claims, wherein the chemotherapy comprises the administration of mitoxantrone, etoposide, cytarabine, or anthracycline.

37. The method according to any one of the prior claims, wherein the chemotherapy comprises a 7-day administration of cytarabine and a 3-day administration of anthracycline.

38. The method according to any one of the prior claims, wherein the cytarabine is administered together with venetoclax, fludarabine, or another B-cell lymphoma 2 (BCL2) inhibitor.

39. The method according to any one of the prior claims, wherein the chemotherapy comprises administering a hypomethylating agent for at least two cycles.

40. The method according to any one of the prior claims, wherein the chemotherapy comprises monotherapy with a hypomethylating agent for at least four cycles.

41. The method according to any one of the prior claims, wherein the subject is at least two years old.

42. The method according to any one of the prior claims, wherein the subject is at least 12 years old.

43. The method according to any one of the prior claims, wherein the subject is at least 18 years old.

44. The method according to any one of the prior claims, wherein one or more symptoms or biomarkers that are improved after treatment are substantially the absence of myeloblasts.

45. The method according to any one of the prior claims, wherein one or more symptoms or biomarkers that are improved after treatment is that the myeloblasts are less than approximately 5%.

46. The method according to any one of the prior claims, wherein one or more symptoms or biomarkers that are improved after treatment are that the percentage of myeloblasts and / or peripheral blood blasts is 5% to 25%.

47. The method according to any one of the prior claims, wherein one or more symptoms or biomarkers that are improved after treatment is that the myeloblasts are less than about 10%.

48. The method according to any one of the prior claims, wherein one or more post-treatment symptoms or biomarkers that are improved is that the myeloblasts are less than approximately 15%.

49. The method according to any one of the prior claims, wherein one or more post-treatment symptoms or biomarkers that are improved is that the myeloblasts are less than approximately 20%.

50. The method according to any one of the prior claims, wherein one or more post-treatment symptoms or biomarkers that are improved is that the myeloblasts are less than approximately 25%.

51. The method according to any one of the prior claims, wherein one or more symptoms or biomarkers that are improved after treatment are substantially absent peripheral hemoblasts.

52. The method according to any one of the prior claims, wherein one or more symptoms or biomarkers that are improved after treatment is that the peripheral hemoblasts are less than approximately 5%.

53. The method according to any one of the prior claims, wherein one or more symptoms or biomarkers that are improved after treatment is that the peripheral hemoblasts are less than about 10%.

54. The method according to any one of the prior claims, wherein one or more symptoms or biomarkers that are improved after treatment is that the peripheral hemoblasts are less than approximately 15%.

55. The method according to any one of the prior claims, wherein one or more symptoms or biomarkers that are improved after treatment is that the peripheral hemoblasts are less than approximately 20%.

56. The method according to any one of the prior claims, wherein one or more symptoms or biomarkers that are improved after treatment is that the peripheral hemoblasts are less than approximately 25%.

57. The method according to any one of the prior claims, wherein the one or more symptom or biomarker improved after treatment is at least a 50% reduction in the pre-treatment myeloblast rate based on the AML European Leukemia Net (ELN) 2022 response criteria.

58. The method according to any one of the prior claims, wherein one or more symptoms or biomarkers that are improved after treatment are the absence of extramedullary lesions.

59. One or more of the above symptoms or biomarkers that improve after treatment are 0.5 × 10 ９ The method according to any one of the prior claims, wherein the absolute neutrophil count (ANC) is 1 / liter or more.

60. One or more of the above symptoms or biomarkers that improve after treatment are 1.0 × 10 ９ The method according to any one of the prior claims, wherein the absolute neutrophil count (ANC) is 1 / liter or more.

61. One or more of the above symptoms or biomarkers that improve after treatment are 50 × 10 ９ The method according to any one of the prior claims, wherein the platelet count is / liter or more.

62. One or more of the above symptoms or biomarkers that improve after treatment are 100 × 10 ９ The method according to any one of the prior claims, wherein the platelet count is / liter or more.

63. The method according to any one of the prior claims, wherein the measurable residual disease (MRD) after treatment is less than approximately 0.1%.

64. The method according to claim 63, wherein MRD is measured by flow cytometry of bone marrow cells and / or blood.

Images