Compositions and methods for treating acute myeloid leukemia resistant to ven / aza

Abstract

The present disclosure describes T cells expressing chimeric antigen receptors (CARs), as well as pharmaceutical compositions comprising the T cells, and methods of making and using such T cells. In particular, the present disclosure describes T cells expressing CARs that bind CD64, and methods of using the same in the treatment of acute myeloid leukemia.

Description

[0001] Cross-reference of related applications

[0002] This application claims the benefit of U.S. Patent Application Serial No. 63 / 610,215, filed December 14, 2023. Previous application disclosures are considered part of this application disclosure (and are incorporated herein by reference). Background of the Invention

[0004] Acute myeloid leukemia (AML) is a blood cancer in which the body's bone marrow produces abnormal myeloblasts, red blood cells, or platelets. AML is one of the most common forms of acute leukemia in adults. The accumulation of AML cells in the bone marrow and blood can rapidly lead to infection, anemia, excessive bleeding, and death. The BCL-2 inhibitor venetoclax has recently become an important component of treatment for AML. Currently, the FDA-approved standard of care for most patients who are elderly or unsuitable for aggressive chemotherapy is venetoclax in combination with a hypomethylating agent, such as azacitidine ("venetoclax / azacitidine (ven / aza) therapy") or decitabine. It is estimated that about 70% of these patients achieve complete remission (CR) after receiving ven / aza therapy. However, it is estimated that about 30% of patients do not respond to ven / aza therapy and do not achieve CR. Furthermore, most of the patients who initially achieve CR will relapse within the following two years. Therefore, there is a need in the field for alternative treatments for individuals who do not respond to ven / aza treatment or who relapse after treatment. Summary of the Invention

[0005] This disclosure provides a polypeptide comprising: a) a first domain comprising an amino acid sequence of one of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; b) a second domain comprising an amino acid sequence of SEQ ID NO: 4; c) a third domain comprising an amino acid sequence of SEQ ID NO: 5; d) a fourth domain comprising an amino acid sequence of SEQ ID NO: 6; and e) a fifth domain comprising an amino acid sequence of SEQ ID NO: 7.

[0006] In some respects, the first domain also contains the amino acid sequence of SEQ ID NO: 23.

[0007] In some respects, the polypeptide comprises an amino acid sequence of one of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

[0008] This disclosure provides a polypeptide comprising: a) a first domain comprising an amino acid sequence of one of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; b) a second domain comprising an amino acid sequence of SEQ ID NO: 8; c) a third domain comprising an amino acid sequence of SEQ ID NO: 9; d) a fourth domain comprising an amino acid sequence of SEQ ID NO: 10; and e) a fifth domain comprising an amino acid sequence of SEQ ID NO: 7.

[0009] In some respects, the first domain also contains the amino acid sequence of SEQ ID NO: 23.

[0010] In some respects, the polypeptide comprises an amino acid sequence of one of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0011] This disclosure provides nucleic acid molecules comprising a nucleic acid sequence encoding a polypeptide of this disclosure. In some aspects, the nucleic acid molecule comprises a nucleic acid sequence of one of SEQ ID NO: 17-22.

[0012] This disclosure provides a vector containing the nucleic acid molecules of this disclosure, preferably wherein the vector is a viral vector, and preferably wherein the viral vector is a lentiviral vector.

[0013] This disclosure provides cells comprising the polypeptides, nucleic acid molecules, and / or vectors of this disclosure.

[0014] In some respects, the cells are immune cells, preferably T cells or natural killer (NK) cells.

[0015] This disclosure provides a cell population of the cells disclosed herein.

[0016] This disclosure provides a method for treating acute myeloid leukemia (AML) in a therapeutic subject, the method comprising administering the cell population described in this disclosure.

[0017] In some respects, the subjects suffering from AML have a population of AML cells with CD64+.

[0018] In some respects, the subjects suffering from AML have a population of monocytic leukemia stem cells (mLSC).

[0019] In some respects, the subject has previously been given at least one therapy targeting AML, preferably wherein the therapy targeting AML includes administration of a combination of venetoclax and azacitidine.

[0020] In some respects, the subject relapses after receiving treatment with the at least one targeted AML therapy, and / or the subject becomes resistant to the at least one targeted AML therapy.

[0021] Any of the foregoing aspects and implementations, or the aspects and implementations described herein, may be combined with any other aspects and implementations.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In this specification, singular forms also include plural forms unless the context clearly indicates otherwise; for example, the terms “an,” “a,” and “the” are to be understood as singular or plural, and the term “or” is to be understood as inclusive (i.e., “and / or”). For example, “an element” refers to one or more elements. Throughout this specification, the word “comprising” or variations such as “including” or “containing” are to be understood as implying inclusion of the stated element, integer, or step, or group of elements, integers, or steps, but not excluding any other element, integer, or step, or group of elements, integers, or steps. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless the context clearly indicates otherwise, all numerical values ​​provided herein are modified by the term “about.”

[0023] Although similar or equivalent methods and materials described herein may be used in the practice or testing of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. References cited herein should not be considered prior art to the claimed invention. In case of conflict, this specification (including definitions) shall prevail. Furthermore, the materials, methods, and examples described are illustrative only and not intended to be limiting. Other features and advantages of this disclosure will become apparent from the following detailed description and claims.

[0024] Brief description of the attached figures

[0025] Figure 1A-C series of graphs shows the antigen-dependent effector activity of CD64 CAR T cells with the CD28 co-stimulatory domain and the m22 single-chain antibody variable fragment (scFv) (CD64-28z) against AML cell lines. A) CD64 expression in AML cell lines K562 (CD64 negative), MOLM14 (CD64 low expression), and THP1 (CD64 high expression) used in downstream experiments (top). AML cell lines were co-incubated with activated but untransduced T cells from healthy donors (simulated, negative control) or with T cells transduced with CD64 CARs containing the CD28 co-stimulatory domain (CD64 CAR). AML cells and T cells were co-incubated in triplicate at a 1:1 ratio for 24 hours and evaluated by flow cytometry. B) Quantification of the percentage of AML cells remaining viable after 24 hours of co-incubation using flow cytometry. The results confirmed that CD64-negative K652 cells were not reduced by CD64 CAR T cells, but CD64-low expressing MOLM14 and CD64-high expressing THP1 AML cells were significantly reduced by CD64-28z CAR T cells (labeled as CD64 CAR). C) CD64-negative K562 cells and CD64-high expressing THP1 cells were co-incubated with CD19 CAR T cells (CD19-28z, used as an irrelevant CAR as a negative control) or CD64 CAR T cells (CD64-28z) at a 1:1 ratio for 24 hours to measure antigen-dependent cytokine secretion from CD64-28z CAR T cells. CAR T cells cultured alone served as a negative control. All co-incubation / culture was performed in triplicate. Twenty-four hours later, the supernatant from all wells was collected and cytokines were assessed using enzyme-linked immunosorbent assay (ELISA). This confirmed that CD64-28z CAR T cells only significantly produced interferon-γ (IFNG) and interleukin-2 (IL-2) in the presence of CD64+ AML cells. Statistical comparisons between CD64-28z and negative controls were performed using Mann-Whitney analysis.

[0026] Figure 2A -B series of graphs shows that CD64-28z CAR T cells (containing m22 scFv) have in vivo therapeutic effects against CD64+ AML cell lines in a xenograft model. A) On day -4, NSG mice were inoculated with 10 cells via tail vein injection. 6 THP1 cells expressing luciferase were observed. Bioluminescence imaging (BLI) confirmed the engraftment of THP1 cells on day -1. On day 0, mice received 5 x 10⁻⁶ THP1 cells. 6Mice were treated with CD64-28z CAR T cells or received no treatment. Leukemia burden was monitored once or twice weekly via BLI, and survival was monitored based on the development of pre-specified end-stage leukemia endpoint events. Compared to no treatment, CD64-28z CAR T cells significantly reduced leukemia progression and prolonged survival. B) On day -4, NSG mice were inoculated with 10 CD64-28z CAR T cells via tail vein injection. 6 MOLM14 cells expressing luciferase were observed. Bioluminescence imaging (BLI) confirmed the engraftment of MOLM14 cells on day -1. On day 0, mice received 5 x 10⁻⁶ MOLM14 cells. 6 Mice were treated with CD64-28z CAR T cells or received no treatment. BLI was monitored twice weekly to assess leukemia burden, and survival was monitored based on the development of pre-specified end-stage leukemia endpoint events. Compared to no treatment, CD64-28z CAR T cells significantly reduced leukemia progression and prolonged survival. Statistical analyses of survival in all experiments were performed using the log-rank test, where p-values ​​are less than 0.01.

[0027] Figure 3A -C series of graphs shows the cytotoxicity of CD64-28z CAR T cells (containing m22 scFv) against patient-derived monocytic AML in vitro. A) CD64+ monocytic AML cells with monocytic leukemia stem cells (mLSC) were obtained from the University of Colorado Biobank. BC) Patient AML cells were co-incubated in vitro with either mimic T cells or CD64-28z CAR T cells derived from the same healthy donor in triplicate for 24 or 48 hours, using different effector cell (T cell) to target cell (AML cell) ratios (E:T ratio). B) As measured by flow cytometry, co-incubation with CD64-28z CAR T cells resulted in significant and dose-dependent clearance of live AML cells at all E:T ratios. C) Flow cytometry evaluation of remaining AML cells at 24 and 48 hours confirmed that CD64-28z CAR T cells were significantly superior to mimic T cells in specifically clearing CD64+ AML cells at all E:T ratios. The clearance of CD64+ AML cells by CD64-28z CAR T cells occurred in a dose-dependent manner, with increases from 24 to 48 hours at E:T ratios of 1:4 and 1:16, while at an E:T ratio of 1:1, all CD64+ AML cells were cleared within the first 24 hours. Statistical comparisons were performed using two-way ANOVA and Tukey's multiple comparison test. ns = No significant difference. = p-value less than 0.001, = p value is less than 0.0001.

[0028] Figure 4A -C series of graphs shows the cytotoxicity of CD64-28z CAR T cells (containing m22 scFv) against patient-derived AML with a mixed population of blast cells and monocytes in vitro. A) Patient-derived AML with a mixed population of CD64- (blast) and CD64+ (monocyte) leukemia stem cells (LSCs) obtained from the University of Colorado Biobank. BC) Patient-derived AML cells were co-incubated in vitro with mimic T cells or CD64-28z CAR T cells derived from the same healthy donor in triplicate for 24 or 48 hours, using different effector (T cell) to target (AML cell) ratios (E:T ratio). B) As measured by flow cytometry, co-incubation with CD64-28z CAR T cells resulted in significant and dose-dependent clearance of live AML cells at all E:T ratios. C) Flow cytometry evaluation of remaining AML cells at 24 and 48 hours confirmed that CD64-28z CAR T cells were significantly more effective than mimic T cells in specifically clearing CD64+ AML cells at all E:T ratios. Clearance of CD64+ AML cells by CD64-28z CAR T cells occurred in a dose-dependent manner, increasing from 24 to 48 hours at E:T ratios of 1:4 and 1:16, while at an E:T ratio of 1:1, all CD64+ AML cells were cleared within the first 24 hours. Statistical comparisons were performed using two-way ANOVA and Tukey's multiple comparison test. ns = No significant difference. = p-value less than 0.05, = p value is less than 0.0001.

[0029] Figure 5A -D series of graphs show that CD64-28z CAR T cells (containing m22 scFv) can deplete monocytic leukemia stem cells (mLSCs) from patient-derived AML samples. Patient-derived AML samples were obtained from the University of Colorado Biobank. The samples were predominantly monocytic AML samples ( Figures 5A-5B ) and mixed primitive / monocyte AML patient samples ( Figures 5C-5D AML cells were co-incubated with CD64-28z CAR T cells or paired T-cell mimics in vitro at a 1:2 effector:target ratio for 24 hours. T cells were depleted by antibody, and the remaining AML cells were injected into NSG-S mice (10 cells per mouse). 6AML cells; n = 7-10 mice per group). Mice were allowed to engraft over the next 10 weeks, at which point they were sacrificed, and engraftment of human AML cells and CD64+ human AML cells in the bone marrow was assessed by flow cytometry. We observed that engraftment of mononuclear AML was significantly reduced after pre-incubation with CD64-28z CAR T cells compared to T-mimicking cells. Figure 5A ), and CD64+ AML cells were significantly reduced in the implantation population ( Figure 5B We also observed that, compared to simulated T cells, engraftment of mixed primitive / monocyte-type AML cells was reduced after pre-incubation with CD64-28z CAR T cells. Figure 5C At the same time, CD64+ AML cells were also significantly reduced. Figure 5D Overall, the reduction in the total cell population and CD64+ AML cell engraftment after in vitro CAR incubation confirms that CD64 CAR T cells can target mLSCs carrying leukemia cell engraftment potential. Statistical comparisons were performed using Mann-Whitney analysis, as shown.

[0030] Figure 6A -C series of graphs show that CD64 CAR (CD64-BBz) containing m22 scFv and 4-1BB co-stimulatory domains and CD64 CAR (CD64-28z) containing m22 scFv and CD28 co-stimulatory domains have in vivo therapeutic effects against CD64+ AML cell lines in xenograft models. AB) On day -4, NSG mice were inoculated with 10 CAR T cells via tail vein injection. 6 MOLM14 cells expressing luciferase were observed. Bioluminescence imaging (BLI) confirmed the engraftment of MOLM14 cells on day -1. On day 0, mice received 3 x 10⁻⁶ MOLM14 cells. 6 One CD64-28z CAR T-cell therapy, or receive 3x10 6 Mice were treated with CD64-BBz CAR T cells or received an equivalent amount of mimic T cells. Mice were treated with mimic or CAR T cells from three independent T cell donors (5 mice per donor per treatment). BLI was monitored one to two weeks weekly to assess leukemia burden. Figure 6A The survival of mice was monitored based on the development of pre-set terminal leukemia endpoint events. Figure 6BCompared to T-cell analogs, both CD64-28z and CD64-BBz CAR T cells significantly reduced leukemia progression and prolonged survival, with CD64-BBz CAR T cells showing a superior survival advantage over CD64-28z CAR T cell therapy. Statistical analysis of survival across donor groups was performed using the log-rank test. = p-value less than 0.05, and = p-value less than 0.0001. C) Quantitative bioluminescence signal as total flux (photons / second) per mouse was used to quantify the leukemia burden of mice over time. Mice treated with CD64-BBz and CD64-28z CAR-T cells showed a reduced leukemia burden over time compared with mice receiving simulant treatment. Statistical analysis was performed by two-way ANOVA, where = p-value less than 0.05, and = p-value less than 0.0001. The difference between CD64-28z and CD64-BBz CAR T-cell therapy was 0.06 according to two-way ANOVA.

[0031] Figure 7A -B's series of graphs show CD64 expression in various leukemia models. A) Series of histograms showing CD64 expression as measured by flow cytometry in K562, MOLM14, and THP-1 AML cell lines. B) Series of histograms and contour plots showing the characterization of CD64 expression in primary AML samples.

[0032] Figure 8A-D's series of schematic diagrams and graphs show that different forms of CD64 CAR can be expressed in healthy donor T cells. A) Schematic diagrams of exemplary CD64-28-m22, CD64-BB-m22, and CD64-BB-611 CAR constructs. CD64-28-m22 consists of an m22 scFv, an extracellular and transmembrane (EC / TM) domain derived from CD28, a CD28 co-stimulatory domain, and a CD3z (CD3-ζ) signaling domain. CD64-BB-m22 consists of an m22 scFv, an extracellular and transmembrane (EC / TM) domain derived from CD8, a 4-1BB co-stimulatory domain, and a CD3z (CD3-ζ) signaling domain. CD64-BB-611 consists of a 611 scFv, an extracellular and transmembrane (EC / TM) domain derived from CD8, a 4-1BB co-stimulatory domain, and a CD3z (CD3-ζ) signaling domain. B) Histogram showing the transduction efficiency and expression of CD64-28-m22, CD64-BB-m22, and CD64-BB-611 CAR constructs in healthy donor human T cells. C) Chart showing the CD4 to CD8 T cell ratio in the CD64-BB-611, CD64-BB-m22, and CD64-28-m22 CAR T cell populations. D) Bar chart comparing T cell phenotypes in the CD64-BB-611, CD64-BB-m22, and CD64-28-m22 CAR T cell populations. Statistical analysis was performed using two-way ANOVA. = p-value less than 0.01, = p-value less than 0.001 and = p value is less than 0.0001.

[0033] Figure 9A -E series of graphs show that CD64 CAR T cells are cytotoxic to CD64+ AML cells in vitro. A) Scatter plot of flow cytometry results, which evaluated K562, MOLM14, and THP-1 AML cell lines remaining after co-culturing with CD64-BB-611, CD64-BB-m22, and CD64-28-m22 CAR T cells. B) As shown in the triple replicates at two different E:T ratios, the cytotoxicity of CD64 CAR T cells to CD64+ AML cells in vitro. Figure 9A Quantitative analysis of AML cell line cytotoxicity was performed, demonstrating antigen-dependent cytotoxicity of all CD64 CAR constructs. Statistical analysis was conducted using a two-way ANOVA. = p-value less than 0.01, = p-value less than 0.001 and = p-value < 0.0001. C) Luciferase-based killing assays were used to evaluate the killing effect of CD64-BB-611, CD64-BB-m22, and CD64-28-m22 CAR T cells on K562, MOLM14, and THP-1 AML cell lines. Results showed that each CD64 CAR construct produced antigen-dependent cytotoxicity. D) Scatter plot showing the reduction in the killing effect of CD64-BB-611, CD64-BB-m22, and CD64-28-m22 CAR T cells on CD64+ AML cells of patient-derived monocyte-type and mixed monocyte / blastocyte-type (“mixed”) AML. E) Quantification of the killing effect of CD64-BB-611, CD64-BB-m22, and CD64-28-m22 CAR T cells on monocyte-type and mixed monocyte / blastocyte-type AML patient samples after co-culturing for 24 hours at various E:T ratios. Cytotoxicity was calculated based on normalized AML cells co-cultured with an equal volume of simulated T cells at the same E:T ratio, and analyzed by flow cytometry at the end of 24 hours of co-culture.

[0034] Figure 10A The series of graphs (-F) shows the release of T cell effector cytokines when CD64 CAR T cells bind to CD64+ AML cells. The AC) bar graph shows the response of CD64-BB-m22, CD64-28-m22, and CD64-BB611 CAR T cells to antigen-dependent interleukin-2 (IL-2) after 24 hours of co-culturing with AML cell lines K562, MOLM14, and THP1. Figure 10A ), interferon-γ (IFNg)( Figure 10B ) and tumor necrosis factor a (TNFa) Figure 10C The secretion of IL-2. DF) bar graph, which shows the secretion of CD64-BB-m22, CD64-28-m22, and CD64-BB611 CAR T cells in response to antigen-dependent IL-2 after co-culturing with patient-derived monocytic or mixed primitive / monocytic AML samples for 24 hours. Figure 10D ), IFNg( Figure 10E ) and TNFa ( Figure 10F The secretion of ). All statistical analyses in Figure 10 were performed using two-way ANOVA, where = p-value less than 0.05, = p-value less than 0.01, = p-value less than 0.001 and = p value is less than 0.0001.

