Methods of using venetoclax to enhance t cells

Abstract

Methods for treating T cells with venetoc to enhance T cell-mediated cytotoxicity and / or T cell-mediated antitumor activity are described. Enhanced T cell populations and their related methods and uses in cancer treatment are also described.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 971,534, filed February 7, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to immunotherapy for the treatment of cancer, and more specifically, to the use of venetoclax to enhance T cells for the treatment of cancer. Background Technology

[0004] Adoptive cell therapy (ACT) has significantly improved outcomes for patients with certain cancer types, such as B-cell leukemia and melanoma (1, 2). While these successes demonstrate the efficacy of ACT, similar clinical efficacy has not yet been achieved in other cancer types. For example, acute myeloid leukemia (AML) is a highly heterogeneous disease both within and between patients, and despite investigations into various ACT approaches to improve outcomes for patients with this highly lethal disease, ACT for AML has not yet achieved clinical success (3). Therefore, there remains a need for improved ACT therapies for cancer treatment.

[0005] One form of ACT utilizes a specific subset of T cells defined as CD4- and CD8-double-negative T cells (DNT). In preclinical models, unlike many other T-cell therapies, infusion of allogeneic DNT cells expanded from healthy volunteers does not induce allogeneic reactivity in normal cells and generates resistance to recipient immune rejection, supporting its potential as a "off-the-shelf" ACT (3-6). However, the anticancer activity of DNT cells is not complete (5, 6), therefore, methods to further enhance the antitumor activity of DNT cells could lead to better patient outcomes. Summary of the Invention

[0006] On the one hand, it has been determined that venetoclax enhances the efficacy of T-cell therapy by increasing T-cell-mediated cytotoxicity.

[0007] T cells were pretreated with compounds from a library of 269 drugs approved for various clinical uses, and then the compound-treated cells were used as effectors against human AML cell lines. Surprisingly, the Bc1-2 inhibitor venetoc maximally increased the cytotoxicity of T cells. Figure 1 ).

[0008] As described in the examples, venetoclax-pretreated T cells exhibited enhanced T cell-mediated cytotoxicity against AML in vitro. Furthermore, venetoclax-pretreated T cells demonstrated increased antitumor activity in xenograft models. Venetoclax enhanced T cell cytotoxicity, which other Bcl-2 family protein inhibitors did not. Compared to untreated T cells, venetoclax-pretreated T cells showed higher expression of T cell activation markers CD25 and CD69, as well as higher expression of effector molecules NKG2D and DNAM-1. Venetoclax-pretreated T cells also exhibited increased levels of reactive oxygen species (ROS) compared to untreated cells. Treatment-associated concentrations of venetoclax were also demonstrated to increase T cell effector function without decreasing T cell viability. Additionally, T cells isolated from patients receiving venetoclax showed increased ROS levels.

[0009] Therefore, in one embodiment, a method for enhancing T-cell therapeutic efficacy is provided, the method comprising contacting T cells with venetoclax to generate functionally enhanced T cells.

[0010] As described herein, pretreatment of T cells with venetoclax generates enhanced T cells that possess several characteristics that enable the cells to treat cancer more effectively. For example, in one embodiment, venetoclax is used to increase T cell-mediated cytotoxicity. In one embodiment, venetoclax is used to increase T cell-mediated antitumor activity. In one embodiment, contacting the T cells with venetoclax increases the relative proportion of T cells in an effector memory state.

[0011] In one embodiment, the T cells are conventional T cells (CD4+). + or CD8 + In one embodiment, the T cells are unconventional T cells, such as double-negative T cells (CD4+). - CD8 - ).

[0012] In one embodiment, the method includes contacting the T cells with venetoclax at a concentration of at least 50 nM. In another embodiment, the method includes contacting the T cells with venetoclax at a concentration of at least 100 nM, at least 200 nM, at least 300 nM, or at least 400 nM; alternatively, the method includes contacting the T cells with venetoclax at a concentration of about 100 nM to about 1 μM.

[0013] In one embodiment, the method includes exposing the T cells to venetoclax for at least about 30 minutes, at least about 45 minutes, or at least about 60 minutes. In one embodiment, the method includes exposing the T cells to venetoclax for at least 1 hour, at least 1.5 hours, at least 2 hours, or at least 4 hours. In one embodiment, the method includes exposing the T cells to venetoclax for at least 6 hours, at least 8 hours, or at least 12 hours, optionally from about 1 hour to about 7 days. In one embodiment, the method includes exposing the T cells to venetoclax for at least 1 hour and less than about 14 days, 10 days, 9 days, 8 days, 7 days, 6 days, or 5 days. In one embodiment, the method includes exposing the T cells to venetoclax for a period of time sufficient, relative to unexposed control cells, to increase the expression levels of one or more of CD25, CD69, NKG2D, DNAM-1, and NRF2 in the T cells. In one embodiment, the method includes exposing the T cells to venetoclax for a period of time sufficient to increase the level of reactive oxygen species (ROS) in the cells, relative to control cells not exposed to venetoclax. In one embodiment, the T cells are in vitro. In another embodiment, the T cells are in vivo or ex vivo.

[0014] The enhanced T cells described herein are easily distinguishable from T cells that have not been pretreated with venetoclax. In one embodiment, contacting the T cells with venetoclax increases the expression levels of one or more of CD25, CD69, NKG2D, DNAM-1, and NRF2. In one embodiment, contacting the T cells with venetoclax increases the levels of cellular reactive oxygen species (ROS).

[0015] This document also provides an enhanced T cell population generated using the methods described herein. In one embodiment, the enhanced T cells exhibit increased expression levels of one or more of CD25, CD69, NKG2D, DNAM-1, and NRF2 compared to unexposed control T cells. In one embodiment, the enhanced T cells exhibit increased levels of reactive oxygen species (ROS) compared to unexposed control T cells.

[0016] In one implementation, the ratio of effector memory state T cells to naive state T cells in the enhanced T cell population is increased compared to the ratio of effector memory state T cells to naive state T cells in the control T cell population that has not been exposed to venetoclax.

[0017] In one embodiment, a composition is provided comprising T cells and venetoclax. A pharmaceutical composition is also provided comprising enhanced T cells treated with venetoclax as described herein.

[0018] Also provided is the use of enhanced T cells, compositions, and / or combinations of T cells with venetoclax as described herein for treating cancer in a subject with this need. In one embodiment, a method of treating cancer in a subject with this need is provided, the method comprising administering to the subject the enhanced T cells, compositions, and / or combinations of T cells with venetoclax as described herein. In one embodiment, the cancer is leukemia, optionally, acute myeloid leukemia (AML).

[0019] Other features and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that while showing preferred embodiments of this disclosure, the detailed description and specific examples are for illustrative purposes only, as various changes and modifications made within the spirit and scope of this disclosure will become apparent to those skilled in the art through this detailed description. Attached Figure Description

[0020] One or more embodiments of this disclosure will now be described in conjunction with the accompanying drawings, in which:

[0021] Figure 1 Drug screening assays identified venetoclax as the drug of choice for enhancing T-cell cytotoxicity against AML. The schematic diagram of the drug screening assay was performed to identify clinically approved drugs that could be used in combination with DNT cells to produce synergistic antitumor activity. DNT cells were treated overnight at 400 nM with 269 different clinically approved drugs. Subsequently, the compound-treated cells were washed and then co-cultured with AML cells for two hours. A dot plot illustrates the change in the degree of DNT cell-mediated cytotoxicity against AML cells relative to untreated DNT cells.