[0035] Figure 11A-E's series of graphs show the preparation and efficacy of patient-derived CD64 CAR T cells relative to healthy donor CD64 CAR T cells. A) Line graph, showing T cell proliferation during CD64-BB-m22 CAR T cell preparation. This data confirms that the proliferation kinetics of patient-derived T cells are comparable to those of healthy donor-derived T cells during CD64-BB-m22 CAR T cell preparation. B) Histogram, showing the transduction efficiency of CD64-BB-m22 CAR constructs from two AML patient donors (pt-AML03 and pt-AML06) compared to those from two healthy donors (healthy donors 42 and 20). C) Bar graph, comparing the proliferation of patient-derived CD64-BB-m22 CAR T cells and healthy donor-derived CD64-BB-m22 CAR T cells after 24 hours of co-culturing with MOLM14 AML cells. D) A luciferase killing assay evaluated the overnight killing effect of two patient-derived CD64-BB-m22 CAR T cell products on MOLM14 AML cells, confirming the cytotoxicity of patient-derived CAR T cells. E) Scatter plots and histograms showing the clearance of CD64+ AML cells from monocytic and mixed-type patient-derived AML samples by autologous patient-derived CD64-BB-m22 CAR T cells.

[0036] Figure 12A A series of bioluminescent images and graphs from -F show CD64 CAR T cells clearing CD64+ AML cell lines in a xenograft model. A) 10 cells were implanted on day -4. 6 10 THP1 cells and received 5 x 10 6 Bioluminescent images of THP-1 leukemia burden in NSG mice treated with CD64-BB-611, CD64-BB-m22, or CD64-28-m22 (labeled CD64-28) CAR T cells or equal-dose mimic T cells from the same T cell donor. This experiment was performed in triplicate using three independent healthy T cell donors. B) Survival analysis of mice treated with CD64-BB-611, CD64-BB-m22, or CD64-28-m22 CAR T cells or control mice treated with mimic T cells. Compared with mice treated with mimic T cells and CD64-28-m22 CAR T cells, both CD64-BB-611 and CD64-BB-m22 CAR T cells significantly delayed leukemia progression and prolonged survival. Statistical analysis of survival across donor pooling groups was performed using the log-rank test, where... = p-value less than 0.01, = p-value less than 0.001 and = p-value less than 0.0001. C) Quantitative bioluminescence signal as total flux (photons / second) per mouse was used to quantify the leukemia burden of mice over time. Mice treated with CD64-BB-m22 and CD64-BB-611 CAR-T cells showed a reduced leukemia burden compared to mice that had undergone simulant treatment. Statistical analysis was performed using two-way ANOVA, where... = p-value less than 0.05, = p-value less than 0.01, = p-value less than 0.001 and = p-value less than 0.0001. D) Single T cell donor replication experiments with data shown in Figure 6, including treatment with CD64-BB-611 in NSG mice implanted with MOLM14 leukemia. Compared with the control group receiving a mimic treatment, those receiving 3x10 6 Bioluminescence imaging of MOLM14 leukemia burden in NSG mouse xenograft models treated with CD64-BB-611, CD64-BB-m22, and CD64-28-m22 CAR T cells. E) Survival analysis of mice treated with CD64-BB-611, CD64-BB-m22, or CD64-28-m22 CAR T cells or control mice treated with a mimicry treatment showed that survival was prolonged after treatment with any form of CD64 CAR compared with mimicry T cells. Statistical analysis of survival across donor groups was performed using a log-rank test, where = p-value less than 0.01, = p-value less than 0.001 and = p-value less than 0.0001. F) Quantitative bioluminescence signal was used as the total flux (photons / second) per mouse to quantify the leukemia burden of mice over time. Mice treated with CD64-28-m22, CD64-BB-m22, and CD64-BB-611 CAR-T cells showed a reduced leukemia burden compared to mice that had undergone simulant treatment. Statistical analysis was performed using two-way ANOVA, where... = p-value less than 0.05, = p-value less than 0.01, = p-value less than 0.001 and = p value is less than 0.0001.

[0037] Figure 13A -F series of charts show the clearing of CD64 in xenograft models. +CD64 CAR T cells persist after leukemia. A) A graph showing the percentage and absolute number of total CD64-BB-m22 and CD64-28-m22 CAR T cells, and their CD4+ and CD8+ subsets, persisting in the bone marrow 35 days after infusion and after MOLM14 leukemia clearance. Statistical analysis was performed by Mann-Whitney. = p-value less than 0.05, = p-value less than 0.01, =p value less than 0.001 and = p-value less than 0.0001. B) Bar graph, comparing the T cell phenotypes of persistent CD64-BB-m22 and CD64-28-m22 CAR T cells 35 days after infusion and after MOLM14 leukemia clearance. C) Bar graph, showing the percentage of persistent CD64-BB-m22 and CD64-28-m22 CAR T cells expressing PD1 and TIM3 depletion markers 35 days after infusion and after MOLM14 leukemia clearance. D) Graph, showing the percentage and absolute number of total CD64-BB-611, CD64-BB-m22, and CD64-28-m22 CAR cells, and their CD4+ and CD8+ subsets, persistent in the bone marrow 105 days after infusion and after MOLM14 leukemia clearance. Statistical analysis was performed by one-way ANOVA, where = p-value less than 0.05, = p-value less than 0.01, = p-value less than 0.001 and = p-value less than 0.0001. E) Bar graph, which compares the T cell phenotypes of persistent CD64-BB-611, CD64-BB-m22, and CD64-28-m22 CAR T cells at 105 days post-infusion and after MOLM14 leukemia clearance. Statistical analysis was performed by two-way ANOVA, where = p value less than 0.05. F) Bar graph showing the percentage of persistent CD64-BB-611, CD64-BB-m22 and CD64-28-m22 CAR T cells expressing PD1 and TIM3 depletion markers at 105 days post-infusion and after MOLM14 leukemia clearance.

[0038] Figure 14A-E series of graphs showing the cytotoxicity of CD64 CAR T cells to monocytes. A) Flow cytometry scatter plot showing the clearance of healthy donor-derived monocytes by CD64-BB-611, CD64-BB-m22, and CD64-28-m22 CAR T cells after overnight co-culture. B) Quantification of monocyte killing in two independent monocyte / T cell donors, where CD64-28-m22, CD64-BB-m22, and CD64-BB-611 CAR T cells were co-cultured overnight with autologous monocytes at different E:T ratios, and cytotoxicity to monocytes was calculated by flow cytometry compared with a negative control of simulated T cells. CE) Bar graph showing the response of CD64-BB-m22, CD64-28-m22, and CD64-BB-611 CAR T cells to antigen-dependent IL-2 ( ) after overnight co-culture with monocytes. Figure 14C ), IFNg( Figure 14D ) and TNFa ( Figure 14E The secretion of CAR T cells. Three independent healthy T cell donors were used for CAR T cell production (20, 28, and 42). Statistical analysis was performed by two-way ANOVA, where... = p-value less than 0.05, = p-value less than 0.01, = p-value less than 0.001 and = p value is less than 0.0001.

[0039] Figure 15A -F series of graphs show the additive clearance of CD64+ primary AML by CD64 CAR T cells in combination with venetoclax and azacitidine under in vitro conditions. AB) graphs show the remaining viable AML population in mixed mononuclear / primitive AML cells treated with venetoclax and azacitidine (ven / aza), CD64-BB-m22 CAR T cells, a combination of ven / aza and CD64-BB-m22 CAR T cells, or neither ven / aza nor CD64-BB-m22 CAR T cells (DMSO was used as a negative control for ven / aza, and T-cell mimicry was used as a negative control for CAR-T cells). Figure 15A And the expression of CD33, CD34 and CD64 ( Figure 15BC) Graph showing the quantification of residual CD64+ and CD64- AML cells in samples from primary mixed monocytic / primitive AML patients after treatment with ven / aza, CD64-BB-m22 CAR T cells, a combination of ven / aza and CD64-BB-m22 CAR T cells, or neither ven / aza nor CD64-BB-m22 CAR T cells (DMSO / dummy control). DE) Graph showing the remaining viable AML population after treatment with samples from monocytic AML patients with ven / aza, CD64-BB-m22 CAR T cells, a combination of ven / aza and CD64-BB-m22 CAR T cells, or neither ven / aza nor CD64-BB-m22 CAR T cells (DMSO / dummy control). Figure 15D ) and the expression of CD33, CD34 and CD64 on these residual AML cells ( Figure 15E ). F) Graph showing the quantification of CD64+ and CD64-AML cells from primary monocytic AML patient samples after treatment with ven / aza, CD64-BB-m22 CAR T cells, a combination of ven / aza and CD64-BB-m22 CAR T cells, or DMSO and simulated T cell negative control. Figure 15C and 15F Statistical analysis was performed using a two-way ANOVA, where = p-value less than 0.05, = p-value less than 0.01, = p-value less than 0.001 and = p value is less than 0.0001. Detailed Implementation

[0040] Acute myeloid leukemia (AML) is a blood cancer and one of the most commonly diagnosed types of leukemia in adults. An estimated 11,000 people died from AML in the United States in 2020, with 20,000 new cases diagnosed. The average age of people diagnosed with AML is approximately 68 years, with most cases occurring after age 45. However, AML is also diagnosed in younger patients and is the second most common leukemia in children. The prognosis for patients diagnosed with AML is generally poor, with long-term survival rates of only 40-50% for younger adults and a median overall survival of less than one year for older patients. New therapies designed to complement the standard induction remission regimen of cytarabine combined with intermittent anthracyclines have not yet yielded significant clinical benefit. Therefore, there is a need for more specific and personalized treatment options, particularly in older patients who are ineligible for induction therapy.

[0041] Recent studies have shown that acute myeloid leukemia (AML) exhibits a high level of biological heterogeneity, which may explain the difficulty in finding effective treatment strategies for AML. Furthermore, it has recently been recognized that leukemia stem cells (LSCs) capable of producing identical and differentiated progeny cells enable AML to persist and maintain the disease, and promote disease relapse.

[0042] As an alternative to high-intensity induction therapy, the current FDA-approved standard of care for elderly patients or those otherwise unsuitable for intensive chemotherapy is treatment with the BCL-2 inhibitor venetoclax in combination with a demethylating agent (HMA) such as azacitidine or decitabine. Specifically, it is estimated that treatment with the venetoclax and azacitidine combination (hereinafter referred to as "ven / aza treatment" or "ven / aza therapy") induces complete remission (CR) in approximately 70% of treated patients. However, this means that approximately 30% of patients ultimately do not respond to ven / aza therapy and therefore do not achieve complete remission. Furthermore, on average, this remission lasts approximately 18 months, and patients eventually relapse and die from leukemia or complications from targeted leukemia therapy. Therefore, there is a need in the art for new therapies for the treatment of AML, particularly for those who do not respond to ven / aza therapy or relapse after ven / aza therapy.

[0043] This disclosure is based in particular on the discovery that the cell surface protein differentiation cluster 64 (“CD64”) can be used for targeted cell therapy to eliminate a specific subset of AML cells. Therefore, this disclosure provides an anti-CD64 cell immunotherapy for use in treating AML subjects.

[0044] This invention typically provides cells, including immune cells such as T cells, B cells, natural killer (NK) cells, monocytes, macrophages, or artificially generated cells with immune effector functions derived from a patient, a healthy donor, differentiated stem cells (including but not limited to induced pluripotent stem cells (iPSCs), embryonic stem cells, hematopoietic and / or other tissue-specific stem cells), or non-human cells, said cells being genetically modified to express an antigen recognition receptor that binds to CD64 (e.g., a chimeric antigen receptor (CAR)), and a method of using them in the treatment of acute myeloid leukemia. Activation of immune cells (e.g., T cells) is mediated by the binding of the CAR molecule to its homologous antigen (i.e., CD64), and when the CAR binds to its respective homolog, a signal amplification occurs, leading to persistence, antigen sensitivity, and enhanced therapeutic efficacy.

[0045] CARs (sometimes referred to as artificial T-cell receptors, chimeric T-cell receptors (cTCRs), T-bodies, or chimeric immune receptors) are engineered receptors known in the art. They are primarily used to transform immune effector cells, particularly T cells, to provide those cells with the desired antigen-specific and effector functional responses. Adoptive cell therapy using CAR T cells is in the research stage in the field of cancer treatment. In these therapies, T cells are taken from a patient, donor, or derived from stem cells and engineered to express CARs that specifically target antigens found in a particular form of cancer. The CAR T cells, capable of recognizing and killing cancer cells, are then reintroduced into the patient, where they undergo proliferative expansion, clear target antigen-positive cells, and, in a minority of patients, transform into a long-lasting, durable cell population that maintains anti-tumor effector functional activity.

[0046] First-generation CARs induce tumor-killing function by providing TCR-like signaling through intracellular signal transduction domains containing the immune receptor tyrosine activation motif (ITAM), most commonly derived from the CD3ζ (CD3z) molecule. However, in the absence of accompanying co-stimulatory signals, binding to the CD3z chain fusion receptor may be insufficient to induce substantial IL-2 secretion and / or T cell proliferation. In physiological T cell responses, optimal lymphocyte activation requires the binding of one or more co-stimulatory receptors (such as CD28 or 4-1BB). Under the suboptimal activation induced by first-generation CARs, in vivo T cell activity is often transient and fails to control malignant tumors.

[0047] Second-generation CARs are constructed to transduce functional antigen-dependent co-stimulatory signals in human primary T cells in addition to antigen-dependent TCR-like signaling, thereby allowing T cells to proliferate while maintaining tumor-killing activity. The most common method for second-generation CARs is to provide co-stimulation using co-stimulatory domains derived from CD28 or 4-1BB (synonymously, co-stimulatory signal transduction regions). Compared to their first-generation counterparts (CD3z signaling only), the combined delivery of co-stimulation and CD3ζ signaling makes second-generation CARs functionally superior. An example of a second-generation CAR can be found in U.S. Patent No. 7,446,190, which is incorporated herein by reference.

[0048] Third-generation CARs have also been fabricated. These combine multiple co-stimulatory domains (synonymous, co-stimulatory signal transduction regions) with TCR-like signal transduction domains in a cis-regulatory manner, such as CD28+4-1BB+CD3z or CD28+OX40+CD3z, to further enhance potency. In third-generation CARs, the co-stimulatory domains are arranged in tandem within the intracellular domains of the CAR and are typically located upstream of CD3z or its equivalents. However, overall, the results obtained using these third-generation CARs are disappointing, showing only minor improvements compared to second-generation configurations, and some third-generation CARs are even inferior to second-generation configurations.

[0049] This invention provides various CARs that bind to CD64. This disclosure also provides immune cells (e.g., T cells) genetically engineered to express the CARs described herein. As demonstrated in the experimental examples herein, these immune cells containing the CARs of this disclosure exhibit excellent activity against acute myeloid leukemia (AML), including AML subtypes resistant to combination therapy with venetoc / azacitidine. Therefore, this invention overcomes the challenges associated with treating patients who ultimately do not respond to venetoc / aza therapy and thus do not achieve complete remission.

[0050] The chimeric antigen receptor (CAR) disclosed herein

[0051] In some aspects, this disclosure provides a polypeptide (i.e., a CAR) comprising: a) a first domain; b) a second domain; c) a third domain; d) a fourth domain; and e) a fifth domain. In some aspects, the polypeptide comprises an antigen recognition domain that specifically binds to CD64.

[0052] In some aspects of the aforementioned polypeptide, the first domain may comprise, consist substantially of, or be composed of an antigen recognition domain.

[0053] In some aspects of the aforementioned polypeptide, the second domain may comprise, consist substantially of, or be composed of hinge domains.

[0054] In some aspects of the aforementioned polypeptide, the third domain may comprise, consist substantially of, or be composed of the transmembrane domains described herein.

[0055] In some aspects of the aforementioned polypeptide, the fourth domain may comprise, consist substantially of, or be composed of the co-stimulatory domains described herein.

[0056] In some aspects of the aforementioned polypeptide, the fifth domain may comprise, consist substantially of, or be composed of the activation domains described herein.

[0057] Antigen recognition domain

[0058] An antigen recognition domain (also referred to as an "antigen recognition portion") is a molecule or part of a molecule that specifically binds to an antigen. The antigen recognition domain disclosed herein can specifically bind to CD64.

[0059] In some embodiments, the antigen recognition domain of the CAR described herein binds (e.g., specifically binds) to the antigen CD64 described in Table 1. When expressed on the cell surface, the antigen-specific CAR specifically redirects immune cells (e.g., T cells) to the corresponding antigen.

[0060] Table 1. Targets of antigen recognition domains

[0061] In some respects, the antigen recognition domain of the CAR disclosed herein is an antibody, an antibody-like molecule, or a fragment thereof, and the antigen is a tumor antigen.

[0062] In some aspects, the antigen recognition domain of the CAR described herein can recognize an epitope containing a shared space between one or more antigens. In some embodiments, the antigen recognition domain comprises a complementarity-determining region (CDR) of a monoclonal antibody, a variable region of a monoclonal antibody, scFv, VH, VHH, a single-domain antibody (e.g., a camel-like single-domain antibody), an antibody mimic, and / or an antigen-binding fragment thereof. In some embodiments, the specificity of the antigen recognition domain is derived from a protein or peptide (e.g., a ligand in a receptor-ligand pair) that specifically binds to another protein or peptide (e.g., a receptor in a receptor-ligand pair). In some embodiments, the antigen recognition domain comprises an aptamer (nucleic acid aptamer), a T-cell receptor (TCR)-like antibody, or a single-chain TCR (scTCR). Almost any portion that binds to a given target (e.g., a tumor-associated antigen (TAA)) with sufficient affinity can be used as an antigen recognition domain. The arrangement of antigen recognition domains can be multimeric, such as a biantibody or a multimer. In some implementations, the multimer can be formed by cross-pairing of variable portions of the light and heavy chains into a biantibody.

[0063] In some embodiments, the antigen recognition domain of the CAR described herein comprises an antibody mimic. The term "antibody mimic" is intended to describe an organic compound that specifically binds to a target sequence and has a structure different from that of naturally occurring antibodies. Antibody mimics may comprise proteins, nucleic acids, or small molecules. The target sequence specifically bound to the antibody mimics of this disclosure may be an antigen. Exemplary antibody mimics include, but are not limited to: affiliates, affilin, affiliates, affitin, alpha bodies, anticalins, avimers (also known as affinity multimers), DARPin (designed ankylosing repeat protein), fynomers, Kunitz domain peptides, monoclonal antibodies, and centyrin.

[0064] In some embodiments, the antigen recognition domain of the CAR provided herein comprises a single-chain antibody variable region fragment (scFv) derived from a monoclonal antibody specifically targeting CD64. Therefore, the antigen recognition domain of the CAR provided herein may comprise any scFv known in the art that specifically binds to CD64. In some embodiments, the antigen recognition domain of the CAR provided herein comprises fragments of the VH and VL chains of a single-chain antibody variable region fragment (scFv) that specifically binds to CD64.

[0065] In some embodiments, the antigen recognition domain of the CAR provided herein comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen recognition domain of the CAR provided herein comprises a single-chain antibody fragment (scFv) comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) of a monoclonal anti-CD64 antibody. Optionally, the VH and VL can be linked by a flexible linker (such as a glycine-serine linker or a Whitlow linker). In some embodiments, the antigen-binding portion may comprise directionally linked VH and VL, for example, VH-linker-VL or VL-linker-VH from the N-terminus to the C-terminus.

[0066] In some embodiments, the antigen recognition domain of the CAR provided herein includes an scFv whose affinity for CD64 has been optimized to induce cytotoxicity against tumor cells producing high or normal levels of CD64. In some embodiments, the antigen recognition domain of the CAR provided herein includes an scFv whose affinity for CD64 has been optimized to induce cytotoxicity against tumor cells producing low levels of CD64.

[0067] In some embodiments, the antigen recognition domain of the CAR described herein includes a complementarity-determining region (CDR) derived from an anti-CD64 antibody and / or a heavy chain variable domain (VH) and a light chain variable domain (VL).

[0068] In some embodiments, the antigen recognition domain of the CAR described herein may comprise, substantially consist of, or consist of an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0069] The antigen recognition domain of the CAR provided herein may comprise a CDR and / or VH and VL derived from an anti-CD64 antibody (or its antigen-binding fragment). The anti-CD64 antibody disclosed herein may comprise any partial light chain sequence and / or any partial heavy chain sequence known in the art. In some embodiments, the antigen recognition domain of the CAR described herein comprises an scFv containing VH and VL, wherein the VH comprises the amino acid sequence of a VH of an anti-CD64 antibody known in the art, and the VL comprises the amino acid sequence of a corresponding VL known in the art.

[0070] In some embodiments, the antigen recognition domain of the CAR described herein comprises an scFv containing VH and VL, wherein the VH comprises CDRH1, CDRH2, and CDRH3, each comprising the amino acid sequences of CDRH1, CDRH2, and CDRH3 of an anti-CD64 antibody known in the art, and wherein the VL comprises CDRL1, CDRL2, and CDRL3, each comprising the amino acid sequences of CDRL1, CDRL2, and CDRL3 of the same anti-CD64 antibody known in the art. The determination of the CDR region is entirely within the skill of the art. It should be understood that in some embodiments, the CDR may be a combination of the Kabat and Chothia CDRs (also referred to as a "combined CDR" or "extended CDR").

[0071] In some embodiments, the CDR is a Kabat CDR. In other embodiments, the CDR is a Chothia CDR. In still other embodiments, the CDR is an IMGT CDR. In other words, in embodiments having more than one CDR, the CDR can be any combination of Kabat, Chothia, IMGT CDRs, or a combination thereof.

[0072] In some embodiments, the antigen recognition domain of the CAR provided herein may comprise: (i) a VH having CDRH1, CDRH2, and CDRH3, wherein CDRH1 has an amino acid sequence as shown in SEQ ID NO:24 (or a variant of SEQ ID NO:24 having one, two, three, or four amino acid modifications), CDRH2 has an amino acid sequence as shown in SEQ ID NO:25 (or a variant of SEQ ID NO:25 having one, two, three, or four amino acid modifications), and CDRH3 has an amino acid sequence as shown in SEQ ID NO:26 (or a variant of SEQ ID NO:26 having one, two, three, or four amino acid modifications); and / or (ii) a VL having CDRL1, CDRL2, and CDRL3, wherein CDRL1 has an amino acid sequence as shown in SEQ ID NO:32 (or a variant of SEQ ID NO:32 having one, two, three, or four amino acid modifications), and CDRL2 has an amino acid sequence as shown in SEQ ID NO:33 (or a variant of SEQ ID NO:34 having one, two, three, or four amino acid modifications). The amino acid sequence shown in SEQ ID NO:33 variant, and the CDRL3 having the amino acid sequence shown in SEQ ID NO:34 (or a variant of SEQ ID NO:34 with one, two, three, or four amino acid modifications). Examples of such antigen recognition domains having these CDRs and having the ability to bind CD64 include, but are not limited to, antigen recognition domains having VH as shown in SEQ ID NO:31 and VL as shown in SEQ ID NO:39.

[0073] In some embodiments, the antigen recognition domain of the CAR provided herein having the following can comprise any suitable backbone region: (i) a VH having CDRH1, CDRH2, and CDRH3, wherein CDRH1 has the amino acid sequence shown in SEQ ID NO:24 (or a variant of SEQ ID NO:24 having one, two, three, or four amino acid modifications), CDRH2 has the amino acid sequence shown in SEQ ID NO:25 (or a variant of SEQ ID NO:25 having one, two, three, or four amino acid modifications), and CDRH3 has the amino acid sequence shown in SEQ ID NO:26 (or a variant of SEQ ID NO:26 having one, two, three, or four amino acid modifications); and / or (ii) a VL having CDRL1, CDRL2, and CDRL3, wherein CDRL1 has the amino acid sequence shown in SEQ ID NO:32 (or a variant of SEQ ID NO:32 having one, two, three, or four amino acid modifications), and CDRL2 has the amino acid sequence shown in SEQ ID NO:33 (or a variant of SEQ ID NO:34 having one, two, three, or four amino acid modifications). The amino acid sequence shown in the NO:33 variant, and the CDRL3 having the amino acid sequence shown in SEQ ID NO:34 (or the SEQ ID NO:34 variant having one, two, three or four amino acid modifications).For example, such antigen recognition domains may include: (i) a VH containing backbone regions 1, 2, 3, and 4, wherein backbone region 1 has an amino acid sequence as shown in SEQ ID NO:27 (or a variant of SEQ ID NO:27 having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), backbone region 2 has an amino acid sequence as shown in SEQ ID NO:28 (or a variant of SEQ ID NO:28 having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), backbone region 3 has an amino acid sequence as shown in SEQ ID NO:29 (or a variant of SEQ ID NO:29 having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), and backbone region 4 has an amino acid sequence as shown in SEQ ID NO:30 (or a variant of SEQ ID NO:30 having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications). (ii) a VL containing backbone regions 1, 2, 3, and 4, wherein backbone region 1 has the amino acid sequence shown in SEQ ID NO:35 (or a variant of SEQ ID NO:35 having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), backbone region 2 has the amino acid sequence shown in SEQ ID NO:36 (or a variant of SEQ ID NO:36 having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), backbone region 3 has the amino acid sequence shown in SEQ ID NO:37 (or a variant of SEQ ID NO:37 having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), and backbone region 4 has the amino acid sequence shown in SEQ ID NO:38 (or a variant of SEQ ID NO:35 having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications). The amino acid sequence shown in variant NO:38 is shown.

[0074] In some embodiments, the antigen recognition domain of the CAR provided herein may comprise: (i) a VH containing an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:31, and / or (ii) a VL containing an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:39. For example, the CAR provided herein may comprise: (i) a VH containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:31, and / or (ii) a VL containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:39. In some cases, the CAR provided herein may comprise: (i) a VH containing an amino acid sequence that is 100% identical to the amino acid sequence shown in SEQ ID NO:31, and / or (ii) a VL containing an amino acid sequence that is 100% identical to the amino acid sequence shown in SEQ ID NO:39.

[0075] In some embodiments, the antigen recognition domain of the CAR provided herein may include: (i) a VH containing an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:31, provided that the VH contains the amino acid sequences shown in SEQ ID NO:24, 25 and 26; and / or (ii) a VL containing an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:39, provided that the VL contains the amino acid sequences shown in SEQ ID NO:32, 33 and 34. For example, the CAR provided herein may comprise: (i) a VH containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:31, provided that the VH contains the amino acid sequences shown in SEQ ID NO:24, 25, and 26; and / or (ii) a VL containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:39, provided that the VL contains the amino acid sequences shown in SEQ ID NO:32, 33, and 34.

[0076] In some embodiments, the antigen recognition domain of the CAR provided herein may comprise: (i) a VH having CDRH1, CDRH2, and CDRH3, wherein CDRH1 has an amino acid sequence as shown in SEQ ID NO:40 (or a variant of SEQ ID NO:40 having one, two, three, or four amino acid modifications), CDRH2 has an amino acid sequence as shown in SEQ ID NO:41 (or a variant of SEQ ID NO:41 having one, two, three, or four amino acid modifications), and CDRH3 has an amino acid sequence as shown in SEQ ID NO:42 (or a variant of SEQ ID NO:42 having one, two, three, or four amino acid modifications); and / or (ii) a VL having CDRL1, CDRL2, and CDRL3, wherein CDRL1 has an amino acid sequence as shown in SEQ ID NO:48 (or a variant of SEQ ID NO:48 having one, two, three, or four amino acid modifications), and CDRL2 has a VL as shown in SEQ ID NO:49 (or a variant of SEQ ID NO:49 having one, two, three, or four amino acid modifications). The amino acid sequence shown in SEQ ID NO:49 variant, and the CDRL3 having the amino acid sequence shown in SEQ ID NO:50 (or a variant of SEQ ID NO:50 with one, two, three, or four amino acid modifications). Examples of such antigen recognition domains having these CDRs and having the ability to bind CD64 include, but are not limited to, antigen recognition domains having VH as shown in SEQ ID NO:47 and VL as shown in SEQ ID NO:55.

[0077] In some embodiments, the antigen recognition domain of the CAR provided herein having each of the following can comprise any suitable backbone region: (i) a VH having CDRH1, CDRH2, and CDRH3, wherein CDRH1 has the amino acid sequence shown in SEQ ID NO:40 (or a variant of SEQ ID NO:40 having one, two, three, or four amino acid modifications), CDRH2 has the amino acid sequence shown in SEQ ID NO:41 (or a variant of SEQ ID NO:41 having one, two, three, or four amino acid modifications), and CDRH3 has the amino acid sequence shown in SEQ ID NO:42 (or a variant of SEQ ID NO:42 having one, two, three, or four amino acid modifications); and / or (ii) a VL having CDRL1, CDRL2, and CDRL3, wherein CDRL1 has the amino acid sequence shown in SEQ ID NO:48 (or a variant of SEQ ID NO:48 having one, two, three, or four amino acid modifications), and CDRL2 has the amino acid sequence shown in SEQ ID NO:49 (or a variant of SEQ ID NO:49 having one, two, three, or four amino acid modifications). The amino acid sequence shown in the NO:49 variant, and the CDRL3 having the amino acid sequence shown in SEQ ID NO:50 (or the SEQ ID NO:50 variant having one, two, three or four amino acid modifications).For example, such antigen recognition domains may include: (i) a VH containing backbone regions 1, 2, 3, and 4, wherein backbone region 1 has an amino acid sequence as shown in SEQ ID NO:43 (or a variant of SEQ ID NO:43 having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), backbone region 2 has an amino acid sequence as shown in SEQ ID NO:44 (or a variant of SEQ ID NO:44 having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), backbone region 3 has an amino acid sequence as shown in SEQ ID NO:45 (or a variant of SEQ ID NO:45 having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), and backbone region 4 has an amino acid sequence as shown in SEQ ID NO:46 (or a variant of SEQ ID NO:44 having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications). (ii) a VL containing backbone regions 1, 2, 3, and 4, wherein backbone region 1 has the amino acid sequence shown in SEQ ID NO:51 (or a variant of SEQ ID NO:51 having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), backbone region 2 has the amino acid sequence shown in SEQ ID NO:52 (or a variant of SEQ ID NO:52 having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), backbone region 3 has the amino acid sequence shown in SEQ ID NO:53 (or a variant of SEQ ID NO:53 having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), and backbone region 4 has the amino acid sequence shown in SEQ ID NO:54 (or a variant of SEQ ID NO:53 having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications). The amino acid sequence shown in the NO:54 variant is as follows.

[0078] In some embodiments, the antigen recognition domain of the CAR provided herein may comprise: (i) a VH containing an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:47, and / or (ii) a VL containing an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:55. For example, the CAR provided herein may comprise: (i) a VH containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:47, and / or (ii) a VL containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:55. In some cases, the CAR provided herein may comprise: (i) a VH containing an amino acid sequence that is 100% identical to the amino acid sequence shown in SEQ ID NO:47, and / or (ii) a VL containing an amino acid sequence that is 100% identical to the amino acid sequence shown in SEQ ID NO:55.

[0079] In some embodiments, the antigen recognition domain of the CAR provided herein may include: (i) a VH containing an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:47, provided that the VH contains the amino acid sequences shown in SEQ ID NO:40, 41 and 42; and / or (ii) a VL containing an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:55, provided that the VL contains the amino acid sequences shown in SEQ ID NO:48, 49 and 50. For example, the CAR provided herein may comprise: (i) a VH containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:47, provided that the VH contains the amino acid sequences shown in SEQ ID NO:40, 41, and 42; and / or (ii) a VL containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:55, provided that the VL contains the amino acid sequences shown in SEQ ID NO:48, 49, and 50.

[0080] In some embodiments, the antigen recognition domain of the CAR provided herein may include: (a) VH, wherein the VH includes (i) CDRH1 consisting of, substantially composed of or composed of, the amino acid sequence shown in SEQ ID NO:24, (ii) CDRH2 consisting of, substantially composed of or composed of, the amino acid sequence shown in SEQ ID NO:25, and (iii) CDRH3 consisting of, substantially composed of or composed of, the amino acid sequence shown in SEQ ID NO:26; and / or (b) VL, wherein the VL includes (i) CDRL1 consisting of, substantially composed of or composed of, the amino acid sequence shown in SEQ ID NO:32, (ii) CDRL2 consisting of, substantially composed of or composed of, the amino acid sequence shown in SEQ ID NO:33, and (iii) CDRL3 consisting of, substantially composed of or composed of, the amino acid sequence shown in SEQ ID NO:34. As used herein, "CDRH1 consisting essentially of the amino acid sequence shown in SEQ ID NO:24" means CDRH1 having zero, one, or two amino acid substitutions within SEQ ID NO:24, CDRH1 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO:24, and / or CDRH1 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO:24, provided that the CAR retains its basic ability to bind CD64. Examples of CDRH1 consisting essentially of the amino acid sequence shown in SEQ ID NO:24 include, but are not limited to, those described in Table 2.

[0081] Table 2. Exemplary CDRH1 consisting essentially of an amino acid sequence as shown in SEQ ID NO:24.

[0082]

[0083] As used herein, "CDRH2 consisting essentially of the amino acid sequence shown in SEQ ID NO:25" means CDRH2 having zero, one, or two amino acid substitutions within SEQ ID NO:25, CDRH2 having zero, one, two, three, four, or five amino acid residues before SEQ ID NO:25, and / or CDRH2 having zero, one, two, three, four, or five amino acid residues after SEQ ID NO:25, provided that the CAR retains its basic ability to bind CD64. Examples of CDRH2 consisting essentially of the amino acid sequence shown in SEQ ID NO:25 include, but are not limited to, those described in Table 3.

[0084] Table 3. An exemplary CDRH2 consisting essentially of an amino acid sequence as shown in SEQ ID NO:25.

[0085]

[0086] As used herein, "CDRH3 consisting essentially of the amino acid sequence shown in SEQ ID NO:26" means CDRH3 having zero, one, or two amino acid substitutions within SEQ ID NO:26, CDRH3 having zero, one, two, three, four, or five amino acid residues before SEQ ID NO:26, and / or CDRH3 having zero, one, two, three, four, or five amino acid residues after SEQ ID NO:26, provided that the CAR retains its basic ability to bind CD64. Examples of CDRH3 consisting essentially of the amino acid sequence shown in SEQ ID NO:26 include, but are not limited to, those described in Table 4.

[0087] Table 4. An exemplary CDRH3 consisting essentially of an amino acid sequence as shown in SEQ ID NO:26.

[0088]

[0089] As used herein, "CDRL1 consisting essentially of the amino acid sequence shown in SEQ ID NO:32" means CDRL1 having zero, one, or two amino acid substitutions within SEQ ID NO:32, CDRL1 having zero, one, two, three, four, or five amino acid residues before SEQ ID NO:32, and / or CDRL1 having zero, one, two, three, four, or five amino acid residues after SEQ ID NO:32, provided that the CAR retains its basic ability to bind CD64. Examples of CDRL1 consisting essentially of the amino acid sequence shown in SEQ ID NO:32 include, but are not limited to, those described in Table 5.

[0090] Table 5. An exemplary CDRL1 consisting essentially of an amino acid sequence as shown in SEQ ID NO:32.

[0091]

[0092] As used herein, "CDRL2 consisting essentially of the amino acid sequence shown in SEQ ID NO:33" means CDRL2 having zero, one, or two amino acid substitutions within SEQ ID NO:33, CDRL2 having zero, one, two, three, four, or five amino acid residues before SEQ ID NO:33, and / or CDRL2 having zero, one, two, three, four, or five amino acid residues after SEQ ID NO:33, provided that the CAR retains its basic ability to bind CD64. Examples of CDRL2 consisting essentially of the amino acid sequence shown in SEQ ID NO:33 include, but are not limited to, those described in Table 6.

[0093] Table 6. An exemplary CDRL2 consisting essentially of an amino acid sequence as shown in SEQ ID NO:33.

[0094]

[0095] As used herein, "CDRL3 consisting essentially of the amino acid sequence shown in SEQ ID NO:34" means CDRL3 having zero, one, or two amino acid substitutions within SEQ ID NO:34, CDRL3 having zero, one, two, three, four, or five amino acid residues before SEQ ID NO:34, and / or CDRL3 having zero, one, two, three, four, or five amino acid residues after SEQ ID NO:34, provided that the CAR retains its basic ability to bind CD64. Examples of CDRL3 consisting essentially of the amino acid sequence shown in SEQ ID NO:34 include, but are not limited to, those described in Table 7.

[0096] Table 7. An exemplary CDRL3 consisting essentially of an amino acid sequence as shown in SEQ ID NO:34.

[0097]

[0098] In some embodiments, the antigen recognition domain of the CAR provided herein may include: (a) VH, wherein the VH includes (i) CDRH1 consisting of, substantially composed of or composed of, the amino acid sequence shown in SEQ ID NO:40, (ii) CDRH2 consisting of, substantially composed of or composed of, the amino acid sequence shown in SEQ ID NO:41, and (iii) CDRH3 consisting of, substantially composed of or composed of, the amino acid sequence shown in SEQ ID NO:42; and / or (b) VL, wherein the VL includes (i) CDRL1 consisting of, substantially composed of or composed of, the amino acid sequence shown in SEQ ID NO:48, (ii) CDRL2 consisting of, substantially composed of or composed of, the amino acid sequence shown in SEQ ID NO:49, and (iii) CDRL3 consisting of, substantially composed of or composed of, the amino acid sequence shown in SEQ ID NO:50. As used herein, "CDRH1 consisting essentially of the amino acid sequence shown in SEQ ID NO:40" means CDRH1 having zero, one, or two amino acid substitutions within SEQ ID NO:40, CDRH1 having zero, one, two, three, four, or five amino acid residues before SEQ ID NO:40, and / or CDRH1 having zero, one, two, three, four, or five amino acid residues after SEQ ID NO:40, provided that the CAR retains its basic ability to bind CD64. Examples of CDRH1 consisting essentially of the amino acid sequence shown in SEQ ID NO:40 include, but are not limited to, those described in Table 8.

[0099] Table 8. An exemplary CDRH1 consisting essentially of an amino acid sequence as shown in SEQ ID NO:40.

[0100]

[0101] As used herein, "CDRH2 consisting essentially of the amino acid sequence shown in SEQ ID NO:41" means CDRH2 having zero, one, or two amino acid substitutions within SEQ ID NO:41, CDRH2 having zero, one, two, three, four, or five amino acid residues before SEQ ID NO:41, and / or CDRH2 having zero, one, two, three, four, or five amino acid residues after SEQ ID NO:41, provided that the CAR retains its basic ability to bind CD64. Examples of CDRH2 consisting essentially of the amino acid sequence shown in SEQ ID NO:41 include, but are not limited to, those described in Table 9.

[0102] Table 9. An exemplary CDRH2 consisting essentially of an amino acid sequence as shown in SEQ ID NO:41.

[0103]

[0104] As used herein, "CDRH3 consisting essentially of the amino acid sequence shown in SEQ ID NO:42" means CDRH3 having zero, one, or two amino acid substitutions within SEQ ID NO:42, CDRH3 having zero, one, two, three, four, or five amino acid residues before SEQ ID NO:42, and / or CDRH3 having zero, one, two, three, four, or five amino acid residues after SEQ ID NO:42, provided that the CAR retains its basic ability to bind CD64. Examples of CDRH3 consisting essentially of the amino acid sequence shown in SEQ ID NO:42 include, but are not limited to, those described in Table 10.

[0105] Table 10. An exemplary CDRH3 consisting essentially of an amino acid sequence as shown in SEQ ID NO:42.

[0106]

[0107] As used herein, "CDRL1 consisting essentially of the amino acid sequence shown in SEQ ID NO:48" means CDRL1 having zero, one, or two amino acid substitutions within SEQ ID NO:48, CDRL1 having zero, one, two, three, four, or five amino acid residues before SEQ ID NO:48, and / or CDRL1 having zero, one, two, three, four, or five amino acid residues after SEQ ID NO:48, provided that the CAR retains its basic ability to bind CD64. Examples of CDRL1 consisting essentially of the amino acid sequence shown in SEQ ID NO:48 include, but are not limited to, those described in Table 11.