[0022] Figure 2 Venetoclax enhances T-cell-mediated cytotoxicity against AML in vitro. (A) To confirm the results of drug screening, in vitro killing assays were performed against AML cell lines OCI-AML2, OCI-AML3, and KG1a using untreated DNT cells or DNT cells pretreated overnight with different concentrations of Ven (50 nM, 100 nM, 200 nM, 400 nM). Data represent four biological replicates. (B) In vitro cytotoxicity assays were performed using DNT cells pretreated with 400 nM Ven as effectors against primary AML patient samples (n = 17). (C) To determine the activity of Ven-treated or untreated DNT cells against leukemia initiating cells, a methylcellulose-based colony formation assay was performed at 10 mg / mL. 3Cells were seeded with untreated AML or AML treated with untreated DNT cells or Ven-treated DNT cells, and the number of colonies formed was determined after 10 days. This experiment used OCI-AML2 and KG1a, as well as patient samples 140372, 100857, 110162, and 141065. (D) The increased effector activity against the three AML cell lines OCI-AML2, OCI-AML3, and KG1a after venetoclax treatment remained for at least 4 days after drug removal from DNT cells. This experiment used DNT cells from two different donors (UPN119 and UPN38). (E) Correlation between AML sensitivity to DNT cells and the degree of increase in DNT cell-mediated cytotoxicity after Ven treatment. (F) DNT cells expanded from 11 donors, either untreated or treated with 400 nM venetoclax for 18 hours. Subsequently, the cells were cultured with OCI-AML2 at DNT:AML ratios of 1:1, 2:1, or 4:1, and the viability of the AML cells was determined by annexin V staining and flow cytometry. Each pair symbol represents a DNT from a single donor.

[0023] Figure 3 In xenograft models, pretreatment of DNT cells with Ven enhances their antitumor activity. To determine whether Ven-pretreated DNT cells induce higher antileukemia activity in xenograft models, tumors reaching 100 mm in size were pretreated. 3 (As indicated by the arrow) 2x10 subcutaneous grafts were then performed. 6 NOD / SCID mice with OCI-AML2 cells were administered PBS (●) and 2x10 cells intravenously. 7 Untreated DNT cells (■) or 2x10 7 Ven-treated DNT cells (▲). Tumor volume was monitored until the PBS-treated group reached the humanitarian endpoint (A), and tumor weight was measured on day 20 post-leukemia inoculation (B). The results shown represent three independent experiments using DNT cells from three different donors. (C) NSG mice that received systemic infusion of KG1a were treated with PBS, DNT cells, or VenDNT cells. Bone marrow transplantation of KG1a was compared between the groups. Mice treated with VenDNT showed significantly lower levels of KG1a transplantation compared to the PBS and DNT cell-treated groups, further supporting the superior anti-leukemic activity of VenDNT cells even against those resistant in other ways. (D) Primary AML cells (ID: 130607), treated with untreated or DNT-treated or Ven-treated DNT at a 2:1 DNT:AML ratio for 2 hours, were injected intrafemorally into NOD / SCID mice (1.6 x 10⁻⁶ cells per mouse). 61,000 cells; n = 6 per group). Six weeks after injection, the number of AML transplanted (human CD45) cells in the bone marrow of each group was measured by flow cytometry. + CD33 + Percentage of cells. (E) Primary AML cells (n=4; 2–5x10⁻⁵) were injected intravenously into NSG mice subjected to sublethal radiation. 6 / mice). Two weeks later, the mice were administered three infusions (mediator control or 1.5–2 x 10⁻⁶ per infusion). 7 Mice were treated with DNT cells (or Ven-treated DNT cells) at 3-4 day intervals. Five weeks after AML injection, primary AML cells (human CD45, low CD33) were measured by flow cytometry. + Bone marrow transplantation status with or without CD34 expression. (Left) Representative contour plots of BM cells stained with CD45 and CD33 in each group. (Right) Summary results of patient-derived xenograft experiments using samples from four different primary AML patients. Horizontal bars represent the mean BM AML transplantation level normalized to the mediator control group, each symbol represents an individual mouse, and error bars represent SD. Data represent the mean ± SEM decrease in bone marrow leukemia levels relative to the PBS group. Statistical analysis was performed using Student's t-test or one-way ANOVA. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0024] Figure 4 (A). Venetok Enhanced CD4 + or CD8 + Anti-leukemic activity of conventional T cells. In vitro expanded T cells, either untreated or treated with different concentrations of Ven (25 nM, 50 nM, 100 nM, 200 nM, or 400 nM). conv Cells were used as effector cells against AML cell lines (OCI-AML2, OCI-AML3, and KG1a). The results shown represent four biological replicates. Figure 4 (B) Venetoclax rapidly and directly increases T cell cytotoxicity against AML. DNT (top image) and T cells treated with venetoclax (100 nM and 400 nM) for 4 h, 18 h, and 3 days, respectively, are shown. conv Cells (see image below). Subsequently, their cytotoxicity against OCI-AML2 was determined. Data represent the mean ± SEM values ​​from four different donor T cells. Figure 4 (C) DNT and T without treatment or treated with venetotke (100 nM or 400 nM) for 4 hours conv Cells. Their viability was then measured. Data are presented as mean ± SEM values ​​from results obtained from four different donor T cells.

[0025] Figure 5 Venetoclax enhanced the anti-leukemic activity of DNT cells, while obacolax and ABT-737 did not. (A) DNT cells were pretreated overnight with different concentrations of obacolax, ABT-737, or venetoclax, and then used as effector cells against OCI-AML2. (B) Results show the rate of change in DNT-mediated cytotoxicity compared to the killing effect induced by untreated DNTs. (C) Expression of Bcl-xL and Bcl-2 in DNT cells expanded in vitro from three donors (UPN38, UPN108, and UPN134) and in AML cell lines OCI-AML2, TEX, NB4, and K562 was determined by Western blotting. Tubulin was used as a loading control.

[0026] Figure 6 Ven increases the expression of activation markers and effector molecules on DNT cells. Ex vivo expanded DNT cells were stained for the expression of T cell activation markers CD25 and CD69 (A) and effector molecules (NKG2D and DNAM-1) before and after Ven treatment. Each pair of dots represents DNT cells from the same donor before and after Ven treatment. This experiment was performed using DNT cells from four (A) or six (B) different donors. (C) Granzyme B expression in DNT cells treated with different concentrations of Ven. The results shown represent two biological replicates. (D) A dose-dependent increase in the expression of CD25, NKG2D, and DNAM-1 was also observed in Ven-treated CD8+ T cells.

[0027] Figure 7 Ven increases cellular ROS levels in DNT cells and enhances their cytotoxic activity. (A) via CellROX TM DNT cells (left) or CD8 cells treated with different concentrations of Ven as detected by staining. +(B) (Left) Relative expression of the transcription factor Nrf2, regulated by cellular ROS levels, as determined by qPCR. (Right) Immunoblot assay of Nrf2 protein in the cytoplasm and nucleus of DNTs treated with or without 400 nM Ven to determine the location of the Nrf2 protein. DNT generation data from three different donors (UPN38, UPN108, and UPN134) were used. (C) To determine the functional relevance of increased ROS levels in Ven-treated DNTs, ROS levels in DNTs treated with 400 nM Ven were measured in the presence of different concentrations of the ROS scavenger N-acetylcysteine ​​(NAC), and these cells were used as effector cells against AML in an in vitro killing assay. The results shown represent three independent experiments. (D) To determine the source of ROS generation in Ven-treated DNTs, non-denaturing gel and immunoblotting assays were performed on untreated DNTs or DNTs treated with 400 nM to detect components of the electron transport chain supercomplex subunits (NDUFA9, UQCRC2, and MTCO1). The results shown represent three independent experiments using DNTs derived from two different donors. (e and f) For DNT cells (E) and CD8 + T conv (F) Ven increased the proportion of cells in the effector memory phase while decreasing the frequency of central memory T cells. (G) Ven had no significant effect on glycolysis, glycolytic capacity, and basal oxygen consumption of DNT cells. (HK) DNT (H and I) or T conv Cells (J and K) were treated with 0 nM, 100 nM, or 400 nM venetoclax for 4 h, 18 h, and 2 days, respectively. Cells were stained with CellROX (H and J) or MitoSOX (I and K). The mean fractional filtration rate (MFI) of cellular or mitochondrial (mt) ROS was measured by flow cytometry. Data represent the mean ± SEM of results from four different donor T cells. (L) DNTs were treated with 400 nM venetoclax with or without 2 mM NAC for 18 h. Flow cytometry histograms show cellular ROS levels as measured by flow cytometry. The MFI of CD25 and CD69 was measured by flow cytometry. Experiments were repeated three times, and the data shown represent two independent experiments using DNTs from two different donors. (M) DNT cells were treated with 400 nM venetoclax for 18 h. After processing, mitochondria were isolated, and the levels of respiratory chain complex subunits were measured by SDS-PAGE gel electrophoresis and by immunoblotting using antibodies against NDUFB8 (complex I), SDHA (complex II), UQCRC2 (complex III), and MTCO1 (complex IV).

[0028] Figure 8In patients receiving Ven+Aza treatment, the proportion of T cell subsets associated with cytotoxic activity was increased. Peripheral blood samples were collected from patients before Ven+Aza treatment and on day 4 of treatment, and the frequency of different T cell subsets, expression of effector molecules, and cellular ROS levels were determined by flow cytometry. (A) Comparison of CD8 levels between samples obtained before and after Ven+Aza treatment. + The frequency of DNT cells and (BE) CD8. + Effector memory T cell subsets (CD45RA) in T (b and c) and DNT (D and E) cell populations - CD62L - The frequency of ), expression level of NKG2D, and cellular ROS levels were analyzed. The chart shown is a summary of the results from samples from four patients.

[0029] Figure 9 It can be seen that the killing effect of Ven-treated and untreated DNT cells on autologous and allogeneic PBMCs was not significant.

[0030] Figure 10 Venetoclax enhanced its cytotoxicity against AML without killing DNTs. (A) Viability of DNTs and OCI-AML2 cells treated with 400 nM venetoclax for 18 hours was determined by annexin V staining and flow cytometry. (B and C) DNTs were treated with escalating concentrations of venetoclax for 18 hours. Subsequently, viability (B) and cytotoxicity (C) against OCI-AML2 and two primary AML cell lines (090765 and 110162) were determined. Statistical analysis was performed using ANOVA. ****p<0.0001.

[0031] Figure 11 For both diagnostic and relapsed / refractory AML samples, venetoclax exhibited similar effects on DNT-mediated cytotoxicity. DNTs treated with 400 nM venetoclax or untreated samples were co-cultured with diagnostic (n=12) or relapsed / refractory (n=4) primary AML samples at a 2:1 ratio for 2 hours. An increase in DNT-mediated cytotoxicity following venetoclax treatment was determined for each patient sample type.

[0032] Figure 12 DNT induces superior antileukemic activity in the presence of venetoclax. (A) KG1a and OCI-AML2 cells untreated or treated with venetoclax (100 nM) in the presence or absence of DNT. (B) Percentage reduction in AML count induced by DNT in the presence or absence of venetoclax (100 nM).

[0033] Figure 13Ven-treated DNTs induced a greater reduction in total AML count without increasing T-cell transplantation in the bone marrow. Intravenous injection of KG1a cells (2 x 10⁻⁶) into NSG mice subjected to sublethal radiation (250 cGy) 6 (cells / mouse) or primary AML cells. Two weeks later, via three infusions (mediator control (PBS) or 1.5-2x10 cells per infusion). 7 Mice were treated with DNT cells (or Ven-treated DNT cells) at 3-4 day intervals. Five weeks after injection of AML, the AML cell count (A) and T cell frequency in the bone marrow were determined by staining bone marrow cells with anti-human CD45, CD3, CD33, and CD34 antibodies and flow cytometry analysis.

[0034] Figure 14 Untreated DNT and venetoclax-treated DNT did not cause tissue damage. Intravenous injection of KG1a cells (2 x 10⁻⁶ cells) into NSG mice subjected to sublethal radiation (250 cGy) resulted in tissue damage. 6 Two weeks later, the mice were administered the vaccine via three infusions (mediator control (PBS) or 1.5–2 x 10⁻⁶ cells / mouse). 7 Mice were treated with DNT cells (either DNT cells or Ven-treated DNT cells) at 3-4 day intervals. On day 35, liver (top) and lung (bottom) tissues were stained with hematoxylin and eosin (H&E) (50x magnification). PV - portal vein; ALV - alveoli; BR - bronchioles.

[0035] Figure 15 Effects of other known ROS-inducing agents on DNT viability, ROS levels, and anti-AML cytotoxicity. DNTs were treated for 18 hours with escalating concentrations of cytarabine (0–3 μM), antimycin (0–250 nM), or daunorubicin (0–10 μM). Subsequently, cellular ROS levels in DNTs (A), DNT viability (B), and anti-OCI-AML2 cytotoxicity (C) were measured.

[0036] Figure 16 Venetoclax does not affect the expression of electron transport chain (ETC) complex subunits. Relative protein levels were normalized to the loading control MnSOD and expressed relative to the control (typically set to 1.0). Representative immunoblotting is shown. Data are presented as mean ± SD of three independent experiments. Detailed Implementation

[0037] Pretreatment of T cells with venetoclax has been shown to increase T cell-mediated cytotoxicity and antitumor activity both in vitro and in vivo. Therefore, T cells in contact with venetoclax and related compositions, as well as combinations of T cells and venetoclax, are expected to be used to treat cancer patients.

[0038] I. Methods to enhance T cells and T cell populations

[0039] In one embodiment, a method for enhancing T-cell therapeutic efficacy is provided, the method comprising contacting T cells with venetoclax to generate enhanced T cells.