[0108] Table 11. An exemplary CDRL1 consisting essentially of an amino acid sequence as shown in SEQ ID NO:48.

[0109]

[0110] As used herein, "CDRL2 consisting essentially of the amino acid sequence shown in SEQ ID NO:49" means CDRL2 having zero, one, or two amino acid substitutions within SEQ ID NO:49, CDRL2 having zero, one, two, three, four, or five amino acid residues before SEQ ID NO:49, and / or CDRL2 having zero, one, two, three, four, or five amino acid residues after SEQ ID NO:49, provided that the CAR retains its basic ability to bind CD64. Examples of CDRL2 consisting essentially of the amino acid sequence shown in SEQ ID NO:49 include, but are not limited to, those described in Table 12.

[0111] Table 12. An exemplary CDRL2 consisting essentially of an amino acid sequence as shown in SEQ ID NO:49.

[0112]

[0113] As used herein, "CDRL3 consisting essentially of the amino acid sequence shown in SEQ ID NO:50" means a CDRL3 having zero, one, or two amino acid substitutions within SEQ ID NO:50, a CDRL3 having zero, one, two, three, four, or five amino acid residues before SEQ ID NO:50, and / or a CDRL3 having zero, one, two, three, four, or five amino acid residues after SEQ ID NO:50, provided that the CAR retains its basic ability to bind CD64. Examples of CDRL3 consisting essentially of the amino acid sequence shown in SEQ ID NO:50 include, but are not limited to, those described in Table 13.

[0114] Table 13. An exemplary CDR3 consisting essentially of an amino acid sequence as shown in SEQ ID NO:50.

[0115]

[0116] The representative number of antigen recognition domains of the CARs provided in this article are further described in Table 14.

[0117] Table 14. Representative antigen recognition domains

[0118] signal peptide

[0119] In some embodiments, any CAR provided herein may further comprise a signal peptide (also referred to in the art as a signal peptide, signal sequence, signal peptide sequence, leader peptide, and leader peptide sequence). In some embodiments, the first domain of the CAR described herein comprises a signal peptide or leader peptide sequence. Exemplary signal sequences include, but are not limited to, CD8α signal sequences or IgG signal sequences. In some embodiments, the CAR described herein does not comprise a signal peptide.

[0120] In some implementations, the CAR (e.g., the first domain of the CAR) may contain a human CD8α signal sequence.

[0121] In some embodiments, the CAR (e.g., the first recognition domain of the CAR) may comprise a human IgG signaling sequence. In some aspects, the hinge domain of the CAR of this disclosure may comprise, or consist substantially of, an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity with SEQ ID NO: 23.

[0122] Hinge structural domain

[0123] In some embodiments, a hinge domain (also referred to in the art as a spacer region or stalk region) is located between the antigen recognition domain and the transmembrane domain of the CAR. Specifically, the stalk region provides greater flexibility and accessibility to the extracellular antigen recognition domain. In some embodiments, the hinge domain may contain up to about 300 amino acids. In some embodiments, the hinge length comprises about 10 to about 100 amino acids. In some embodiments, the hinge length comprises about 25 to about 50 amino acids. In some embodiments, the hinge domain establishes an optimal effector cell-target cell membrane distance. In some embodiments, the hinge domain provides flexibility for the antigen recognition domain to bind to the target antigen. Any stable and / or dimerizing protein can be used for this purpose.

[0124] The hinge domain may be derived from all or part of a naturally occurring molecule, such as from all or part of the extracellular region of CD8α or CD28, or from all or part of the constant region of the antibody heavy chain. Alternatively, the hinge domain may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or it may be a fully synthetic hinge sequence.

[0125] In some embodiments, the hinge domain is a portion of the human CD8α chain (e.g., NP_001139345.1). In some embodiments, the hinge domain of the CAR described herein comprises a wild-type form or a mutated subsequence of CD8α or CD28 to avoid binding to the Fc receptor, specifically the hinge domain of either CD8α or CD28. In some embodiments, the stalk region comprises a human CD8α hinge or a human CD28 hinge.

[0126] In some embodiments, the hinge domain of the CAR of this disclosure may contain an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity with SEQ ID NO: 4, or be substantially composed of such amino acid sequence, or be composed of such amino acid sequence.

[0127] In some embodiments, the hinge domain of the CAR of this disclosure may contain an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identity with SEQ ID NO: 8, or be substantially composed of such amino acid sequence, or be composed of such amino acid sequence.

[0128] Transmembrane domain

[0129] The transmembrane domains of the CARs disclosed herein have the following capabilities: (a) expression on the cell surface, in some embodiments of which are immune cells, such as, for example, T cells; and / or (b) interaction with ligand-binding domains and intracellular signal transduction domains to guide a cellular response of the immune cell against a predetermined target cell. The transmembrane domains may be derived from natural or synthetic sources. The transmembrane domains may be derived from any membrane-binding or transmembrane protein. The transmembrane domains may include transmembrane regions of CD8α and / or CD28. In some embodiments, the transmembrane domains comprise a CD8α transmembrane domain. In some embodiments, the transmembrane domains comprise a CD28 transmembrane domain.

[0130] Alternatively, the transmembrane domain may be synthetic and may contain hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine is found at one or both ends of the synthetic transmembrane domain. Optionally, short oligonucleotides or peptide linkers (between 2 and 10 amino acids in length in some embodiments) may form a link between the transmembrane domain and the intracellular domain of the CAR. In some embodiments, the linker is a glycine-serine linker.

[0131] In some embodiments, the transmembrane domain of the CAR of this disclosure may comprise, or consist substantially of, an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) the same as SEQ ID NO: 5.

[0132] In some embodiments, the transmembrane domain of the CAR of this disclosure may comprise, or consist substantially of, an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) the identity with SEQ ID NO: 9.

[0133] Co-stimulatory domain

[0134] The intracellular domains of the CAR described herein may include one or more co-stimulatory domains. The co-stimulatory domains may include 4-1BB (CD137) and / or CD28 co-stimulatory domains, fragments thereof, or combinations thereof. In some embodiments, the CAR described herein includes a CD28 co-stimulatory domain or a fragment thereof. In some embodiments, the CAR described herein includes a 4-1BB (CD137) co-stimulatory domain or a fragment thereof.

[0135] In some embodiments, the co-stimulatory domain of the CAR of this disclosure may comprise, or consist substantially of, an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) the same as SEQ ID NO: 6.

[0136] In some embodiments, the co-stimulatory domain of the CAR of this disclosure may comprise, or consist substantially of, an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) the same as SEQ ID NO: 10.

[0137] Activated domain

[0138] In some embodiments, the activation domain of the CAR disclosed herein is responsible for activating at least one normal effector function of an immune cell (e.g., a T cell) in which the CAR is expressed. The terms “intracellular signal transduction domain” or “intracellular domain” are used interchangeably and refer to a domain comprising a co-stimulatory domain and / or an activation domain. The term “effector function” refers to a specialized function of the cell. For example, the effector function of a T cell may be cytolytic activity or an auxiliary activity including the secretion of cytokines. The term “activation domain” refers to a portion of a protein that transduces effector function signals and directs the cell to perform its specialized function. While the entire activation domain can generally be used, in many cases it is not necessary to use the entire chain. In cases where a truncated portion of the activation domain is used, such a truncated portion can be used in place of the complete chain as long as it transduces effector function signals. Therefore, the term “activation domain” is intended to include any truncated portion of the activation domain sufficient to transduce effector function signals. In some embodiments, the activation domain further comprises a signal transduction domain for T cell activation. In some instances, the signal transduction domain for T cell activation comprises an intracellular domain derived from CD3ζ (CD3z). In some embodiments, the CAR described herein includes at least one (e.g., one, two, three or more) activation domains selected from CD3ζ or fragments thereof. In some embodiments, the CAR described herein has activation domains comprising domains derived from CD3ζ (CD3z).

[0139] In some embodiments, the activation domain of the CAR of this disclosure may comprise, or consist substantially of, an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) the identity with SEQ ID NO: 7.

[0140] In some embodiments, the CD3ζ activating domain comprises a mutation in the ITAM domain. Examples of mutations in the ITAM domain of CD3ζ are provided in Feucht et al., NatMed. 2019; 25(1): 82–88. In some embodiments, two tyrosine residues in one or more of the ITAM1, ITAM2, or ITAM3 domains of the CD3ζ activating domain are each point-mutated to phenylalanine residues. In some embodiments, the CD3ζ activating domain comprises a deletion of one or more of the ITAM1, ITAM2, or ITAM3 domains.

[0141] Table 15 provides exemplary amino acid sequences that can be used for the domains in the CARs described herein. In some embodiments, the CARs provided herein comprise one or more domains, or fragments or portions thereof, as described in Table 15.

[0142] Table 15. Exemplary amino acid sequences of the CAR domain

[0143] This disclosure of nucleic acid

[0144] Nucleic acids encoding any of the CARs described herein are also provided. The nucleic acids encoding the CARs may be humanized. In some embodiments, the nucleic acids encoding the CARs provided herein are codon-optimized for expression in human cells. In some embodiments, this disclosure provides a full-length CAR cDNA or coding region. The scope of this invention includes nucleic acid sequences encoding functional portions of the CARs described herein. Functional portions encompass, for example, those portions of the CAR that retain, to a degree similar to, the same as, or greater than that of the parent CAR, the ability to recognize target cells or to detect, treat, or prevent disease.

[0145] Exemplary anti-CD64 CAR construct

[0146] This document discloses a CD64-specific CAR. In some embodiments, the CAR comprises an antigen recognition domain that specifically binds to human CD64, a hinge domain comprising or composed of a CD8α hinge domain or a CD28 hinge domain, a transmembrane domain comprising or composed of a CD8α transmembrane domain or a CD28 transmembrane domain, a costimulatory domain comprising or composed of a 4-1BB costimulatory domain or a CD28 costimulatory domain, and an intracellular signal transduction domain comprising or composed of a CD3ζ activation domain. The document also discloses the nucleic acid sequence encoding the CAR. In some embodiments, the T cells or T cell populations described herein are genetically modified to express at least one of the exemplary anti-CD64 CAR constructs described herein.

[0147] 611 scfv CAR construct

[0148] An exemplary amino acid sequence of the anti-CD64 CAR “611-41BBz” is shown below. CD8α signal peptide CD64 scFv(611), CD8α hinge, CD8α transmembrane domain , 4-1BB signal transduction domain , CD3z signal transduction domain )

[0149] In some embodiments, the anti-CD64 CAR provided herein may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 11, or be substantially composed of the amino acid sequence, or be composed of the amino acid sequence.

[0150] An exemplary polynucleotide sequence of the anti-CD64 CAR “611-41BBz” is shown below. CD8α signal peptide CD64 scFv (611), CD8α hinge, CD8α transmembrane domain , 4-1BB signal transduction domain , CD3z signal transduction structure domain )

[0151] In some embodiments, the anti-CD64 CAR provided herein is encoded by a polynucleotide sequence comprising, or substantially comprising, a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO: 17.

[0152] An exemplary amino acid sequence of the anti-CD64 CAR “611-CD28z” is shown below. CD8α signal peptide CD64scFv (611), CD28 hinge, CD28 transmembrane domain , CD28 signal transduction domain , CD3z signal transduction domain )

[0153] In some embodiments, the anti-CD64 CAR provided herein may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 14, or be substantially composed of the amino acid sequence, or be composed of the amino acid sequence.

[0154] An exemplary polynucleotide sequence of the anti-CD64 CAR “611-CD28z” is shown below. CD8α signal peptide CD64 scFv (611), CD28 hinge, CD28 transmembrane domain , CD28 signal transduction domain , CD3z signal transduction structure domain )

[0155] In some embodiments, the anti-CD64 CAR provided herein is encoded by a polynucleotide sequence comprising, or substantially comprising, a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO: 18.

[0156] H22 scfv CAR construct

[0157] An exemplary amino acid sequence of the anti-CD64 CAR “H22-41BBz” is shown below. CD8α signal peptide CD64scFv (H22), CD8α hinge, CD8α transmembrane domain , 4-1BB signal transduction domain CD3z signal transduction domain

[0158] In some embodiments, the anti-CD64 CAR provided herein may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 12, or be substantially composed of the amino acid sequence, or be composed of the amino acid sequence.

[0159] An exemplary polynucleotide sequence of the anti-CD64 CAR “H22-41BBz” is shown below. CD8α signal peptide CD64 scFv (H22), CD8α hinge, CD8α transmembrane domain , 4-1BB signal transduction domain , CD3z signal transduction structure domain )

[0160] In some embodiments, the anti-CD64 CAR provided herein is encoded by a polynucleotide sequence comprising, or substantially comprising, a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO: 19.

[0161] An exemplary amino acid sequence of the anti-CD64 CAR “H22-CD28z” is shown below. CD8α signal peptide CD64scFv (H22), CD28 hinge, CD28 transmembrane domain , CD28 signal transduction domain, CD3z signal transduction domain )

[0162] In some embodiments, the anti-CD64 CAR provided herein may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 15, or be substantially composed of the amino acid sequence, or be composed of the amino acid sequence.

[0163] An exemplary polynucleotide sequence of the anti-CD64 CAR “H22-CD28z” is shown below. CD8α signal peptide CD64 scFv (H22), CD28 hinge, CD28 transmembrane domain , CD28 signal transduction domain, CD3z signal transduction structure domain )

[0164] In some embodiments, the anti-CD64 CAR provided herein is encoded by a polynucleotide sequence comprising, or substantially comprising, a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO: 20.

[0165] M22 scfv CAR construct

[0166] An exemplary amino acid sequence of the anti-CD64 CAR “M22-41BBz” is shown below. CD8α signal peptide CD64scFv (M22), CD8α hinge, CD8α transmembrane domain , 4-1BB signal transduction domain, CD3z signal transduction domain )

[0167] In some embodiments, the anti-CD64 CAR provided herein may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 13, or be substantially composed of the amino acid sequence, or be composed of the amino acid sequence.

[0168] An exemplary polynucleotide sequence of the anti-CD64 CAR “M22-41BBz” is shown below. CD8α signal peptide CD64 scFv (M22), CD8α hinge, CD8α transmembrane domain 4-1BB signal transduction structure domain, CD3z signal transduction structure domain )

[0169] In some embodiments, the anti-CD64 CAR provided herein is encoded by a polynucleotide sequence comprising, or substantially comprising, a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO: 21.

[0170] An exemplary amino acid sequence of the anti-CD64 CAR “M22-CD28z” is shown below. CD8α signal peptide CD64scFv (M22), CD28 hinge, CD28 transmembrane domain , CD28 signal transduction domain, CD3z signal transduction domain )

[0171] In some embodiments, the anti-CD64 CAR provided herein may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 16, or be substantially composed of the amino acid sequence, or be composed of the amino acid sequence.

[0172] An exemplary polynucleotide sequence of the anti-CD64 CAR “M22-CD28z” is shown below. CD8α signal peptide CD64 scFv (M22), CD28 hinge, CD28 transmembrane domain , CD28 signal transduction domain, CD3z signal transduction structure domain )

[0173] In some embodiments, the anti-CD64 CAR provided herein is encoded by a polynucleotide sequence comprising, or consisting of, a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO: 22.

[0174] In some embodiments, the genetically engineered cells include an additional CAR, including an activating or stimulating CAR, a co-stimulating CAR (see, for example, PCT Publication No. WO 2014 / 055668), and / or an inhibitory CAR (iCAR, see, for example, Fedorov et al., 2013). The CAR typically includes an extracellular antigen (or ligand) recognition domain that is linked in some respects to one or more intracellular signaling components via a linker and / or one or more transmembrane domains. Such molecules typically mimic or approximate signals transmitted through natural antigen receptors, signals transmitted through such receptors in combination with co-stimulating receptors, and / or signals transmitted through co-stimulating receptors alone. For example, once an antigen is recognized by the extracellular antigen recognition domain, the intracellular signaling component delivers an activation signal to the T cell, thereby inducing the T cell to destroy target tumor cells.

[0175] In embodiments described herein, the CAR contains additional amino acids at the amino-terminus or carboxyl-terminus, or both, of the portion, which are not present in the amino acid sequence of the parent CAR. Ideally, these additional amino acids do not interfere with the biological function of the functional portion, such as recognizing target cells, detecting cancer, treating or preventing cancer, etc. More ideally, these additional amino acids enhance the biological activity of the CAR compared to the biological activity of the parent CAR.

[0176] As used herein when referring to CAR, the term "functional variant" refers to a CAR, polypeptide, or protein that has substantial or significant sequence identity or similarity to the CAR encoded by a nucleic acid sequence, and that retains the biological activity of the CAR from which it is derived as a variant. Functional variants, for example, encompass those variants of the CAR (parental CAR) described herein that retain the ability to recognize target cells to a degree similar to, the same as, or greater than that of the parental CAR. When referring to the nucleic acid sequence encoding the parental CAR, the nucleic acid sequence of the functional variant encoding the CAR may, for example, have approximately 10%, 25%, 30%, 50%, 65%, 80%, 90%, 95%, or 99% identity with the nucleic acid sequence encoding the parental CAR.

[0177] The CARs described herein (including their functional portions and functional variants) include CARs that have undergone glycosylation, amidation, carboxylation, phosphorylation, esterification, N-acylation, cyclization via, for example, disulfide bond cyclization, or conversion to acid addition salts, and / or optionally dimerization or polymerization.

[0178] As described herein, the amino acid sequences described herein may contain amino acid modifications (e.g., modifications of a specified number of amino acids). Such amino acid modifications may include, but are not limited to, amino acid substitutions, amino acid deletions, amino acid additions, and combinations thereof. In some cases, amino acid modifications may be made to improve binding and / or contact with the antigen, and / or to improve the functional activity of the CAR provided herein. In some cases, amino acid substitutions within a specified sequence identifier may be conserved amino acid substitutions. For example, conserved amino acid substitutions can be made by replacing one amino acid residue with another amino acid residue having a similar side chain. A family of amino acid residues with similar side chains can include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0179] In some cases, amino acid substitutions within an explicitly stated sequence identifier can be nonconservative amino acid substitutions. Nonconservative amino acid substitutions can be made by replacing one amino acid residue with another amino acid residue having a different side chain. Examples of nonconservative substitutions include, but are not limited to: (a) replacing a hydrophilic residue (e.g., serine or threonine) with a hydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine, or alanine); (b) replacing cysteine ​​or proline with any other residue; (c) replacing a residue with a basic side chain (e.g., lysine, arginine, or histidine) with a residue with an acidic side chain (e.g., aspartic acid or glutamic acid); and (d) replacing a residue with a large side chain (e.g., phenylalanine) with glycine or other residues with small side chains.

[0180] Methods for generating amino acid sequence variants (e.g., amino acid sequences containing one or more modifications relative to a well-defined sequence identifier) ​​may involve site-directed or random mutagenesis (e.g., by PCR) of nucleic acids encoding antibodies or fragments thereof. See, for example, Zoller, Curr. Opin. Biotechnol. 3: 348-354 (1992). Both naturally occurring and non-naturally occurring amino acids (e.g., artificially derived amino acids) can be used to generate the amino acid sequence variants provided herein.

[0181] The immune cells disclosed herein

[0182] This disclosure provides cells and cell populations comprising the above-described CAR constructs and / or nucleic acid molecules encoding the CAR constructs.

[0183] Therefore, this disclosure provides a genetically modified population of immune cells (e.g., T cells) engineered to express the aforementioned CAR and / or polynucleotides encoding said CAR. These immune cells may be T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or γ-δ T cells), NK cells, invariant NK cells, NKT cells, or stem cells (e.g., mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs)). In some embodiments, the cells are monocytes or granulocytes, such as bone marrow-derived cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. Methods for preparing and engineering these immune cells, as well as methods for using and administering these cells to a subject, are also provided, in which case the cells may be autologous or allogeneic. Therefore, these immune cells can be used as immunotherapies, such as for targeting cancer cells.