[0040] As used herein, the term "venetoclax" or "Ven" refers to a molecule capable of binding to and inhibiting Bcl-2. In one embodiment, venetoclax is the drug Venclexta TM Or the drug Venclyxto TM .

[0041] In one embodiment, the method further includes contacting cancer cells with azacitidine, or administering or using azacitidine in combination with the enhanced T cells described herein. The terms “azacitidine”, “azacitidine”, or “5-azacitidine” as used herein refer to a compound that is a pyrimidine nucleoside analog of cytidine with antitumor activity. Correct chemical names for azacitidine include 4-amino-1-β-D-ribofuranosyl-1,3,5-triazin-2(1H)-one or 4-amino-1-[3,4-dihydroxy-5-(hydroxymethyl)oxalazine-2-yl]-1,3,5-triazin-2-one.

[0042] As used herein, the term "T cell" includes thymocytes, immature T lymphocytes, mature T lymphocytes, quiescent T lymphocytes, or activated T lymphocytes. T cells can be helper T cells (Th), such as helper T cell 1 (Th1) or helper T cell 2 (Th2) cells. T cells can be obtained by those skilled in the art. T cells can be conventional T cells (T... conv The T cells may be non-standard T cells, such as double-negative T cells (DNT), γ-δ T cells, or NKT cells. In one embodiment, the T cells are activated T cells. In another embodiment, the T cells are cells that have been expanded and / or activated in vitro or in vitro.

[0043] T cells can be readily obtained and / or isolated from biological sources such as blood samples or cell cultures. For therapeutic applications, T cells can be autologous T cells or allogeneic T cells. In one embodiment, the T cells are autologous T cells obtained from a subject (e.g., a subject with cancer or suspected of having cancer). In another embodiment, the T cells are allogeneic T cells, such as T cells obtained from one or more subjects without cancer. In one embodiment, the T cells are obtained from one or more healthy donors.

[0044] DNTs can be obtained by enrichment using a mixture of CD4 and CD8 depleted antibodies. In one embodiment, the DNT does not express CD4 and CD8. In one embodiment, the DNT has the phenotype CD3+, γδ-TCR+ or αβ-TcR+, CD4-, CD8-, α-Ga1-, CTLA4-. In one embodiment, the DNT has the phenotype CD3+, γδ-TCR+ or αβ-TcR+. In one embodiment, the DNT can be obtained from a sample containing peripheral blood mononuclear cells (PBMCs). In one embodiment, the sample is a blood sample. In one embodiment, the sample is an apheresis sample or an enriched leukocyte apheresis product, such as leukopak. In one embodiment, the sample is a bone marrow sample.

[0045] In one embodiment, the T cells are expanded in vitro or ex vivo prior to contact with venetoclax. Exemplary methods for isolating and expanding DNTs are described in U.S. Patent No. 6,953,576, “Methods for Modulating Tumor Immunity,” PCT Publication No. WO2007 / 056854, “Methods for Expanding Double-Negative T Cells,” and PCT Publication No. WO2016 / 023134, “Immunotherapy for the Treatment of Cancer,” the entire contents of which are incorporated herein by reference.

[0046] As used herein, the term "enhanced T cells" refers to a single T cell or population of T cells that exhibits increased cytotoxicity and / or antitumor activity after exposure to venetoclax compared to control T cells that have not been exposed to venetoclax. Optionally, enhanced T cells may be DNT or conventional T cells (T cells). conv In one embodiment, enhanced T cells can be distinguished from other T cells and / or control T cells based on physiological activity and / or gene expression. For example, in one embodiment, compared with unexposed venetoclax control T cells, enhanced T cells show increased expression levels of one or more of CD25, CD69, NKG2D, DNAM-1, and NRF2. In one embodiment, compared with unexposed venetoclax control T cells, enhanced T cells show increased expression levels of 2, 3, 4, or 5 genes selected from CD25, CD69, NKG2D, DNAM-1, and NRF2.

[0047] As used herein, the term "contacted" or "contacting" refers to any method of exposing T cells to venetoclax to generate enhanced T cells. "Contact" includes both "incubation" and "exposure," and does not imply any specific time or temperature requirements unless otherwise stated. In one embodiment, T cells are contacted with venetoclax in vitro, for example, by combining venetoclax with a culture medium and exposing or incubating the T cells in the medium. T cells can be "contacted" with venetoclax in vitro by in vitro incubation or by administration to a subject or in combination with other administrations.

[0048] In one embodiment, T cells are contacted in vitro, ex vivo, or in vivo with venetoclax at concentrations of at least 25 nM, 50 nM, or 100 nM. In one embodiment, T cells are contacted with venetoclax at concentrations of at least 100 nM, at least 200 nM, at least 300 nM, or at least 400 nM. In one embodiment, T cells are contacted with venetoclax at concentrations of about 10 nM to 10 μM, optionally about 50 nM to 500 nM, about 50 nM to 800 nM, or about 100 nM to about 1 μM.

[0049] In another embodiment, T cells are exposed to venetoclax for at least about 30 minutes, 45 minutes, 60 minutes, or 90 minutes. In one embodiment, T cells are exposed to venetoclax for at least about 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 48 hours. In one embodiment, T cells are exposed to venetoclax for about 1 hour to 14 days, optionally 2 hours to 30 days, about 4 hours to 14 days, about 4 hours to 6 days, about 4 hours to 48 hours, or about 6 hours to about 24 hours. In one embodiment, T cells are exposed to venetoclax for less than about 14 days, 10 days, 9 days, 8 days, 7 days, 6 days, or 5 days.

[0050] In one embodiment, T cells are exposed to a sufficient concentration of venetoclax for a sufficient duration to increase the expression of one or more of CD25, CD69, NKG2D, DNAM-1, and NRF2. In another embodiment, T cells are exposed to a sufficient concentration of venetoclax for a sufficient duration to increase cellular ROS levels.

[0051] In some implementations, T cells are contacted with venetoclax to become enhanced T cells, some or all of the venetoclax may be removed, or enhanced T cells may be isolated to reduce the concentration or decrease extracellular venetoclax.

[0052] As described herein, contacting T cells with venetoclax generates an enhanced T cell population that possesses several characteristics that make them particularly suitable for treating cancer. For example, in one embodiment, venetoclax increases T cell-mediated antitumor activity. In another embodiment, venetoclax increases T cell-mediated cytotoxicity.

[0053] As used herein, the term "antitumor activity" refers to any activity that kills tumor cells and / or inhibits tumor growth. In one embodiment, "antitumor activity" includes reducing tumor cell colony formation.

[0054] The term “cytotoxicity” as used in this article refers to the property of cells that causes cell death, makes cells cytotoxic, and / or prevents cell proliferation.

[0055] II. Products, Compositions, and Kits

[0056] On the other hand, enhanced T cell populations generated according to the methods described herein are provided. Compositions comprising enhanced T cells as described herein are also provided. For example, in one embodiment, the enhanced T cells are optionally included in the pharmaceutical composition along with a pharmaceutically acceptable carrier.