[0184] In some embodiments, the cell comprises one or more nucleic acids encoding one or more antigen receptors, introduced through genetic engineering, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acid is heterologous. In some embodiments, the nucleic acid is non-naturally occurring, such as nucleic acids not found in nature (e.g., chimeric).

[0185] The immune cells can be isolated from a subject, specifically a human subject. The immune cells can be obtained from a target subject, such as a subject suspected of having a specific disease or condition, a subject suspected of being susceptible to a specific disease or condition, or a subject receiving treatment for a specific disease or condition. The immune cells can be enriched / purified from any tissue in which they are located, including but not limited to blood (including blood collected from a blood bank or cord blood bank), spleen, bone marrow, tissue removed and / or exposed during surgery, and tissue obtained through a biopsy procedure. The tissue / organ from which the immune cells are enriched, isolated, and / or purified can be isolated from both living and non-living subjects, where the non-living subject is an organ donor. The isolated immune cells can be used directly or can be stored for a period of time, for example, by freezing. In some embodiments, the immune cells are isolated from blood, such as peripheral blood or cord blood. In some embodiments, immune cells isolated from cord blood have enhanced immunomodulatory capabilities, measured, for example, by inhibition of CD4-positive or CD8-positive T cells. In certain aspects, the immune cells are isolated from pooled blood, particularly pooled cord blood, to obtain enhanced immunomodulatory capabilities. The mixed blood can come from two or more sources, such as three, four, five, six, seven, eight, nine, ten or more sources (e.g., donors).

[0186] The immune cell population can be obtained from a subject requiring treatment or suffering from a disease associated with reduced immune cell activity. Therefore, the cells will be autologous to the subject requiring treatment. Alternatively, the immune cell population can be obtained from a donor. The immune cell population can be collected from the peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue in which the immune cells reside, of the subject or donor. The immune cells can be isolated from a mixture of subjects and / or a mixture of donors, for example, from mixed umbilical cord blood. The immune cell population can be derived from induced pluripotent stem cells (iPSCs) and / or any other stem cells known in the art. In some aspects, the iPSCs and / or stem cells used to derive the immune cell population can be obtained from a subject requiring treatment or suffering from a disease associated with reduced immune cell activity, thus these iPSCs and / or stem cells will be autologous to the subject requiring treatment. Alternatively, the iPSCs and / or stem cells can be obtained from a donor, thus being allogeneic to the subject requiring treatment.

[0187] When the immune cell population is obtained from a donor different from the subject, the donor is preferably allogeneic, provided that the obtained cells are compatible with the subject so that they can be introduced into the subject. Allogeneic donor cells may or may not be human leukocyte antigen (HLA) compatible. To make them compatible with the subject, allogeneic cells can be treated to reduce immunogenicity.

[0188] T cells

[0189] T cells play a major role in cell-mediated immunity (without antibody involvement). Their T cell receptors (TCRs) distinguish them from other lymphocyte types. The thymus (a specialized organ of the immune system) is primarily responsible for T cell maturation. There are six types of T cells: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T cells, or cytotoxic T cells), and memory T cells (i) stem cell-like memory TCM cells, similar to primordial / immature cells, expressing CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Ra+, but they also express large amounts of CD95, IL-2R, CXCR3, and LFA-1, and exhibit many functional properties specific to memory cells; (ii) central memory TCM cells expressing L-selectin and CCR7, secreting IL-2 but not IFNg or IL-4, and (iii) Effector memory TEM cells, however, do not express L-selectin or CCR7, but produce effector cytokines such as IFNg and IL-4, regulatory T cells (Treg, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and γ-δ T cells.

[0190] T cells for immunotherapy can be derived from any source known in the art. For example, T cells can be differentiated from hematopoietic stem cell populations in vitro, or T cells can be obtained from the subject. T cells can be obtained from, for example, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from unit blood collected from the subject using any number of techniques known to those skilled in the art, such as FICOLL™ isolation and / or component apheresis. Other methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, the entire contents of which are incorporated herein by reference.

[0191] CAR expression level

[0192] This disclosure provides an engineered T cell population wherein a plurality of engineered T cells in the population contain any chimeric stimulatory receptor (CAR) disclosed herein. This disclosure also provides a composition comprising a T cell population wherein a plurality of T cells in the population contain a non-naturally occurring CAR comprising, being primarily composed of, or consisting of a chimeric antigen receptor (CAR) containing an antigen recognition domain, a transmembrane domain, and a signal transduction domain that binds to CD64. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population contains any chimeric stimulatory receptor (CAR) disclosed herein. In some embodiments, the CAR peptide is expressed at a copy number of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 copies per cell. In some embodiments, the nucleic acid encoding the CAR is integrated into the genome at a copy number of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 copies per cell.

[0193] In some embodiments, the genetically engineered T cell population disclosed herein exhibits T cell functions (e.g., effector functions). In some embodiments, this cell population is cytotoxic to cells expressing CD64 (e.g., CD64-positive tumor cells, CD64-low expression tumor cells). For example, the effector function of the T cells can be cell lysis activity or helper activity including cytokine secretion. In some embodiments, the cell population exhibits levels of one or more T cell effector functions that are at least 3-4 times higher than those exhibited by a T cell population that does not express CAR.

[0194] Method for preparing the cells disclosed herein

[0195] Chimeric antigen receptors can be readily inserted into and expressed by immune cells (e.g., T cells). In some embodiments, the cells (e.g., immune cells such as T cells) are obtained from a donor subject. In some embodiments, the donor subject is a human patient with cancer or a tumor. In other embodiments, the donor subject is a human patient without cancer or a tumor. In some embodiments, the engineered cells are autologous to the subject. In some embodiments, the engineered cells are allogeneic to the subject.

[0196] The cells disclosed herein can be obtained from any source known in the art. For example, T cells can be differentiated in vitro from hematopoietic stem cell populations, or T cells can be obtained from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from unit blood collected from a subject using any number of techniques known to those skilled in the art (e.g., FICOLL™ isolation and / or apheresis). In some embodiments, cells collected by apheresis are washed to remove plasma components and placed in a suitable buffer or culture medium for subsequent processing. In some embodiments, cells are washed with PBS. It is understood that a washing step can be used, for example, by using a semi-automated flow centrifuge, such as the Cobe™ 2991 cell processor, Baxter CytoMate™, or similar devices. In some embodiments, the washed cells are resuspended in one or more biocompatible buffers or other salt solutions containing or without buffers. In some embodiments, unwanted components are removed from the component apheresis sample. Other methods for isolating T cells for T-cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, the entire contents of which are incorporated herein by reference.

[0197] In some embodiments, T cells are isolated from PBMCs by lysing red blood cells and removing monocytes (e.g., by centrifugation via a PERCOLL™ gradient). In some embodiments, specific subsets of T cells (e.g., CD4+, CD8+, CD28+, CD45RA+, and CD45RO+ T cells) are further isolated using positive or negative selection techniques known in the art. For example, enrichment of T cell populations by negative selection can be achieved using a combination of antibodies targeting surface markers specific to negatively selected cells. In some embodiments, selection can be performed using cell sorting and / or by negative magnetic immunoadhesion or flow cytometry, which uses a mixture of monoclonal antibodies targeting cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody mixture typically contains antibodies against CD8, CD11b, CD14, CD16, CD20, and HLA-DR. In some embodiments, flow cytometry and cell sorting are used to isolate cell populations of interest for use in this disclosure.

[0198] In some embodiments, PBMCs are used directly for the genetic modification of immune cells (e.g., CARs or TCRs) using the methods described herein. In some embodiments, after isolating PBMCs, T lymphocytes are further isolated, and cytotoxic and helper T lymphocytes are sorted into primary, memory, and effector T cell subsets before or after genetic modification and / or expansion.

[0199] In some embodiments, CD8+ cells are further sorted into primary, central memory, and effector cells by identifying various cell surface antigens associated with these types of CD8+ cells. In some embodiments, the expression of phenotypic markers for central memory T cells includes CCR7, CD3, CD28, CD45RO, CD62L, and CD127, and they are granzyme B negative. In some embodiments, central memory T cells are CD8+, CD45RO+, and CD62L+ T cells. In some embodiments, effector T cells are CCR7, CD28, CD62L, and CD127 negative, while granzyme B and perforin are positive. In some embodiments, CD4+ T cells are further sorted into subsets. For example, CD4+ T helper cells can be sorted into primary, central memory, and effector cells by identifying cell populations possessing cell surface antigens.

[0200] In some embodiments, immune cells (e.g., T cells) are genetically modified using known methods after isolation, or activated and expanded in vitro (or differentiated in the case of progenitor cells) prior to genetic modification. In another embodiment, immune cells (e.g., T cells) are genetically modified with chimeric antigen receptors as described herein (e.g., transduced with a viral vector containing one or more nucleotide sequences encoding a CAR), and then activated and / or expanded in vitro. Methods for activating and expanding T cells are known in the art and described, for example, in U.S. Patent Nos. 6,905,874, 6,867,041, and 6,797,514 and PCT Publication No. WO 2012 / 079000, the entire contents of which are incorporated herein by reference. Typically, such methods involve contacting PBMCs or isolated T cells with stimulants and co-stimulants (e.g., anti-CD3 and anti-CD28 antibodies), typically attached to beads or other surfaces, in a culture medium containing appropriate cytokines (e.g., IL-2). Anti-CD3 and anti-CD28 antibodies attached to the same bead act as "alternative" antigen-presenting cells (APCs). One example is the Dynabeads® system, a CD3 / CD28 activator / stimulator system for the physiological activation of human T cells. In other embodiments, methods are used, such as those described in U.S. Patent Nos. 6,040,177 and 5,827,642 and PCT Publication No. WO 2012 / 129514, to activate and stimulate T cell proliferation using feeder cells and appropriate antibodies and cytokines, the entire contents of which are incorporated herein by reference.

[0201] Methods of gene delivery and cell modification

[0202] Those skilled in the art will have the ability to construct vectors (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996, both incorporated herein by reference) using standard recombination techniques for expressing the antigen receptors of this disclosure. Vectors include, but are not limited to, plasmids, granules, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC), such as retroviral vectors (e.g., vectors derived from Moloney mouse leukemia virus (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., vectors derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenovirus (Ad) vectors (including those with replication capability, those with replication defects, and those without enterotype), adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, Epstein-Barr virus vectors, herpesvirus vectors, vaccinia virus vectors, Harvey mouse sarcoma virus vectors, mouse mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, Malaba virus vectors, and group B adenovirus EnAd (enadenotucirev) vectors.

[0203] Viral vector

[0204] Viral vectors encoding antigen receptors, cytokines, and / or functional effector elements may be provided in some aspects of the methods disclosed herein. In the production of recombinant viral vectors, non-essential genes are typically replaced by genes or coding sequences of heterologous (or non-native) proteins. A viral vector is an expression construct that utilizes a viral sequence to introduce nucleic acids, and possibly proteins, into a cell. Certain viruses possess the ability to infect cells or enter cells via receptor-mediated endocytosis and integrate into the host cell genome, stably and efficiently expressing viral genes, making them attractive candidates for transferring exogenous nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver nucleic acids for certain aspects of the present invention are described below.

[0205] In some embodiments of the methods disclosed herein, nucleic acid sequences and / or genome editing constructs are introduced into immune cells in vitro, in vivo, in vitro, or in situ, including the use of viral vectors. In some embodiments, the viral vector is a non-integrating, non-chromosomal vector. Exemplary non-integrating, non-chromosomal vectors include, but are not limited to, adeno-associated virus (AAV), adenovirus, and herpesvirus. In some embodiments, the viral vector is an integrated chromosomal vector. Integrated chromosomal vectors include, but are not limited to, adeno-associated virus (AAV), lentivirus, and gamma retrovirus.

[0206] In some embodiments of the methods disclosed herein, nucleic acid sequence and / or genome editing constructs are introduced into immune cells, either ex vivo, in vivo, in vitro, or in situ, including the use of combinations of vectors. Exemplary, non-limiting vector combinations include viral and non-viral vectors, multiple non-viral vectors, or multiple viral vectors. Exemplary but non-limiting vector combinations include: combinations of DNA-derived and RNA-derived vectors, combinations of RNA and reverse transcriptase, combinations of transposons and transposases, combinations of non-viral vectors and endonucleases, and combinations of viral vectors and endonucleases.

[0207] In some embodiments of the methods disclosed herein, the genomic modification of introducing nucleic acid sequences and / or genome editing constructs into immune cells, either ex vivo, in vivo, in vitro, or in situ, can stably integrate nucleic acid sequences, transiently integrate nucleic acid sequences, produce site-directed integration of nucleic acid sequences, or produce biased integration of nucleic acid sequences. In some embodiments, the nucleic acid sequence is a transgenic sequence.

[0208] In some embodiments of the methods disclosed herein, genomic modifications including the introduction of nucleic acid sequences and / or genome editing constructs into immune cells, either in vitro, in vivo, or in situ, can stably integrate nucleic acid sequences. In some embodiments, stable chromosomal integration can be random integration, site-directed integration, or biased integration. In some embodiments, site-directed integration can be unassisted or assisted. In some embodiments, assisted site-directed integration is co-delivered with a site-specific nuclease. In some embodiments, the site-specific nuclease comprises a transgene with 5' and 3' nucleotide sequence extensions containing a percentage of homology to upstream and downstream regions of the genome integration site. In some embodiments, the transgene with homologous nucleotide extensions can achieve genome integration via homologous recombination, microhomologous-mediated end joining, or non-homologous end joining. In some embodiments, site-directed integration occurs at a safe harbor site. Genome safe harbor sites accommodate the integration of new genetic material in a manner that ensures the newly inserted genetic element functions reliably (e.g., expressed at therapeutically effective levels) and does not cause harmful alterations to the host genome that pose a risk to the host organism. Potential genomic safe harbors include, but are not limited to, intron sequences of the human albumin gene, adeno-associated virus site 1 (AAVS1) (the integration site naturally generated by AAV on chromosome 19), sites of the chemokine (CC motif) receptor 5 (CCR5) gene, and human homolog sites of the mouse Rosa26 locus.

[0209] In some embodiments, site-directed transgene integration occurs at sites that disrupt target gene expression. In some embodiments, disruption of target gene expression occurs through site-directed integration at introns, exons, promoters, genetic elements, enhancers, repressors, start codons, stop codons, and response elements. In some embodiments, exemplary target genes targeted by site-directed integration include, but are not limited to, any immunosuppressive genes and genes involved in allogeneic rejection.

[0210] In some embodiments, site-directed transgene integration occurs at sites that lead to enhanced expression of the target gene. In some embodiments, the enhancement of target gene expression occurs through site-directed integration at introns, exons, promoters, genetic elements, enhancers, repressors, start codons, stop codons, and response elements.

[0211] Control element

[0212] The expression cassette contained in the vectors used in this disclosure specifically includes (in a 5' to 3' orientation) a eukaryotic transcription promoter operatively linked to a protein-coding sequence, a splicing signal including an insertion sequence (intron), and a transcription termination / polyadenylation sequence. Promoters and enhancers controlling protein-coding gene transcription in eukaryotic cells consist of multiple genetic elements. Cellular machinery is able to collect and integrate the regulatory information delivered by each element, thereby allowing different genes to evolve unique, and often complex, transcriptional regulatory patterns.

[0213] Promoters used in the context of this disclosure include constitutive, inducible, and tissue-specific promoters.

[0214] Copy start point

[0215] To propagate the vector within a host cell, it may contain one or more origins of replication (often referred to as "ori"), such as a nucleic acid sequence corresponding to the oriP of EBV as described above, or a genetically engineered oriP with similar or enhanced function programmed in the programming, which is the specific nucleic acid sequence that initiates replication. Alternatively, a replication origin or autonomous replication sequence (ARS) of other extrachromosomal replicating viruses as described above may be used.

[0216] Other methods of nucleic acid delivery

[0217] In addition to viral delivery of nucleic acids encoding antigen receptors, the following are other methods for delivering recombinant genes to a given cell (e.g., NK cells), and are therefore considered in this disclosure.

[0218] Introducing nucleic acids (e.g., DNA or RNA) into the immune cells of this disclosure can be achieved using any method suitable for nucleic acid delivery to transform cells, as shown herein or as is known to those skilled in the art. Such methods include, but are not limited to, direct delivery of DNA, such as by in vitro transfection, by injection (including microinjection); by electroporation; by calcium phosphate precipitation; by the use of DEAE-glucan followed by polyethylene glycol; by direct sonic loading; by liposome-mediated and receptor-mediated transfection; by microbolite bombardment; by stirring with silicon carbide fibers; by Agrobacterium-mediated transformation; by drying / inhibition-mediated DNA uptake; and any combination of such methods. By applying techniques of this kind, organelles, cells, tissues, or organisms can be stably or transiently transformed.

[0219] Typically, gene transfer systems can include transposon-based or virus-based integration systems.

[0220] In some embodiments, the gene transfer system includes a transposon system. DNA transposons can be translocated via a non-replicating "cut and paste" mechanism. This mechanism requires a catalytic enzyme (i.e., a transposase) to recognize two inverted terminal repeat sequences (ITRs), which can cleave its target and subsequently release the DNA transposon from its donor template. After excision, the DNA transposon can then be integrated into recipient DNA cleaved by the same transposase. In some of their native structures, the DNA transposon is flanked by two ITRs and may contain a gene encoding a transposase that catalyzes the transposition. Those skilled in the art will appreciate that transposon systems offer numerous advantages for nucleic acid integration compared to viral vectors. For example, transposons can carry larger cargoes, which is advantageous for delivering one or more CARs, functional effector elements, and / or cytokines disclosed herein to immune cells (e.g., NK cells). Furthermore, transposons can contain, for example, CRISPR tools (e.g., along with the cargo), thereby allowing for multiple engineering of the cells.

[0221] How to use

[0222] In some embodiments, this disclosure provides methods of immunotherapy comprising administering an effective dose of the immune cells of this disclosure. In one embodiment, a medical condition or disorder is treated by transferring a population of immune cells that elicit an immune response. In some embodiments of this disclosure, cancer or infection is treated by transferring a population of immune cells that elicit an immune response. This document provides methods for treating or delaying the progression of cancer in an individual, comprising administering an effective dose of antigen-specific cell therapy to the individual. This method can be applied to the treatment of immune dysfunction, solid tumors, hematologic malignancies, and viral infections.

[0223] The tumors to which this treatment is useful include any malignant cell type, such as those found in solid tumors or hematologic malignancies. In some embodiments, the cancer is acute myeloid leukemia (AML). In some embodiments, the cancer is monocytic AML. In some embodiments, the cancer is mixed monocytic / blastic AML. In some embodiments, the cancer is CD64-positive cancer. In some embodiments, the cancer has low levels of CD64 expression (e.g., low CD64 expression cancer). In some embodiments, the cancer has low levels of CD64 expression and transforms into CD64-positive cancer. In some embodiments, the cancer had low levels of CD64 expression (e.g., low CD64 expression cancer) before treatment with at least one AML-targeted therapy (e.g., treatment with venetoclax in combination with a demethylating agent such as azacitidine), but the remaining cancer after treatment with at least one AML-targeted therapy has higher levels of CD64 expression (e.g., CD64-positive cancer). In some embodiments, the cancer has little or no CD64 expression and transforms into CD64-positive cancer. In some embodiments, the cancer expressed little or no CD64 before treatment with at least one targeted therapy for acute myeloid leukemia (e.g., treatment with venetoclax in combination with a demethylating agent, such as azacitidine) (e.g., CD64-negative cancer), but after treatment with at least one targeted therapy for acute myeloid leukemia, the remaining cancer expressed CD64 (e.g., CD64-positive cancer).