[0057] In another embodiment, a composition is provided comprising T cells and Venetotok. In one embodiment, the composition further comprises a cell culture medium.

[0058] A kit is also provided comprising T cells and venetoclax. In one embodiment, the kit further includes instructions for use to perform the methods described herein, such as for generating enhanced T cells, for treating cancer, or for slowing tumor growth or proliferation. In one embodiment, the T cells and venetoclax are in separate containers. In another embodiment, the T cells and venetoclax are in the same container, optionally as a composition containing a pharmaceutically acceptable carrier.

[0059] The use of the products, compositions or kits described herein for the treatment of cancer or for the preparation of medicaments for the treatment of cancer is also provided.

[0060] III. Methods and applications for treating cancer and slowing tumor growth and proliferation

[0061] Compared to untreated T cells, enhanced T cells generated by the method described herein exhibited increased cytotoxicity against AML cells in vitro. As shown in Example 2, AML cells treated with enhanced T cells showed more specific killing of AML cells and less colony formation compared to AML cells treated with control T cells. Furthermore, Example 3 demonstrates that enhanced T cells possess stronger antitumor activity in a tumor xenograft model.

[0062] Therefore, in one embodiment, a method for treating cancer in a subject with this need is provided. In one embodiment, the method includes administering an effective amount of enhancing T cells to the subject. In one embodiment, as described herein, enhancing T cells are generated by contacting T cells with venetoclax. In one embodiment, the method includes administering T cells and venetoclax to the subject, the T cells and venetoclax optionally combined with a pharmaceutically acceptable carrier to form a composition, wherein the T cells are enhanced by contact with venetoclax in vivo.

[0063] A method for slowing tumor growth and / or proliferation is also provided. In one embodiment, the method includes contacting the tumor with an effective amount of enhanced T cells. In one embodiment, as described herein, enhanced T cells are generated by contacting T cells with venetoc.

[0064] Also provided is the use of enhanced T cells, compositions, and / or kits as described herein for treating cancer in subjects with this need. In one embodiment, enhanced T cells are generated according to the methods described herein. In one embodiment, enhanced T cells, compositions, and / or kits are used to manufacture a medicament for treating cancer. In one embodiment, use includes administering or applying enhanced T cells to a subject. In another embodiment, the use includes administering or applying venetoclax and T cells to a subject at the same or different times.

[0065] It also provides uses for slowing tumor growth and proliferation. In one embodiment, the enhanced T cells, compositions, and / or kits described herein are used to slow tumor growth and proliferation. In one embodiment, the enhanced T cells, compositions, and / or kits are used to manufacture a drug for slowing tumor growth and proliferation. In one embodiment, the enhanced T cells and / or compositions are used to manufacture a drug for slowing tumor growth and proliferation. In one embodiment, T cells and venetoc are used to manufacture a drug for slowing tumor growth and proliferation.

[0066] As used herein, the term "cancer" refers to one of a range of diseases caused by uncontrolled, abnormally growing cells that can spread to adjacent tissues or other parts of the body. In one implementation, cancer is leukemia, such as acute myeloid leukemia (AML).

[0067] The term "cancer cell" refers to a cell or a cell derived from such a cell, characterized by its uncontrolled, abnormal growth and ability to invade another tissue. For example, cancer cells include primary cancer cells obtained from a cancer patient or cell lines derived from such cells. In one embodiment, cancer cells are leukemia cells, such as AML cells.

[0068] As used in this article, "leukemia" refers to any disease involving a progressive increase in the number of abnormal white blood cells normally found in the blood, in hematopoietic tissues or other organs. "Leukemia cells" refers to abnormally proliferating white blood cells characterized by an increase in cell count. Leukemia cells can be obtained from subjects diagnosed with leukemia.

[0069] The term "acute myeloid leukemia" or "acute myelogenous leukemia" ("AML") refers to a myeloid blood cell cancer characterized by the accumulation of rapidly growing, abnormal white blood cells in the bone marrow, interfering with the production of normal blood cells. Pre-leukemic conditions such as myelodysplastic syndromes or myelodysplastic syndromes can also develop into AML.

[0070] The term "tumor" refers to a collection of cancer cells. In one implementation, a tumor is a leukemic tumor, such as AML cells. In another implementation, a tumor is a hematologic malignancy.

[0071] As used herein, the term "subject" includes all members of the animal kingdom, including mammals, and where appropriate, humans. Optionally, the term "subject" includes mammals that have been diagnosed with cancer or are in remission. In one implementation, the subject has received treatment or is currently receiving chemotherapy (optionally using cytarabine and / or azacytidine).

[0072] In one implementation, the methods and uses described herein involve administering or using an effective amount of enhanced T cells, or an effective amount of T cells and venetok.

[0073] As used herein, the phrase "effective amount" or "therapeutic effective amount" refers to an amount effective for the dose and time period required to achieve the desired effect. For example, in the case of treating cancer, an effective amount, compared to an untreated response, is, for example, an amount that induces remission, reduces tumor burden, and / or prevents tumor spread or cancer cell growth. In one embodiment, an effective amount of venetoclax refers to an amount that increases T cell-mediated antitumor activity and / or increases T cell-mediated cytotoxicity. In one embodiment, an effective amount of enhancing T cells refers to an amount that has sufficient cytotoxicity against cancer and / or tumor cells, either in vitro or in vivo.

[0074] Effective doses can vary depending on factors such as the animal's disease state, age, sex, and weight. The amount of a given dose corresponding to such a dose can vary depending on various factors, such as the drug formulation, route of administration, type of disease or ailment, and the identity of the subject or host receiving treatment, but can generally still be determined by those skilled in the art. In one embodiment, enhanced T cells or T cells and venetoclax are administered to the subject by injection. In one embodiment, the injection is intravenous. In one embodiment, the injection is subcutaneous, optionally at the tumor site.

[0075] In one embodiment, enhanced T cells or a combination of T cells and venetoclax can be used to slow the growth or proliferation of cancer cells in vitro, ex vivo, or in vivo. As used herein, "slowing the growth or proliferation of cancer cells" means reducing the number of cells derived from cancer cells due to cell growth or cell division, including cell death. The term "cell death" as used herein includes all forms of cell killing, including cell lysis, necrosis, and / or apoptosis. In one embodiment, enhanced T cells or a combination of T cells and venetoclax can be used to kill cancer cells in vitro, ex vivo, or in vivo.

[0076] In one implementation, enhanced T cells or T cells and / or venetoclax may be formulated using pharmaceutically acceptable formulations known in the art for use in or after preparation in subjects. For example, routine procedures and ingredients for selecting and preparing suitable formulations are described in Remington Pharmaceuticals (20th edition, 2003) and the United States Pharmacopeia: National Formulary (USP 24NF19), published in 1999. The term "pharmaceuticalally acceptable" means compatible with treatment in animals, and particularly humans.

[0077] In one embodiment, T cells and venetoclax are administered to the subject simultaneously, optionally as a composition comprising T cells and venetoclax, or as two separate doses. In one embodiment, T cells and venetoclax are used or administered to the subject at different times. For example, in one embodiment, T cells are used or administered before or after venetoclax administration. In one embodiment, T cells are used or administered before or after venetoclax administration at intervals less than about 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 8 hours, 10 hours, 12 hours, 16 hours, or 24 hours. In one embodiment, T cells are used or administered before or after venetoclax administration at intervals less than about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

[0078] In one embodiment, venetoclax is used or administered to achieve a concentration of at least 25 nM, 50 nM, or 100 nM in the subject. In another embodiment, venetoclax is used or administered to achieve a concentration of at least 100 nM, at least 200 nM, at least 300 nM, or at least 400 nM in the subject. In one embodiment, the concentration of venetoclax is optionally established at least 25 nM, 50 nM, 200 nM, 300 nM, or 400 nM concurrently with the administration or use of exogenous T cells or DNT.

[0079] In one embodiment, venetoclax is used or administered at a daily dose of 50 mg to 800 mg, optionally 100 mg to 600 mg. For example, in one embodiment, venetoclax is used or administered to a subject in combination with T cells, thereby enhancing T cells in vivo by venetoclax.

[0080] The following non-limiting embodiments are used to illustrate this disclosure.

[0081] Example 1: Venetoclax enhances the potency of T cell-mediated cytotoxicity

[0082] To identify molecules that enhance T-cell-mediated cytotoxicity against AML, in vitro expanded DNT cells were used as a substitute for anti-leukemia T cells and pretreated with a library of 269 compounds approved for various clinical uses. The compound-treated cells were then used as effectors for the anti-human AML cell line OCI-AML2. The Bcl-2 inhibitor (Ven) maximally increased the cytotoxicity of DNT cells. Figure 1 ).

[0083] Ven is primarily used to treat chronic lymphocytic leukemia (CLL) and small lymphocytic leukemia, where Ven inhibits the activity of the anti-apoptotic molecule Bcl-2, promoting apoptosis in malignant tumor cells. In relapsed / refractory CLL patients, the overall response rate of Ven as monotherapy is 64.8–79.4% (9). Recently, Ven has been approved by the FDA for use in combination with the demethylating agents azacitidine or decitabine for the treatment of AML patients, as these drugs significantly improve treatment outcomes in patients who are unsuitable for other conventional therapies. The prognosis of AML patients (18, 20), although the underlying mechanism is unclear. Furthermore, Ven has not previously been reported to have immunostimulatory activity.

[0084] Example 2: Ven pretreatment increases the cytotoxicity of DNT cells against three different AML cell lines in a dose-dependent manner.

[0085] To confirm the results of drug screening, DNT cells were pretreated with different concentrations of Ven. Ven pretreatment increased the cytotoxicity of DNT cells against three different AML cell lines, AML2-OCI, AML3-OCI, and KG1a, in a dose-dependent manner. Figure 2 A). Compared with untreated DNT, Ven-treated DNT also showed higher cytotoxicity in 16 out of 17 primary AML samples. Figure 2 B). Notably, Ven-treated DNT effectively killed four DNT-resistant samples (090271, 080043, 290985, and 150099). Ven-treated DNT was also effective in killing AML cells in patients at the diagnostic stage and in relapsed / refractory patients after induction chemotherapy. Figure 11 Furthermore, venetoc-treated DNT cells more effectively reduced colony formation in AML cell lines AML2-OCI and KG1a, as well as primary AML blast cells, demonstrating its effect on leukemia initiating cells. Figure 2 C)(9–12).

[0086] like Figure 2 As shown in Figure A, DNT cells pretreated with different concentrations of Ven showed a dose-dependent increase in cytotoxicity against three AML cell lines, OCI-AML2, OCI-AML3, and KG1a. This increased effector activity, enhanced by venetoclax treatment, was maintained for at least 4 days after drug removal from DNT cells. Figure 2 D). A significant negative correlation was observed between the sensitivity of AML to DNT cells and the increased cytotoxicity mediated by Ven-treated DNT cells. Figure 2 E). Venetoclax increases DNT-mediated killing of AML cells ( Figure 2 A), but it did not reduce the activity of DNT ( Figure 10 As shown in the figure, the antileukemic activity of venetoclax-treated DNT (Ven-treated DNT) was increased in DNT from all eleven tested DNT donors, with an average increase of 61.25 ± 31%. Figure 2 F).

[0087] To determine the antileukemic activity of DNT in the presence of venetoclax, KG1a and OCI-AML2 cells were treated with venetoclax, DNT, or both. Treatment of AML cells with both DNT and venetoclax produced fewer viable AML cells than either treatment alone. Figure 12 ).

[0088] Example 3: Compared to untreated DNT cells, Ven-treated DNT cells induced a significantly greater reduction in tumor volume and tumor weight.

[0089] Next, we will use a xenotransplantation model to investigate in vivo whether the ex vivo treatment of expanded T cells with Ven can improve its efficacy. Immunodeficient mice were subcutaneously inoculated with human leukemia cells. After tumor formation (size >100mm)... 3 Mice were given a single intravenous infusion of either non-drug-treated or Ven-treated DNT cells, and tumor growth was monitored. While DNT cell treatment effectively targeted leukemia (4–6), VenDNT cell treatment further reduced tumor volume (26.15% ± 5.724% in the DNT group and 52.23% ± 8.468% in the VenDNT-treated group on day 20); Figure 3 A). Similarly, compared with mice treated with PBS or DNT cells, mice treated with VenDNT cells showed a significant reduction in tumor weight (A). Figure 3 B). These data suggest that VenDNT cells can target AML cells more effectively in vivo. Given that AML is primarily located in the bone marrow (BM), the next step was to investigate whether VenDNT cells could target AML from bone marrow transplantation more effectively. Previous reports have shown that KG1a exhibits high resistance to DNT cell treatment in xenograft models (6). Although the effect of DNT cells was minimal, the KG1a transplantation level in mice treated with VenDNT cells was significantly reduced compared to the PBS and DNT cell treatment groups, further supporting the excellent antileukemic activity of VenDNT cells even against cells resistant in other ways (B). Figure 3 C).

[0090] The amount of primary AML samples transplanted using Ven-treated DNTs in vitro was less than the amount of the same cells treated with DNTs alone. Figure 3 D). The effect of Ven-treated DNT on primary AML sample transplantation was further investigated. Mice were treated with DNT or Ven-treated DNT after intravenous injection of primary AML cells. Compared with mice treated with the vector control or DNT, mice treated with Ven-treated DNT showed reduced AML transplantation and counting (D). Figure 3 E and Figure 13 A). Similar T cell frequencies were detected in the DNT group and the Ven-treated DNT group. Figure 13 B), indicating that Ven-treated DNTs exhibit enhanced antileukemic activity, attributed to functional improvements rather than enhancements in persistence or proliferative capacity. Importantly, no significant toxicity was observed from these treatments. Figure 14 ).