[0224] Therefore, this disclosure provides a method for treating acute myeloid leukemia in a subject, the method comprising administering at least one dose of the immune cells of this disclosure to the subject. This disclosure provides the immune cells of this disclosure for treating acute myeloid leukemia in a subject. This disclosure provides the use of the immune cells of this disclosure in the preparation of a medicament for treating acute myeloid leukemia in a subject.

[0225] Specific implementations relate to methods for treating acute myeloid leukemia in subjects with this need. Acute myeloid leukemia (AML) is a blood cancer in which the bone marrow of a subject produces abnormal myeloblasts, red blood cells, or platelets. AML is one of the most common forms of acute leukemia in adults. The accumulation of AML cells in the bone marrow and blood can rapidly lead to infection, anemia, excessive bleeding, and death. In some implementations, the AML has a population of CD64+ AML cells. In some implementations, the AML lacked a population of CD64-positive AML cells prior to treatment with at least one targeted AML therapy (e.g., treatment with venetoclax in combination with a demethylating agent such as azacitidine), but has a population of CD64-positive AML cells after treatment with at least one targeted AML therapy. In some implementations, the AML has a population of monocytic leukemia stem cells (mLSCs). In some implementations, the subject has previously been given at least one targeted AML therapy. In some implementations, the targeted AML therapy includes administration of a combination of venetoclax and azacitidine. In some embodiments, the subject relapses after treatment with at least one targeted AML therapy, and / or the subject is resistant to at least one targeted AML therapy. In some embodiments, a subject who has previously been given at least one targeted AML therapy (e.g., treatment with venetoclax in combination with a demethylating agent, such as azacitidine) has a CD64-positive AML cell population, whereas the subject lacked a CD64-positive AML cell population prior to being given at least one targeted AML therapy.

[0226] Specifically, the CD64-specific CAR described herein is administered to subjects with acute myeloid leukemia (AML). Specifically, the CD64-specific CAR described herein is administered to subjects with CD64+ AML cell populations and / or monocytic leukemia stem cell (mLSC) populations. Specifically, the CD64-specific CAR described herein is administered to subjects who have relapsed after treatment with at least one targeted AML therapy, and / or who are resistant to at least one targeted AML therapy. Specifically, the CD64-specific CAR described herein is administered to subjects who have CD64-positive AML cell populations after treatment with at least one targeted AML therapy (e.g., treatment with venetoclax in combination with a demethylating agent such as azacitidine), but who lacked CD64-positive AML cell populations prior to treatment with at least one targeted AML therapy.

[0227] In some embodiments of this disclosure, immune cells are delivered to an individual in need, such as someone with cancer or an infection. The cells then enhance the individual's immune system to attack or directly attack the corresponding cancer cells or pathogenic cells. In some cases, one or more doses of immune cells are provided to the individual. When two or more doses of immune cells are provided to the individual, the interval between administrations should be sufficient to allow time for proliferation within the individual, and in specific embodiments, the interval between doses is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or longer.

[0228] In another embodiment, the subject is a recipient of a transplanted organ or stem cells, and immune cells are used to prevent and / or treat rejection. In a specific embodiment, the subject has graft-versus-host disease (GVHD) or is at risk of developing GVHD. GVHD is a possible complication of any transplantation procedure that uses or includes stem cells from a related or unrelated donor. There are two types of GVHD: acute and chronic. Acute GVHD occurs within the first three months after transplantation. Signs of acute GVHD include a red rash on the hands and feet that may spread and become more severe, accompanied by peeling or blistering of the skin. Acute GVHD can also affect the stomach and intestines, in which case cramps, nausea, and diarrhea may occur. Yellowing of the skin and eyes (jaundice) indicates that acute GVHD has affected the liver. Chronic GVHD is graded according to its severity: stage / grade 1 is mild; stage / grade 4 is severe. Chronic GVHD occurs three months or later after transplantation. The symptoms of chronic GVHD are similar to those of acute GVHD, but in addition, chronic GVHD may also affect the mucous glands in the eyes, the salivary glands in the mouth, and the glands that lubricate the stomach lining and intestines. Any of the immune cell populations disclosed herein can be utilized. Examples of transplanted organs include solid organ transplants such as kidneys, livers, skin, pancreas, lungs, and / or hearts, or cell transplants such as islets of Langerhans, hepatocytes, myoblasts, bone marrow, or hematopoietic stem cells or other stem cells. Transplantation can be a composite transplant, such as facial tissue. Immune cells can be administered before, concurrently with, or after transplantation. In some embodiments, immune cells are administered before transplantation, for example, at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 1 month before transplantation. In a particular, non-limiting example, a therapeutically effective amount of immune cells is administered 3–5 days before transplantation.

[0229] In some implementations, the subject may be given non-myeloablative lymphoablative chemotherapy prior to immunotherapy. This non-myeloablative lymphoablative chemotherapy can be any suitable such therapy and can be administered via any suitable route. The non-myeloablative lymphoablative chemotherapy may comprise, for example, administration of venetoclax and azacitidine.

[0230] Therapeutic amounts of immune cells can be administered via a variety of routes, including parenteral administration, such as intravenous, intraperitoneal, intramuscular, intrasternal, or intra-articular injection or infusion.

[0231] The therapeutically effective amount of immune cells used in adoptive cell therapy is the amount that achieves the desired effect in the treated subject. For example, this could be the amount of immune cells needed to suppress progression or cause regression of autoimmune or alloimmune diseases, or the amount needed to alleviate symptoms caused by autoimmune diseases (such as pain and inflammation). It could be the amount needed to alleviate inflammation-related symptoms (such as pain, edema, and fever). It could also be the amount needed to reduce or prevent transplant rejection. It could be the amount needed to reduce the number of cancer cells in the subject. It could be the amount needed to increase the subject's survival time.

[0232] Immune cell populations can be administered in a treatment regimen consistent with the disease, such as a single or few doses given over one to several weeks to improve the disease state, or periodic doses given over a longer period to suppress disease progression and prevent relapse. The precise dosage used in the formulation will also depend on the route of administration and the severity of the disease or disorder. The therapeutically effective amount of immune cells will depend on the subject being treated, the severity and type of the ailment, and the method of administration. In some embodiments, the dosage range for treating human subjects is at least 3.8 x 10⁻⁶. 4 At least 3.8x10 5 At least 3.8x10 6 At least 3.8x10 7 At least 3.8x10 8 At least 3.8x10 9 Or at least 3.8x10 10 immune cells / m 2 In one specific implementation, the dosage range for treating human subjects is approximately 3.8 x 10⁻⁶. 9 To approximately 3.8x10 10 immune cells / m 2 (For example, approximately 3.8 x 10) 9 To approximately 3.3x10 9 Approximately 3.8 x 10 9 To approximately 2.8x10 10 Approximately 3.8 x 10 9 To approximately 2.2x1010 Approximately 3.8 x 10 9 To approximately 1.7x10 10 Approximately 3.8 x 10 9 From approximately 1.4x10 10 Approximately 3.8 x 10 9 To approximately 1x10 10 Approximately 3.8 x 10 9 Approximately 7.1x10 10 Approximately 7.1 x 10 9 To approximately 3.8x10 10 Approximately 1x10 10 To approximately 3.8x10 10 Approximately 1.4 x 10 10 To approximately 3.8x10 10 Approximately 1.7 x 10 10 To approximately 3.8x10 10 Approximately 2.2 x 10 10 To approximately 3.8x10 10 Approximately 2.8 x 10 10 To approximately 3.8x10 10 Approximately 3.3 x 10 9 To approximately 3.8x10 10 Approximately 7.1 x 10 9 To approximately 3.3x10 10 Approximately 1x10 10 To approximately 2.8x10 10 or approximately 1.4 x 10 10 To approximately 2.2x10 10 In an additional embodiment, a therapeutically effective amount of immune cells can be approximately 5 x 10⁻⁶. 6 Cells / kg body weight approximately 7.5 x 10⁻⁶ 8 Variation between cells per kg of body weight, for example, approximately 2 x 10-1 7 One cell to approximately 5 x 10 8 Cells / kg body weight, or approximately 5 x 10 7 One cell to approximately 2 x 10 8 Cells / kg body weight, or approximately 5 x 10 6 One cell / kg body weight to approximately 1x10 7 Cells / kg body weight. In some embodiments, the therapeutically effective amount of immune cells ranges from approximately 1 x 102. 5 1 cell / kg body weight to approximately 1 x 10⁹ cells / kg body weight (e.g., approximately 1 x 10⁹ cells / kg body weight) 5 Cells / kg body weight approximately 8.8 x 10⁻⁶ 9 Approximately 1x10 5 To approximately 7.5x10 9 Approximately 1x105 Cells / kg body weight approximately 6.3 x 10⁻⁶ 9 Approximately 1x10 5 Cells / kg body weight approximately 5 x 10 9 Approximately 1x10 5 Cells / kg body weight approximately 3.8 x 10⁻⁶ 9 Approximately 1x10 5 Cells / kg body weight approximately 2.5 x 10⁻⁶ 9 Approximately 1x10 5 Cells / kg body weight approximately 1.3 x 10⁻⁶ 9 Approximately 1.3 x 10 9 To approximately 1x10 10 Approximately 2.5 x 10 9 To approximately 1x10 10 Approximately 3.8 x 10 9 To approximately 1x10 10 Approximately 5x10 9 To approximately 1x10 10 Approximately 6.3 x 10 9 To approximately 1x10 10 Approximately 7.5 x 10 9 To approximately 1x10 10 Approximately 1.3 x 10 9 To approximately 8.8x10 9 Approximately 2.5 x 10 9 To approximately 7.5x10 9 or approximately 3.8 x 10 9 To approximately 6.3x10 9 The exact number of immune cells can be easily determined by those skilled in the art based on the subject's age, weight, sex, and physiological condition. The effective dose can be inferred from dose-response curves derived from in vitro or animal model testing systems.

[0233] Immune cells can be administered in combination with one or more other therapeutic agents to treat immune-mediated disorders. Combination therapies may include, but are not limited to: one or more antimicrobial agents (e.g., antibiotics, antivirals, and antifungals), antitumor agents (e.g., fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immune scavengers (e.g., fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressants (e.g., azathioprine or glucocorticoids such as dexamethasone or prednisone), anti-inflammatory agents (e.g., glucocorticoids such as hydrocortisone, dexamethasone, or prednisone, or nonsteroidal anti-inflammatory agents such as acetylsalicylic acid, ibuprofen, or naproxen), cytokine antagonists (e.g., anti-TNF and anti-IL-6), cytokines (e.g., interleukin-10 or transforming growth factor-β), hormones (e.g., estrogens), or vaccines. In addition, immunosuppressants or tolerance-inducing agents may be administered, including but not limited to calcineurin inhibitors (e.g., cyclosporine and tacrolimus); mTOR inhibitors (e.g., rapamycin); mycophenolate mofetil, antibodies (e.g., those recognizing CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., methotrexate, trooseltamivir, busulfan); radiation; chemokines, interleukins, or their inhibitors (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors); or immune checkpoint inhibitors (e.g., anti-PD1 antibodies, anti-PDL1 antibodies, anti-PDL2 antibodies, anti-LAG3 antibodies, and anti-CTLA4 antibodies). These additional drug agents may be administered before, during, or after the administration of immune cells, depending on the desired outcome. These cells and drugs may be administered via the same or different routes and at the same or different sites.

[0234] Pharmaceutical Composition

[0235] This document also provides pharmaceutical compositions and formulations comprising immune cells (e.g., T cells) and pharmaceutically acceptable carriers disclosed herein.

[0236] In some embodiments, the pharmaceutical composition comprises about 1x10 5 1 x 10 T cells to approximately 1 x 10 9 A dose of T cells (e.g., approximately 1 x 10⁻⁶) 5 Approximately 8.75 x 10 T cells. 8 Approximately 5x10 5 To approximately 7.5x10 8 Approximately 5x10 5 To approximately 6.25x10 8 Approximately 5x10 5 Approximately 5x10 8 Approximately 5x10 5 To approximately 3.75x10 8Approximately 5 x 10⁵ to approximately 2.5 x 10⁵ 8 Approximately 5 x 10⁵ to approximately 1.25 x 10⁵ 8 Approximately 1.25 x 10 8 To approximately 1x10 9 Approximately 2.5 x 10 8 To approximately 1x10 9 Approximately 3.75 x 10 8 To approximately 1x10 9 Approximately 5x10 8 To approximately 1x10 9 Approximately 6.25 x 10 8 To approximately 1x10⁹, approximately 7.5x10⁹ 8 To approximately 1x10 9 Approximately 8.75 x 10 8 To approximately 1x10 9 Approximately 1.25 x 10 8 To approximately 8.75x10 8 Approximately 2.5 x 10 8 To approximately 7.5x10 8 or approximately 3.75 x 10 8 To approximately 6.25x10 8 In some implementations, the dose is approximately 1 x 10⁻⁶. 5 1x10 6 1x10 7 1x10 8 Or 1x10 9 10 T cells. In some embodiments, the pharmaceutical composition comprises about 5 x 10 T cells. 5 10 x 10 T cells to approximately 10 x 10 12 A dose of T cells (e.g., approximately 5 x 10) 5 Approximately 8.75 x 10 T cells. 11 Approximately 5x10 5 To approximately 7.5x10 11 Approximately 5x10 5 To approximately 6.25x10 11 Approximately 5x10 5 Approximately 5x10 11 Approximately 5x10 5 To approximately 3.75x10 11 Approximately 5x10 5 To approximately 2.5x10 11 Approximately 5x10 5 To approximately 1.25x10 11 Approximately 1.25 x 10 11 To approximately 1x10 12 Approximately 2.5 x 10 11 To approximately 1x10 12Approximately 3.75 x 10 11 To approximately 1x10 12 Approximately 5x10 11 To approximately 1x10 12 Approximately 6.25 x 10 11 To approximately 1x10 12 Approximately 7.5 x 10 11 To approximately 1x10 12 Approximately 8.75 x 10 11 To approximately 1x10 12 Approximately 1.25 x 10 11 To approximately 8.75x10 11 Approximately 2.5 x 10 11 To approximately 7.5x10 11 or approximately 3.75 x 10 11 To approximately 6.25x10 11 ).

[0237] The pharmaceutical compositions and formulations described herein can be prepared as lyophilized formulations or aqueous solutions by mixing an active ingredient (such as an antibody or peptide) of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 22nd edition, 2012). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants containing ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight (less than about 10 residues). Polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable delivery carriers described herein further comprise interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publications 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans (such as chondroitinase).

[0238] Combination therapy

[0239] In some embodiments, the compositions and methods of this embodiment involve a population of immune cells in combination with at least one adjunctive therapy. This adjunctive therapy may be radiotherapy, surgery (e.g., tumor resection and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. This adjunctive therapy may be in the form of adjuvant therapy or neoadjuvant therapy.

[0240] In some embodiments, the adjunctive therapy is the administration of a small molecule enzyme inhibitor or an anti-metastasis agent. In some embodiments, the adjunctive therapy is the administration of a drug that limits side effects (e.g., a drug designed to reduce the occurrence and / or severity of treatment side effects, such as an antiemetic). In some embodiments, the adjunctive therapy is radiotherapy. In some embodiments, the adjunctive therapy is surgery. In some embodiments, the adjunctive therapy is a combination of radiotherapy and surgery. In some embodiments, the adjunctive therapy is gamma irradiation. In some embodiments, the adjunctive therapy may be one or more chemotherapeutic agents known in the art.

[0241] Immunotherapy can be administered before, during, or after additional cancer therapy, or in various combinations with additional cancer therapy. The interval between administrations can range from simultaneous to minutes, days, or weeks. Various combinations can be used. For example, immunotherapy might be "A," and anticancer therapy might be "B." A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A Administering any compound or therapy of this embodiment to a patient will follow the general protocol for administering such compounds, taking into account the toxicity of the drug (if any). Therefore, in some embodiments, there is a step to monitor for toxicity attributable to the combination therapy.

[0242] Chemotherapy

[0243] According to this embodiment, a variety of chemotherapeutic agents can be used. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapy agent" is used to mean a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within cells, such as whether they affect the cell cycle and at which stage. Alternatively, drugs can be characterized based on their ability to directly crosslink DNA, embed in DNA, or induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

[0244] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedepa, and uredepa; and ethylene imines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, and triethylenethiophosphoramide. iethylenethiophosphoramide and trimethylolomelamine; acetogenins (specifically bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophytic acid (specifically cryptophytic acid 1) Cryptophycin 1 and cryptophycin 8; dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide HCl, melphalan, novoembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard;Nitrosoureas such as carmustine, chlorozotocin, formmustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, specifically calicheamicin γ1I and calicheamicin ω1I); dynemicin (including dynemicin A); bisphosphonates such as clodronate; and esperamicin.And neocarzinostatin chromophore and related chromogenic chromophores, aclacinomysins, actinomycins, authramycin, azaserine, bleomycin, cactinomycin D, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine, doxorubicin (including morpholino-doxorubicin) (in), cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C (mitomycin) C) mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimetrexate;Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, decitabine, dideoxyuridine, doxifluridine, enocitabine, and fluxuridine; and androgens such as calusterone and dromostanolone. Propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenergic drugs such as mitotane and trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate acetate); epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids (such as maytansine and ansamitocin); mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;Losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonicacid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecene (specifically T-2 toxin, verracurin A, roridin A) A) and anguidine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactal; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxanes (e.g., paclitaxel and docetaxel); gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin 1, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids (e.g., retinoic acid); capecitabine;Carboplatin, procarbazine, plicamycin, gemcitabine, navelbine, farnesyltransferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above drugs.

[0245] In some embodiments, azacitidine is administered at 75 mg / m². 2 The dosage is administered subcutaneously.

[0246] In some aspects, the immune cells of this disclosure can be administered in combination with venetoclax. In some aspects, the immune cells of this disclosure can be administered in combination with azacitidine. In some aspects, the immune cells of this disclosure can be administered in combination with both venetoclax and azacitidine. In some aspects, the immune cells of this disclosure can be administered after administration of venetoclax and / or azacitidine.

[0247] Radiation therapy

[0248] Other widely used factors that cause DNA damage include gamma rays, X-rays, and / or direct delivery of radioactive isotopes to tumor cells. Other forms of DNA damage are also considered, such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287), and ultraviolet radiation. These factors are highly likely to cause extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from 50 to 200 roentgens per day over a prolonged period (3 to 4 weeks) to a single dose of 2,000 to 6,000 roentgens. Radioactive isotope dose ranges vary considerably depending on the isotope's half-life, the intensity and type of emitted radiation, and the uptake by tumor cells.

[0249] Dosing regimen

[0250] In one embodiment, immune effector cells (e.g., T cells) are modified by engineering / introducing a chimeric antigen receptor (e.g., anti-CD64 CAR) into the immune effector cells, and then infused into a subject. In some embodiments, immune effector cells are modified by engineering / introducing a chimeric receptor, a functional effector element, and / or cytokines into the immune effector cells, and then infused into a subject within approximately 0 days, approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, or approximately 7 days.