[0091] Example 4: Ven increases the cytotoxicity of conventional T cells

[0092] While treating DNT cells with Ven to enhance their antileukemic activity may be beneficial for DNT therapy, clinical practice has limited effectiveness. conv Cells are more widely used as cancer immunotherapy, so experiments were conducted to determine whether Ven targets CD4. + or CD8 + Conventional T(T) conv Ven has been shown to have anti-leukemic activity in T cells. Therefore, polyclonal activated T cells were pretreated with different concentrations of Ven before co-culturing with AML cell lines. conv Cells. Similar to those observed in DNT cells, T cells were observed. conv The cells showed significantly enhanced cytotoxicity against various AML cell lines. Figure 4 These data indicate that Ven can improve T. conv It also demonstrates the anti-leukemic activity of DNT cells and supports the combined use of Ven with adoptive T-cell therapy to further enhance treatment efficacy.

[0093] Example 5: Compared with other Bcl-2 inhibitors, Ven uniquely increased DNT cytotoxicity.

[0094] It is well known that Bcl-2 protects cells from apoptosis, and inhibition of this pathway is expected to exacerbate T cell apoptosis and weaken T cell function. Given the unexpected finding that Ven increases T cell-mediated cytotoxicity, we then used the pan-inhibitor of Bcl-2 family proteins, obacla, as well as the Bcl-2, Bcl-xL, and Bcl-w inhibitor ABT-737 to determine whether inhibition of other anti-apoptotic Bcl-2 family proteins would have similar effects. In contrast to Ven, these Bcl-2 family protein inhibitors induced DNT cell death or inhibited their cytotoxicity. Figure 5 A and Figure 5 B). The expression level of Bcl-xL in DNT cells was relatively higher than that in AML cells. Figure 5 C) This indicates that DNT cells can develop resistance to Bcl-2 inhibition through the compensatory activity of Bcl-xL.

[0095] Example 6: Ven treatment increases the expression of DNT effector molecules and activation markers, as well as ROS levels.

[0096] To elucidate the potential mechanism by which Ven mediates increased T cell cytotoxicity, the expression of T cell activation markers and effector molecules on Ven-treated and untreated DNT cells was compared. Ven treatment led to the expression of activation markers CD69 and CD25 on DNT cells. Figure 6 A) and higher expression of effector molecules NKG2D and DNAM-1 ( Figure 6 B). DNT cells treated with Ven also expressed higher levels of granzyme B than cells treated with the medium. Figure 6 C). Similarly, on CD8 processed by Ven... + ( Figure 6 D)T conv A dose-dependent increase in the expression of CD25, NKG2D, and DNAM-1 was also observed in cells. Treatment of DNT and T cells with venetoclax... conv Cells, for a minimum of 4 hours and a maximum of 3 days, increased the cytotoxicity of T cells against AML. Figure 4 B), while also increasing T cell activation markers (CD69 and CD25); Figure 6 A) and activating receptors (NKG2D and DNAM-1); Figure 6 The expression of B was not altered, but the activity of T cells was not changed. Figure 4 C). Therefore, Venetoclax directly activates effector T cells to increase their cytotoxicity without depleting naive or suppressor T cell subsets.

[0097] Recent studies have reported that venetoclax increases ROS levels, and ROS play an important role in the cascade amplification of T cell activation signals (9, 13-15). However, it has not been previously reported whether venetoclax increases ROS levels in T cells and enhances T cell activation. To determine the role of ROS in venetoclax-mediated T cell activation and enhancement, DNT cells and CD8+ T cells treated with escalating concentrations of venetoclax were measured. conv Cellular and mitochondrial ROS levels. Venetoclax increases DNT and CD8+ T in a dose-dependent manner. conv Cellular ROS in cells Figure 7 A). Despite a compensatory increase in the expression and nuclear localization of the antioxidant Nrf2, an increase in ROS levels was still observed. Figure 7 B).

[0098] To determine the functional relevance of elevated ROS levels in venetoclax-treated DNT cells, we co-treated DNT cells with venetoclax and escalating concentrations of N-acetylcysteine ​​(NAC). Treatment with venetoclax and NAC reduced cellular ROS levels and eliminated the effect of venetoclax on DNT cell-mediated anti-AML cytotoxicity. Figure 7 C), thus demonstrating the functional relevance of elevated ROS levels in Venetok-DNT cells. Figure 7As shown in L, treatment of DNT with venetoclax and increasing concentrations of the ROS scavenger N-acetylcysteine ​​(NAC) eliminated venetoclax-induced ROS generation and blocked the upregulation of activation markers.

[0099] Venetoclax increased ROS production in malignant tumor cells (9, 21), and ROS plays an important role in T cell activation and differentiation (15, 22-24). To further understand the mechanism by which venetoclax activates T cells, we measured the DNT and T cells after venetoclax treatment. conv ROS production in cells. Venetoclax increases DNT and T at concentrations and durations associated with enhanced T cell effector function. conv Cellular ROS and mitochondrial ROS ( Figure 7 HK).

[0100] To understand the mechanism by which Venetok increases mitochondrial ROS in DNTs, we measured the levels of respiratory chain proteins. No changes were observed in electron transport chain (ETC) complexes I, II, and IV, subunits NDUFA9, UQCRC2, and MTCO1. ROS generation is regulated by the respiratory chain supercomplex, a higher-order quaternary structure containing respiratory chain complexes I, III, and IV. A reduction in the respiratory chain supercomplex may be associated with higher mitochondrial ROS production (16, 17). As measured by non-denaturing gels, Venetok reduced the formation of the respiratory chain supercomplex in DNT cells (…). Figure 7 D).

[0101] To determine the effects of other ROS inducers on DNT-mediated cytotoxicity, DNTs were treated with escalating concentrations of cytarabine, daunorubicin, and antimycin. In DNTs treated with cytarabine and antimycin, ROS levels were observed to increase in a dose-dependent manner, while viability showed little or no loss. Figure 15 A and Figure 15 B). DNTs treated with daunorubicin exhibited lower ROS levels and significantly reduced activity. Figure 15 A and Figure 15 B). Unlike venetoclax treatment, despite increased cellular ROS levels, cytarabine and antimycin did not enhance DNT cytotoxicity, while daunorubicin reduced DNT-mediated anti-AML cytotoxicity. Figure 15 C). These data indicate that the ROS-dependent increase in DNT-mediated cytotoxicity is unique to venetoclax.

[0102] Interestingly, for DNT cells ( Figure 7 E) and CD8 + T conv cell( Figure 7Ven also increased the proportion of effector memory cells while decreasing the frequency of central memory T cells. Since effector memory T cells preferentially depend on glycolysis, while central memory T cells depend on oxidative phosphorylation, and Ven has been shown to inhibit oxidative phosphorylation on AML cells, the glycolytic levels, glycolytic capacity, and oxygen consumption rate (OCR) of DNT and VenDNT cells were compared. However, Ven had no significant effect on glycolysis, glycolytic capacity, and basal oxygen consumption in DNT cells, suggesting that Ven biases DNT cells towards an effector memory phenotype independent of their metabolic pathways. Figure 7 G). To understand the mechanism by which venetoclax increases ROS production, we measured the levels of respiratory chain proteins. No changes were observed in the subunits NDUFA9, UQCRC2, and MTCO1 of electron transport chain (ETC) complexes I, III, and IV. Figure 7 M and Figure 16 ).