[0251] In some embodiments, a specific amount of modified effector cells is administered to the recipient, the amount being determined based on the therapeutic efficacy and the potential to induce cytokine-related toxicity. In another embodiment, the modified effector cells are CAR+ and CD56+ cells. In some embodiments, the specific amount of modified effector cells comprises approximately 10 4 To about 10 9 Modified effector cells / kg. In some cases, a certain amount of modified effector cells contains approximately 10 4 To about 10 5 Modified effector cells / kg. In some cases, a certain amount of modified effector cells contains approximately 10 5 To about 10 6 Modified effector cells / kg. In some cases, a certain amount of modified effector cells contains approximately 10 6 To about 10 7 Modified effector cells / kg. In some cases, a certain amount of modified effector cells contains approximately 10 7 To about 10 8 Modified effector cells / kg. In some cases, a certain amount of modified effector cells contains approximately 10 8 To about 10 9 1 modified effector cell / kg. In some cases, a certain amount of modified effector cells contains approximately 1 x 102 cells. 6 Approximately 2x10 6 Approximately 3x10 6 Approximately 4x10 6 Approximately 5x10 6 Approximately 6x10 6 Approximately 7x10 6 Approximately 8x10 6 Approximately 9x10 6 Approximately 1x10 7 Approximately 2x10 7 Approximately 3x10 7 Approximately 4x10 7 Approximately 5x10 7 Approximately 6x10 7 Approximately 7x10 7 Approximately 8x10 7 Approximately 9x10 7 Approximately 1x10 8 Approximately 2x10 8 Approximately 3x10 8 Approximately 4x10 8 Approximately 5x10 8 Approximately 6x10 8 Approximately 7x10 8 Approximately 8x10 8 Approximately 9x10 8Or approximately 1x10 9 Modified effector cells / kg.

[0252] In some implementations, modified immune effector cells are delivered directly to tumor tissue via regional administration, thereby targeting the cancer. For example, in ovarian or renal cancer, modified immune effector cells can be administered intraperitoneally (IP) into the abdomen or peritoneal cavity. Such IP administration can be performed through a port placed for administering chemotherapy drugs or a pre-existing port. Other regional administration methods of modified immune effector cells may include infusion into the resection cavity via catheter, ultrasound-guided intratumoral injection, hepatic artery infusion, or intrapleural administration.

[0253] In some embodiments, the recipient may begin treatment by administering a first dose of modified immune effector cells administered intravenously (IV), followed by a second dose of modified immune effector cells administered intravenously (IV). In some embodiments, the recipient may begin treatment by administering a first dose of modified immune effector cells administered intraperitoneally (IP), followed by a second dose of modified immune effector cells administered intravenously (IV). In further embodiments, subsequent doses may be administered after the second dose of modified immune effector cells, either intravenously (IV) or intraperitoneally (IP). In some embodiments, the duration between the first dose and the second or subsequent doses may be approximately: 0, 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, or 30 days. In some embodiments, the duration between the first dose and the second or subsequent dose can be from about 0 days to about 30 days (e.g., from about 0 days to about 27 days, from about 0 days to about 24 days, from about 0 days to about 21 days, from about 0 days to about 18 days, from about 0 days to about 15 days, from about 0 days to about 12 days, from about 0 days to about 9 days, from about 0 days to about 6 days, from about 0 days to about 3 days, from about 3 days to about 30 days, from about 6 days to about 30 days, from about 9 days to about 30 days, from about 12 days to about 30 days, from about 15 days to about 30 days, from about 18 days to about 30 days, from about 21 days to about 30 days, from about 24 days to about 30 days, from about 27 days to about 30 days, from about 3 days to about 27 days, from about 6 days to about 24 days, from about 6 days to about 21 days, from about 9 days to about 21 days, from about 9 days to about 15 days, from about 12 days to about 18 days, from about 15 days to about 21 days, from about 18 days to about 24 days, from about 21 days to about 27 days, or from about 24 days to about 30 days). In some embodiments, the duration between the first dose and the second or subsequent dose can be about: 0, 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, 35, or 36 months. In some embodiments, the duration between the first dose and the second or subsequent dose can be about 0 months to about 36 months (e.g., about 0 to about 30, about 0 to about 24, about 0 to about 18, about 0 to about 12, about 0 to about 6, about 6 to about 36, about 12 to about 36, about 18 to about 36, about 24 to about 36, about 30 to about 36, about 6 to about 30, or about 12 to about 24). In some implementations, the duration between the first dose and the second or subsequent doses can be approximately: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years.In some embodiments, the duration between the first dose and the second or subsequent dose can be from about 0 years to about 10 years (e.g., from about 0 years to about 9 years, from about 0 to about 8 years, from about 0 to about 7 years, from about 0 to about 6 years, from about 0 to about 5 years, from about 0 to about 4 years, from about 0 to about 3 years, from about 0 to about 2 years, from about 0 to about 1 year, from about 1 year to about 10 years, from about 2 years to about 10 years, from about 3 years to about 10 years, from about 4 years to about 10 years, from about 5 years to about 10 years, from about 6 years to about 10 years, from about 7 years to about 10 years, from about 8 years to about 10 years, from about 9 years to about 10 years, from about 1 year to about 9 years, from about 2 years to about 8 years, from about 3 years to about 7 years, from about 4 years to about 6 years, from about 0 years to about 2 years, from about 1 year to about 3 years, from about 2 years to about 4 years, from about 3 years to about 5 years, from about 4 years to about 6 years, from about 5 years to about 7 years, from about 6 years to about 8 years, from about 7 years to about 9 years, or from about 8 years to about 10 years).

[0254] In another embodiment, a catheter can be placed at the tumor or metastatic site for subsequent administration of doses 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 of modified immune effector cells. In some cases, the dose of modified effector cells may contain approximately 10... 2 To about 10 9 Modified effector cells / kg (e.g., approximately 1 x 10⁻⁶ cells / kg) 2 The modified effector cells / kg were approximately 8.75 x 10⁻⁶. 8 1 effector cells / kg, approximately 1 x 102 2 To approximately 7.5x10 8 Approximately 1x10 2 To approximately 6.25x10 8 Approximately 1x10 2 Approximately 5x10 8 Approximately 1x10 2 To approximately 3.75x10 8 Approximately 1x10 2 To approximately 2.5x10 8 Approximately 1x10 2 To approximately 1.25x10 8 Approximately 1.25 x 10 8 To approximately 1x10 9 Approximately 2.5 x 10 8 To approximately 1x10 9 Approximately 3.75 x 10 8 To approximately 1x10 9 Approximately 5x10 8 To approximately 1x10⁹, approximately 6.25x10⁹ 8 To approximately 1x10 9 Approximately 7.5 x 10 8 To approximately 1x10 9 Approximately 8.75 x 10 8 To approximately 1x10 9 Approximately 1.25 x 10 8 To approximately 8.75x108 Approximately 2.5 x 10 8 To approximately 7.5x10 8 or approximately 3.75 x 10 8 To approximately 6.25x10 8 In cases where toxicity is observed, the dose of modified effector cells may contain approximately 10. 2 To about 10 5 Modified effector cells / kg (e.g., approximately 1 x 10⁻⁶ cells / kg) 2 Modified effector cells / kg to approximately 7.5 x 10⁻⁶. 4 Modified effector cells / kg, approximately 1 x 10-1 2 Approximately 5x10 4 Approximately 1x10 2 To approximately 2.5x10 4 Approximately 2.5 x 10 4 To approximately 1x10 5 Approximately 5x10 4 To approximately 1x10 5 Approximately 7.5 x 10 4 To approximately 1x10 5 Approximately 2.5 x 10 4 To approximately 7.5x10 4 or approximately 4x10 4 Approximately 6x10 4 In some cases, the dose of modified effector cells can be from approximately 10. 2 Starting with 1 modified effector cells / kg, subsequent doses can be increased to approximately: 10 4 10 5 10 6 10 7 10 8 Or 10 9 Modified effector cells / kg.

[0255] Products or reagent kits

[0256] This document also provides articles or kits containing immune cells. The articles or kits may also include a packaging insert containing instructions on using immune cells to treat or delay cancer progression in an individual, or to enhance the immune function of an individual with cancer. Any antigen-specific immune cells described herein may be included in the articles or kits. Suitable containers include, for example, bottles, vials, bags, and syringes. Containers may be formed from a variety of materials, such as glass, plastics (e.g., polyvinyl chloride or polyolefins), or metal alloys (e.g., stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and a label on or associated with the container may indicate instructions for use. The articles or kits may also contain other materials desirable from a commercial and user perspective, including additional buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use. In some embodiments, the articles further include one or more other pharmaceutical agents (e.g., chemotherapeutic agents and antitumor agents). Suitable containers for one or more pharmaceutical agents include, for example, bottles, vials, bags, and syringes.

[0257] General definition

[0258] As used herein, the term "partial" when used to refer to a polypeptide or peptide means a fragment of that polypeptide or peptide. In some embodiments, a "partial" polypeptide or peptide retains at least one function and / or activity of the full-length polypeptide or peptide from which it is derived. For example, in some embodiments, if a full-length polypeptide binds to a given ligand, a portion of that full-length polypeptide also binds to the same ligand.

[0259] The terms “protein” and “polypeptide” are used interchangeably in this article.

[0260] The term "exogenous," when used to refer to a protein, gene, nucleic acid, or polynucleotide in a cell or organism, means a protein, gene, nucleic acid, or polynucleotide introduced into that cell or organism by artificial or natural means; or, when used to refer to a cell, the term means a cell that has been isolated and subsequently introduced into a population of cells or an organism by artificial or natural means. Exogenous nucleic acids can originate from different organisms or cells, or they can be one or more additional copies of nucleic acids naturally produced within that organism or cell. Exogenous cells can originate from different organisms, or they can originate from the same organism. As a non-limiting example, an exogenous nucleic acid is a nucleic acid located at a different chromosomal location than in a natural cell, or otherwise flanked by a different nucleic acid sequence found in nature. The terms "exogenous" and "heterogeneous" are used interchangeably.

[0261] An "expression construct" or "expression cassette" is a nucleic acid molecule that directs transcription. An expression construct contains at least one or more transcriptional control elements (such as promoters, enhancers, or their functional equivalents) that direct gene expression in one or more desired cell types, tissues, or organs. Additional elements, such as transcription termination signals, may also be included.

[0262] A “vector” or “construct” (sometimes referred to as a gene delivery system or gene transfer “carrier”) is a macromolecule or molecular complex containing a polynucleotide, or a protein expressed by said polynucleotide, to be delivered to a host cell (whether in vitro or in vivo).

[0263] A plasmid is a common type of vector; it is an extrachromosomal DNA molecule that is separate from chromosomal DNA and can replicate independently of chromosomal DNA. In some cases, it is circular and double-stranded.

[0264] An "ori" or "site of replication" is a DNA sequence that, when present in a plasmid within a cell, maintains linkage within the plasmid and / or serves as the site of or near the initiation of DNA synthesis. For example, the ori of EBV (Epstein-Barr virus) contains the FR sequence (20 imperfect copies of a 30 bp repeat sequence) and preferably includes the DS sequence; however, other sites in EBV bind EBNA-1, such as Rep... Sequences can be used in place of DS as the origin of replication (Kirshmaier and Sugden, 1998). Therefore, the origin of replication for EBV includes FR, DS, or Rep. The sequence, or any functionally equivalent sequence obtained through nucleic acid modification or synthetic combinations derived therefrom. For example, the methods of this disclosure may also use genetically engineered EBV replication origins, such as through the insertion or mutation of a single element.

[0265] The terms "gene," "polynucleotide," "coding region," "sequence," "fragment," "segment," or "transgenic," when used to refer to a portion of a nucleic acid molecule that, when placed under the control of a suitable regulatory sequence, is transcribed in vitro or in vivo and optionally translated into a gene product, such as a polypeptide. A coding region can exist as cDNA, genomic DNA, or RNA. When existing as DNA, the nucleic acid molecule can be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of a coding region are defined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. Genes can include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. The transcription termination sequence is typically located at the 3' end of the gene sequence.

[0266] The term "control elements" collectively refers to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (IRES), enhancers, splice junctions, etc., which together ensure the replication, transcription, post-transcriptional processing, and translation of the coding sequence in the recipient cell. All of these control elements are unnecessary if the selected coding sequence can replicate, transcribe, and translate in a suitable host cell.

[0267] The term "promoter" is used herein to refer to a nucleotide region containing a DNA regulatory sequence derived from a gene capable of binding RNA polymerase and allowing transcription initiation of a downstream (3' orientation) coding sequence. It may contain genetic elements that allow regulatory proteins and molecules (such as RNA polymerase and other transcription factors) to bind to initiate nucleic acid sequence-specific transcription. The phrases "operationally positioned," "operationally connected," "controlled," and "transcriptionally controlled" mean that the promoter is in the correct functional position and / or orientation relative to the nucleic acid sequence to control the initiation and / or expression of that sequence.

[0268] An "enhancer" is a nucleic acid sequence that, when located near a promoter, imparts increased transcriptional activity relative to the transcriptional activity produced by the promoter in the absence of the enhancer domain.

[0269] Regarding nucleic acid molecules, "operational linkage" refers to the connection of two or more nucleic acid molecules (e.g., nucleic acid molecules to be transcribed, promoters, and functional effector elements) in a manner that allows the nucleic acid molecules to be transcribed. Regarding peptide and / or polypeptide molecules, "operational linkage" means that two or more peptide and / or polypeptide molecules are connected in a manner that produces a single polypeptide chain (i.e., a fusion polypeptide) having at least one property of the individual peptide and / or polypeptide components being fused. The fusion polypeptide is preferably chimeric, i.e., composed of molecules not found in nature in a single polypeptide.

[0270] The term "homology" refers to the percentage of similarity between nucleic acid residues of two polynucleotides or between amino acid residues of two polypeptides. The correspondence between one sequence and another can be determined using techniques known in the art. For example, homology can be determined by directly comparing sequence information using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that promote the formation of stable double strands between homologous regions, followed by digestion with a single-strand-specific nuclease, and determination of the size of the digested fragments. When determined using the methods described above, two polynucleotide (e.g., DNA) or two polypeptide sequences are "substantially homologous" to each other when at least about 80%, at least about 90%, and most preferably at least about 95% of their nucleotide or amino acid residues match in a molecularly defined length.

[0271] The term "cell" as used in this article, in its broadest sense in the art, refers to a living organism that serves as the structural unit of a multicellular organism, surrounded by a membrane structure that isolates it from the external environment, possesses the ability to self-replicate, and has genetic information and the mechanisms for expressing that genetic information. The cells used in this article may be naturally occurring cells or artificially modified cells (e.g., fused cells, genetically modified cells, etc.).

[0272] The term "stem cell" as used herein refers to cells that, under suitable conditions, can differentiate into multiple specialized cell types, and under other suitable conditions, can self-renew and remain in an essentially undifferentiated pluripotent state. The term "stem cell" also encompasses pluripotent cells, multipotent cells, progenitor cells, and progenitor cells. Exemplary human stem cells can be obtained from hematopoietic or mesenchymal stem cells derived from bone marrow tissue, embryonic stem cells derived from embryonic tissue, or embryonic germ cells derived from fetal reproductive tissue. Exemplary pluripotent stem cells can also be generated by reprogramming somatic cells into a pluripotent state by expressing certain transcription factors associated with pluripotency; these cells are referred to as "induced pluripotent stem cells" or "iPScs," "iPSCs," or "iPS cells."

[0273] "Embryonic stem (ES) cells" are undifferentiated pluripotent cells that are obtained from early embryos, such as the inner cell mass at the blastocyst stage, or produced artificially (e.g., through nuclear transfer), and can produce any differentiated cell type in the embryo or adult, including germ cells (e.g., sperm and eggs).

[0274] "Induced pluripotent stem cells" (iPScs, iPSCs, or iPS cells) are cells generated by reprogramming somatic cells through the expression of either the expression of or a combination of inducing factors (referred to herein as reprogramming factors). iPS cells can be generated from fetal, postnatal, neonatal, juvenile, or adult somatic cells. In some embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct 3 / 4), Sox2, c-Myc, Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed into pluripotent stem cells by expressing at least two, at least three, at least four, at least five, at least six, or at least seven reprogramming factors.

[0275] "Hematopoietic progenitor cells" or "hematopoietic precursor cells" refer to cells that have differentiated and are directed toward the hematopoietic lineage, but possess the ability to further differentiate into hematopoietic cells. These include hematopoietic stem cells, pluripotent hematopoietic stem cells, common myeloid progenitor cells, megakaryocyte progenitor cells, erythrocyte progenitor cells, and lymphoid progenitor cells. Hematopoietic stem cells (HSCs) are pluripotent stem cells capable of producing all blood cell types, including myeloid (monocytes and macrophages, granulocytes (neutrophils, basophils, eosinophils, and mast cells), erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineage (T cells, B cells, NK cells) (see, for example, Doulatov et al., 2012; Notta et al., 2015).

[0276] "Multiple lymphoid progenitor cells" (MLPs) are defined as any progenitor cell that produces all lymphoid lineages (B, T, and NK cells), but may or may not possess other (myeloid) potentials (Doulatov et al., 2010), and is CD45RA+ / CD10+ / CD7+. Any B, T, and NK progenitor cell can be called an MLP. "Common myeloid progenitor cells" (CMPs) are CD45RA+ / CD135+ / CD10+ / CD7+ cells that can produce granulocytes, monocytes, megakaryocytes, and erythrocytes.

[0277] "Pluripotent stem cells" refer to stem cells that have the potential to differentiate into all the cells that make up one or more tissues or organs, or preferably, the potential to differentiate into any of the three germ layers: endoderm (internal stomach lining, gastrointestinal tract, lung), mesoderm (muscle, bone, blood, urogenital system), or ectoderm (epidermal tissue and nervous system).

[0278] As used herein, the term "somatic cell" refers to any cell other than germ cells (such as eggs, sperm, etc.) that does not directly pass on its DNA to the next generation. Somatic cells typically have limited pluripotency or no pluripotency. The somatic cells used in this article can be naturally occurring or genetically modified.

[0279] "Programming" is the process of altering the types of offspring a cell can produce. For example, a cell is programmed when it is altered to the point that it can form offspring of at least one new cell type (whether in culture or in vivo) compared to the offspring it would have formed under the same conditions without programming. This means that after sufficient proliferation, the rate of offspring exhibiting the phenotypic characteristics of the new cell type is measurable if, prior to this, no such offspring could have been formed; or, the rate of offspring exhibiting the characteristics of the new cell type is significantly higher than before programming. This process involves differentiation, dedifferentiation, and transdifferentiation.

[0280] Differentiation is the process by which less specialized cells become more specialized cell types. Dedifferentiation is a cellular process in which partially or terminally differentiated cells revert to an earlier developmental stage, such as pluripotency or multipotency. Transdifferentiation is the process of converting one differentiated cell type into another. Typically, transdifferentiation through programming occurs without the need for an intermediate pluripotency stage—that is, the cell is directly programmed from one differentiated cell type to another. Under certain conditions, the proportion of offspring with the characteristics of the new cell type can be at least approximately 1%, 5%, 25%, or more, in ascending order of preference.

[0281] As used herein, the term “subject” or “subject in need” refers to any mammal, preferably human, male or female, of any age, that requires a therapeutic intervention, cell transplantation, or tissue transplantation. Typically, a subject requires a therapeutic intervention, cell transplantation, or tissue transplantation (also referred to herein as a recipient) due to some disorder or pathology or adverse condition, state, or syndrome, or a physical, morphological, or physiological abnormality that can be treated with a therapeutic intervention, cell transplantation, or tissue transplantation.