[0103] Overall, these results indicate that Ven activates T cells and biases them toward more effector phenotypes.

[0104] Example 7: Ven treatment increased the proportion of cytotoxic CD8+ and DNT cells in the T cell population.

[0105] Recent reports indicate that combination therapy with Ven and Aza significantly improves clinical outcomes in treatment-naïve AML patients with low treatment-related toxicity (9 and 19). To determine whether venetoclax increases T-cell effector activity in patients, we examined T-cell counts in AML patients before and on day 4 after treatment with venetoclax and azacytidine. Compared to pre-treatment levels, we observed an increase in the proportion of CD8+ and DNT cells after treatment with venetoclax and azacytidine (…). Figure 8 A). Consistent with our in vitro findings, the proportion of CD8+ T cells in effector memory / effector state was increased in all patients ( Figure 8 B). Furthermore, after treatment, the expression of NKG2D on CD8+ T cells and the level of cellular ROS both increased (B). Figure 8 C). Similarly, an increased frequency of effector memory / effector subsets was observed in DNT cells ( Figure 8 D), and DNT cells also showed high levels of NKG2D expression and cellular ROS (D). Figure 8 E).

[0106] Example 8: Ven selectively increases the cytotoxic activity of DNT cells against AML.

[0107] To determine whether Ven increases the cytotoxicity of DNT cells against normal blood cells, autologous and allogeneic PBMCs from healthy donors were used as targets. Although strong cytotoxicity against OCI-AML2 was observed, the killing effect of Ven-treated and untreated DNT cells on autologous and allogeneic PBMCs was not significantly different. Figure 9 This indicates that Ven selectively increases the cytotoxic activity of DNT cells against AML.

[0108] While this disclosure has been described in conjunction with what is now considered a preferred embodiment, it should be understood that this disclosure is not limited to the disclosed embodiments. Rather, this disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0109] Just as each individual publication, patent, or patent application is specifically cited in its entirety and incorporated herein by reference, the entirety of all publications, patents, and patent applications is cited in this document.

[0110] References

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Claims

1. An enhancement method for double-negative CD4+ - CD8 - An in vitro approach to the therapeutic effects of T(DNT) cells includes pretreatment of ex vivo expanded DNT cells with venetotak to generate enhanced DNT cells.

2. The in vitro method according to claim 1, wherein the method comprises pretreating the DNT cells with a concentration of at least 50 nM venetoclax.

3. The in vitro method according to claim 1, wherein the method comprises pretreating the DNT cells with venetok at a concentration of at least 100 nM.

4. The in vitro method according to claim 1, wherein the method comprises pretreating the DNT cells with venetok at a concentration of at least 200 nM.

5. The in vitro method according to claim 1, wherein the method comprises pretreating the DNT cells with venetok at a concentration of at least 300 nM.

6. The method of claim 1, wherein the method comprises pretreating the DNT cells with venetok at a concentration of at least 400 nM.

7. The in vitro method according to claim 1, wherein the method comprises pretreating the DNT cells with venetok at a concentration of 50 nM to 1 μM.

8. The in vitro method according to claim 2, wherein the method comprises pretreating the DNT cells with venetotok for at least 30 minutes.

9. The in vitro method according to claim 8, wherein the method comprises pretreating the DNT cells with venetotok for at least 1 hour.

10. The in vitro method of claim 8, wherein the method comprises pretreating the DNT cells with venetok for at least 2 hours.

11. The in vitro method of claim 8, wherein the method comprises pretreating the DNT cells with venetok for at least 4 hours.

12. The in vitro method of claim 8, wherein the method comprises pretreating the DNT cells with venetok for at least 6 hours.

13. The in vitro method of claim 8, wherein the method comprises pretreating the DNT cells with venetotok for at least 8 hours.

14. The in vitro method of claim 8, wherein the method comprises pretreating the DNT cells with venetotok for at least 12 hours.

15. The in vitro method of claim 8, wherein the method comprises pretreating the DNT cells with venetotok for at least 24 hours.

16. The in vitro method of claim 8, wherein the method comprises pretreating the DNT cells with venetotok for less than 14 days.

17. The in vitro method of claim 16, wherein the method comprises pretreating the DNT cells with venetotke for less than 10 days.

18. The in vitro method of claim 16, wherein the method comprises pretreating the DNT cells with venetotok for less than 9 days.

19. The in vitro method of claim 16, wherein the method comprises pretreating the DNT cells with venetotke for less than 8 days.

20. The in vitro method of claim 16, wherein the method comprises pretreating the DNT cells with venetotok for less than 7 days.

21. The in vitro method of claim 16, wherein the method comprises pretreating the DNT cells with venetotok for less than 6 days.

22. The in vitro method of claim 16, wherein the method comprises pretreating the DNT cells with venetotok for less than 5 days.

23. The in vitro method according to any one of claims 1-22, wherein the DNT cells are γ-δT cells.

24. The in vitro method according to any one of claims 1 to 22, the method further comprising removing part or all of the venetokine to prevent it from contacting the enhanced DNT cells.

25. Enhanced DNT cells generated by the in vitro method according to any one of claims 1 to 24.

26. The enhanced DNT cells of claim 25, wherein, relative to unexposed control DNT cells, the expression levels of one or more of CD25, CD69, NKG2D, DNAM-1, and NRF2 are increased in the enhanced DNT cells.

27. The enhanced DNT cells of claim 25, wherein the level of reactive oxygen species in the enhanced DNT cells is increased compared to control DNT cells not exposed to venetoclax.

28. The enhanced DNT cells of claim 25, wherein the ratio of effector-memory DNT cells to naive DNT cells in the enhanced DNT cells is increased compared to the ratio of effector-memory DNT cells to naive DNT cells in control DNT cells not exposed to venetoclax.

29. A pharmaceutical composition comprising enhanced DNT cells according to any one of claims 25 to 28 and a pharmaceutically acceptable carrier.

30. Use of the enhanced DNT cells according to any one of claims 25 to 28 or the pharmaceutical composition according to claim 29 in the preparation of a medicament for treating acute myeloid leukemia in a subject in need.

31. Double-negative CD4+ amplified in vitro - CD8 - The use of the combination of T(DNT) cells and venetoc in the preparation of a medicine for treating acute myeloid leukemia in subjects in need.

32. The use according to claim 31, wherein the DNT cells and the Venetok are used at the same time or at different times.

33. The use according to claim 31, wherein the DNT cells are used within 24 hours of using venetoc.

34. The use according to claim 31, wherein the DNT cells are used within 36 hours of using venetoc.

35. The use according to claim 31, wherein the DNT cells are used within 48 hours of using venetoc.

36. The use according to claim 31, wherein the dose of the venetoclax used is from 50 mg to 800 mg.

37. A composition comprising in vitro amplified double-negative CD4 - CD8 - T(DNT) cells and Venetok.

38. The composition of claim 37, wherein the composition further comprises a pharmaceutically acceptable carrier or culture medium.

39. The composition of claim 37, wherein the concentration of venetoc in the composition is at least 50 nM.

40. The composition according to claim 39, wherein the concentration of venetoc in the composition is from 50 nM to 1 μM.

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