[0282] As used herein, a gene “disruption” or “alteration” refers to a homologous recombination event with a nucleic acid molecule (e.g., an endogenous gene sequence) that results in the elimination or reduction of expression of one or more gene products encoded by said gene in a cell, compared to the expression level of the gene product in the absence of such disruption. Exemplary gene products comprise mRNA and protein products encoded by said gene. Alterations are temporary or reversible in some cases, and permanent in others. Alterations target functional or full-length proteins or mRNAs in some cases, although they may produce truncated or nonfunctional products. In some embodiments herein, the activity or function (rather than expression) of a gene is disrupted. Gene alterations are typically induced by artificial methods, i.e., by the addition or introduction of compounds, molecules, complexes, or compositions, and / or by altering the gene or gene-related nucleic acids (e.g., at the DNA level). Exemplary methods of gene alteration include gene silencing, knockdown, deletion, and / or gene-altering techniques such as gene editing. Examples of gene editing methods include the CRISPR / Cas system, megnuclease system, zinc finger protein (ZFP) and zinc finger nuclease (ZFN) system, and / or transcription activator-like protein (TAL), transcription activator-like effector protein (TALE), or TALE nuclease protein (TALEN) system. Examples of gene alterations also include antisense techniques (such as RNAi, siRNA, shRNA, and / or ribozymes), which typically result in a temporary reduction in expression; and gene editing techniques that cause inactivation or alteration of the target gene (e.g., through inducing breaks and / or homologous recombination). Examples include insertions, mutations, and deletions. These alterations typically result in the suppression and / or complete lack of expression of the normal or “wild-type” product encoded by the gene. Examples of such gene alterations are insertions, frameshifts and missense mutations, deletions, substitutions, knock-ins, and knockouts of a gene or part of a gene (including deletions of the entire gene). Such alterations may occur in coding regions (e.g., in one or more exons) resulting in the inability to produce a full-length product, a functional product, or any product (e.g., through the insertion of a stop codon). Such changes can also occur through alterations in promoters, enhancers, or other regions that affect transcriptional activation, thereby preventing gene transcription. Gene alterations include gene targeting, including targeted gene inactivation via homologous recombination.

[0283] "Immune disorders," "immune-related disorders," or "immune-mediated disorders" refer to impairments in which the immune response plays a crucial role in the occurrence or progression of a disease. Immune-mediated disorders include autoimmune disorders, allogeneic transplant rejection, graft-versus-host disease, and inflammatory and allergic diseases.

[0284] An “immune response” is the reaction of cells of the immune system (such as NK cells, B cells, T cells, or innate immune cells) to a stimulus. In some implementations, this response is specific to a particular antigen (“antigen-specific response”).

[0285] As used herein, the term "antigen" is a molecule that can be bound by antibodies, T-cell receptors, chimeric antigen receptors, and / or engineered immune receptors. Antigens are often used to induce humoral and / or cellular immune responses that result from B and / or T lymphocytes.

[0286] The terms “tumor-associated antigen,” “tumor antigen,” and “cancer cell antigen” are used interchangeably in this document. In each case, these terms refer to proteins, glycoproteins, or carbohydrates that are specifically or preferentially expressed by cancer cells.

[0287] An epitope is a site on an antigen that is recognized by an antibody, determined by the specificity of its amino acid sequence. If two antibodies competitively inhibit (block) the binding of another antibody to an antigen in a competitive binding assay, they are said to bind to the same epitope. Alternatively, if most amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, the two antibodies bind to the same epitope. If each antibody partially inhibits the binding of another antibody to an antigen, and / or if some amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, the two antibodies are said to have overlapping epitopes.

[0288] "Autoimmune diseases" are diseases in which the immune system produces an immune response (such as a B cell or T cell response) against antigens that are part of the normal host (i.e., autoantigens), thereby causing damage to tissues. Autoantigens can originate from host cells or from symbiotic organisms, such as microorganisms that typically colonize the surface of mucous membranes (called symbiotic organisms).

[0289] The term "graft-versus-host disease (GVHD)" refers to a common and serious complication of bone marrow or other tissue transplantation in which donated immune-functional lymphocytes react against the recipient's own tissues. GVHD is a possible complication of any transplantation procedure that uses or includes stem cells from related or unrelated donors. In some implementations, GVHD is chronic GVHD (cGVHD).

[0290] "Parameters of the immune response" are any specific measurable aspect of the immune response, including but not limited to cytokine secretion (IFN-γ, etc.), chemokine secretion, altered migration or cell accumulation, immunoglobulin production, dendritic cell maturation, regulatory activity, number of immune cells, and proliferation of any cells in the immune system. Another parameter of the immune response is any structural damage or functional deterioration of an organ resulting from an immune attack. An increase in any of these parameters can be readily determined by those skilled in the art using known laboratory tests. In a particular non-limiting example, the incorporation of 3H-thymidine can be assessed to evaluate cell proliferation. A "substantial" increase in an immune response parameter means a significant increase in that parameter compared to a control. Specific non-limiting examples of a substantial increase are an increase of at least about 50%, at least about 75%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, and at least about 500%. Similarly, an inhibition or reduction of an immune response parameter means a significant reduction in that parameter compared to a control. Specific, non-limiting examples of substantial reduction are reductions of at least about 50%, at least about 75%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, and at least about 500%. Statistical tests (such as non-parametric ANOVA or t-tests) can be used to compare the difference between the magnitude of a response induced by one agent and the percentage of samples that responded using a second agent. In some instances, p ≤ 0.05 is significant and indicates that any observed increase or decrease in a parameter is less than 5% likely due to random variation. Other useful statistical analyses can be readily identified by those skilled in the art.

[0291] "Treatment" or treatment of a disease or symptom refers to the implementation of a protocol or treatment plan that may involve administering one or more compositions (e.g., the anti-CD64 CAR composition of this disclosure) to a patient in an effort to reduce the signs or symptoms of the disease or a relapse of the disease. Desired therapeutic effects include reducing the rate of disease progression, improving or alleviating the disease state, and achieving remission, prolonging survival, improving quality of life, or improving prognosis. Furthermore, "treatment" or "management" does not require the complete elimination of signs or symptoms, does not require a cure, and particularly includes protocols or treatment plans that have only a minor impact on the patient.

[0292] As used in this article, “prevention,” “avoidance,” and similar terms describe stopping a disease, condition, or disorder, or one or more of its symptoms or complications.

[0293] The terms "treatment benefit" or "treatment effectiveness" as used throughout this application refer to anything that promotes or enhances the well-being of the subject in relation to the medical treatment of the condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of the disease. For example, cancer treatment may involve, for instance, a reduction in tumor size, a reduction in tumor invasiveness, a reduction in the rate of cancer growth, or the prevention of metastasis or recurrence. Cancer treatment may also refer to prolonging the survival of a subject with cancer.

[0294] As used herein, “antibody” refers to either monoclonal or polyclonal antibodies. As used herein, the term “monoclonal antibody” refers to an antibody produced by a single B cell clone and binding to the same epitope. In contrast, “polyclonal antibody” refers to a group of antibodies produced by different B cells and binding to different epitopes of the same antigen. An entire antibody typically consists of four polypeptides: two identical heavy (H) chain copies and two identical light (L) chain copies. Each heavy chain contains an N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains an N-terminal variable (VL) region and a C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH and VL regions have similar general structures, each containing four frame regions whose sequences are relatively conserved. The frame regions are connected by three complementarity-determining regions (CDRs). These three CDRs, referred to as CDR1, CDR2, and CDR3, constitute the “hypervariate region” of the antibody, which is responsible for antigen binding.

[0295] "Antibody-like molecules" can be, for example, proteins belonging to the Ig superfamily that can selectively bind ligands.

[0296] The terms “antibody fragment,” “antibody fragment,” “antibody functional fragment,” and “antigen-binding portion” are used interchangeably in this document to refer to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see Holliger et al. (2005) Nat. Biotech. 23(9):1126-29). Antibody fragments ideally include, for example, one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or combinations thereof.

[0297] Examples of antibody fragments include, but are not limited to: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments comprising two Fab fragments linked by disulfide bonds at the stalk region; (iii) Fv fragments, which consist of the VL and VH domains of an antibody single arm; (iv) single-chain Fv (scFv), which are monovalent molecules consisting of two domains (i.e., VL and VH) of an Fv fragment linked by a synthetic linker that allows the two domains to be synthesized into a single polypeptide chain (see, for example, Bird et al. (1988), Science 242: 423-6; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-83; and Osbourn et al. (1998) Nat. Biotechnol. 16: 778-81); and (v) A diabody is a dimer of polypeptide chains, each containing VH and VL linked by a peptide linker that is too short to allow pairing of VH and VL on the same polypeptide chain. This drives pairing between complementary domains on different VH-VL polypeptide chains to produce a dimer molecule with two functional antigen-binding sites. Antibody fragments are known in the art and are described in more detail, for example, in U.S. Patent Application Publication 2009 / 0093024 A1.

[0298] Chimeric antigen receptors (CARs), also known as artificial cell receptors, chimeric cell receptors, or chimeric immune receptors, are engineered receptors that have been selectively transplanted onto immune effector cells. CARs typically have an extracellular domain, which includes an antigen-binding domain and a stalk region, a transmembrane domain, and an intracellular domain.

[0299] The "handle region" (which encompasses the terms "spacer region" or "hinge domain" or "hinge region") is used to link the antigen-binding domain to the transmembrane domain. As used herein, the term "handle region" generally refers to any oligonucleotide or polypeptide in the polypeptide chain of a CAR that acts to link the transmembrane domain to an extracellular or cytoplasmic domain. In some embodiments, it is flexible enough to allow the antigen-binding domain to be oriented in different directions, thereby facilitating antigen recognition.

[0300] When referring to a CAR, the term "functional part" means any portion or fragment of the CAR described herein that retains the biological activity of the CAR (parental CAR) as a part of it. When referring to the nucleic acid sequence encoding the parental CAR, the nucleic acid sequence encoding the functional part of the CAR may encode a protein containing, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parental CAR.

[0301] As used herein, the term "functional variant" refers to a polypeptide or protein that has substantial or significant sequence identity or similarity to a reference polypeptide and retains the biological activity of the reference polypeptide as a variant. Functional variants include, for example, those variants of the CAR (parental CAR) described herein that retain the ability to recognize target cells to a degree similar to, the same as, or greater than that of the parental CAR. When referring to the nucleic acid sequence encoding the parental CAR, the nucleic acid sequence encoding the CAR functional variant may be, for example, approximately 10%, approximately 25%, approximately 30%, approximately 50%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, or approximately 99% identical to the nucleic acid sequence encoding the parental CAR.

[0302] The phrase "pharmaceutically or pharmacologically acceptable" means a molecular entity or composition that, when properly administered to animals (e.g., humans), will not produce adverse, allergic, or other adverse reactions. For animal (e.g., human) administration, it should be understood that the formulation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA's Office of Biostandards, etc.

[0303] As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcohol / aqueous solutions, saline solutions, parenteral carriers such as sodium chloride, Ringer's dextran, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delay agents, salts, pharmaceuticals, pharmaceutical stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, fluids, and nutritional supplements, as well as combinations thereof. The pH and precise concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0304] The term "T cell" refers to T lymphocytes, including but not limited to γ / δ T cells, α / β T cells, NK T cells, CD4+ T cells, and CD8+ T cells. CD4+ T cells include TH0, Th1, and Th2 cells, as well as regulatory T cells (Tregs). There are at least three types of regulatory T cells: CD4+CD25+ Tregs, CD25 TH3 Tregs, and CD25 TR1 Tregs. "Cytotoxic T cell" refers to a T cell capable of killing another cell. Most cytotoxic T cells are CD8+ MHC class I restricted T cells; however, some cytotoxic T cells are CD4+. In some embodiments, the T cells of this disclosure are either CD4+ or CD8+.

[0305] The activation state of a T cell defines whether it is "resting" (i.e., in the G0 phase of the cell cycle) or "activated" to proliferate after appropriate stimulation (such as recognition of its specific antigen, or stimulation by OKT3 antibodies, PHA, or PMA). The T cell "phenotype" (e.g., naive, central memory, effector memory, lysing effector, helper effector (TH1 and TH2 cells), and regulatory effector) describes the function the cell performs upon activation. Healthy donors possess T cells of each of these phenotypes, and they are primarily in a resting state. Primary T cells proliferate after activation and then differentiate into memory T cells or effector T cells. They can then re-enter a resting state until the next activation to perform their new function and may again alter their phenotype. Effector T cells divide after activation and antigen-specific effector function.

[0306] Natural killer T cells (NKT cells), not to be confused with the natural killer cells of the innate immune system, serve as a bridge between the adaptive and innate immune systems. Unlike regular T cells, which recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by molecules called CD1d. Once activated, these cells can perform functions attributable to Th and Tc cells (i.e., cytokine production and cell lysis / release of cytotoxic molecules). They are also capable of recognizing and eliminating certain tumor cells and cells infected with herpesviruses.

[0307] Natural killer cells (NK cells) are cytotoxic lymphocytes of the innate immune system. In some cases, NK cells provide the first line of defense against viral infections and / or tumor formation. NK cells can detect MHC presented on infected or cancerous cells, triggering cytokine release and subsequently inducing lysis and apoptosis. NK cells can also detect cells under stress in the absence of antibodies and / or MHC, thus allowing for a rapid immune response.

[0308] As used herein, “tumor antigen” refers to any antigenic substance produced, expressed, or overexpressed in tumor cells. For example, it can trigger an immune response in the host. Alternatively, for the purposes of this disclosure, tumor antigens may be proteins expressed by both healthy and tumor cells, but which are suitable therapeutic targets because they identify a certain tumor type. As described herein, in some respects, a tumor antigen may be CD64.

[0309] The term "antigen-presenting cell (APC)" refers to a class of cells capable of presenting one or more antigens in the form of peptide-MHC complexes that are recognizable by specific immune system effector cells, thereby inducing an effective cellular immune response against the presented antigens. APCs can be intact whole cells, such as macrophages, B cells, endothelial cells, activated T cells, and dendritic cells; or other molecules, whether naturally occurring or synthetic, such as purified MHC class I molecules complexed with β2-microglobulin.

[0310] The term "culture" refers to the in vitro maintenance, differentiation, and / or expansion of cells in a suitable culture medium. "Enrichment" refers to a composition containing a higher percentage of cells in the total number of cells than the percentage found in tissues in which such cells are present in an organism.

[0311] "Anti-cancer" drugs can have a negative effect on cancer cells / tumors in subjects, for example, by promoting the killing of cancer cells, inducing cancer cell apoptosis, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, shrinking tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting immune responses against cancer cells or tumors, preventing or inhibiting cancer progression, or prolonging the lifespan of cancer patients.

[0312] Example

[0313] General Method

[0314] Generate CD64 chimeric antigen receptor construct

[0315] CD64-directed chimeric antigen receptors (CARs) are constructed using a single-stranded variable region fragment (scFv) that binds to human CD64. One form of this CAR consists of an scFv linked to the extracellular domain of CD28, which is continuous with the transmembrane and intracellular signaling domains of the CD28 protein. The CD28 signaling domain connects to the intracellular signaling domain of the CD3-ζ chain, together forming the CD64-28z CAR. Another form of this CAR consists of an scFv linked to the extracellular domain of CD8, which is continuous with the transmembrane domain of CD8. The CD8 transmembrane domain connects to the intracellular signaling domain of the 4-1BB co-stimulatory receptor. The 4-1BB signaling domain connects to the intracellular signaling domain of the CD3-ζ chain, together forming the CD64-BBz CAR.

[0316] CD64 CAR T cell generation

[0317] Lenti-X 293T cell lines (Takara Bio) encoding the CAR transgene were generated by transiently transfecting Lenti-X 293T cell lines (Takara Bio) with transfer and packaging plasmids (pRSV-Rev, pMDLg / pRRe, and pMD2.G) in Opti-MEM (Thermo-Fisher) using Lipofectamine 3000 (Life Technologies). The medium was changed 6 hours after transfection, and the lentiviral supernatant was collected at 24 and 56 hours post-transfection, clarified by centrifugation at 2000G for 10 minutes, aliquoted, and frozen at -80°C. Human T cells were cultured at 0.5 x 10⁻⁶ cells / cells. 6 Cells were thawed at a rate of 3:1 per cell to expand CD3 / CD28 Dynabeads (T-expander CD3 / CD28 Dynabeads) (Life Technologies) in AIM-V medium supplemented with 5% heat-inactivated FBS, 100 U / mL penicillin, 100 U / mL streptomycin, 5 mL GlutaMax™, 10 mM HEPES, and 40 IU / mL IL-2. T cells were transduced at an MOI of 2–10 by spinfection at 32°C for 2 hours at 1000g centrifugation in the presence of 40 IU / mL IL-2 and 10 μg / mL protamine sulfate. Cells were incubated overnight at 37°C, and magnetic beads were removed the following day. CAR T cells were grown at a rate of 0.5 x 10⁻⁶ cells / mL. 6 CAR cells were resuspended in 100 IU / mL IL-2 and expanded in vitro for 4 days. CAR expression was validated by flow cytometry. CAR T cells were then cryopreserved 8 days after activation. CAR T cells were thawed one day before the start of in vitro and in vivo assays.

[0318] In vitro assay for CD64 CAR T cell cytotoxicity against AML cell lines

[0319] AML cell lines K562, MOLM14, and THP1 were stained with PE-conjugated anti-CD64 to demonstrate CD64 expression on their surface. 10 5 AML cell line cells and 10 5 1 CD64-28z CAR T cells or 10 5 Mock T cells (CAR-activated and expanded but not transduced) were co-incubated in 200 μL of medium in the wells of 96-well plates. All co-incubations were performed in triplicate in a tissue culture incubator at 37°C and 10% CO2. After 24 hours, cells were collected from the co-incubator, stained with anti-CD3 antibody, and analyzed by flow cytometry. AML cells and T cells were distinguished from each other within the viable cell phylum based on CD3 staining (a T cell marker) and FSC (K562) or GFP expression (MOLM14 and THP1). The percentage of AML cell lines within the viable cell phylum was quantified by flow cytometry, and the mean value of the triplicate samples was taken. Mann-Whitney analysis was performed to determine statistical significance between the CD64 CAR-treated group and the mock T cell-treated group.

[0320] In vitro assay of CD64 CAR T cytokine production in response to AML cell lines

[0321] 10 5 K562 or THP1 cells with 10 5 1 CD64-28z CAR T cells or 10 5 10 CD19-28z CAR T cells (an unrelated CAR targeting an antigen not expressed on any cell line) were co-incubated. Additionally, 10 5 One CD64-28zCAR T cell or 10 5CD19-28z CAR T cells were added to wells containing only culture medium as a negative control. Co-incubation was performed in 96-well plates with 200 μL of culture medium in triplicate at 37°C and 8% CO2 for 24 hours. At the end of co-incubation, cells were pelleted by centrifugation at 300 g for 6 minutes, and the supernatant was collected from all wells and analyzed by ELISA (R&D systems) to quantify secreted interferon-γ (IFNG) and interleukin-2 (IL-2). The quantifications of each cytokine were averaged from the triplicate samples, and Mann-Whitney analysis was performed to determine statistical significance between the CD64-28z CAR group and the CD19-28z CAR T cell group.

[0322] In vivo testing of CD64 CAR T cells targeting AML cell lines

[0323] NOD / SCID / γ chain deletion (NSG) immunodeficient mice were injected with 10 mg of ... 6 THP1 or MOLM14 cells expressing luciferase were used. AML cell engraftment was confirmed by intraperitoneal (ip) injection of D-luciferin followed by bioluminescence imaging (BLI) on a Xenogen in vivo imaging system (IVIS) on day -1. Mice were injected with 3-5 x 10 cells via tail vein on day 0. 6 CAR T cells or T-cell analogs. In some experiments, no treatment was given on day 0 as a negative control. Treatment was performed with CD64-28z CAR T cell doses or without treatment. During in vivo experiments, BLI was repeated one to two times weekly to quantify leukemia burden. Mice were monitored 2–3 times weekly for the development of predefined endpoint symptoms and euthanized according to institutional protocol upon reaching the endpoint. Survival was recorded and statistically compared using the log-rank test.

[0324] In vitro assay of CD64 CAR T cells in patient-derived AML samples

[0325] Frozen patient-derived AML samples obtained from the University of Colorado Biobank were thawed, and cell viability and concentration were determined using the trypan blue exclusion assay. 400,000 patient-derived AML cells were co-cultured with CD64 CAR T cells or an equivalent volume of simulated T cell plates. CAR T cells were added at varying E:T ratios. Co-incubation of CAR T cells with AML cells was performed in a tissue culture incubator at 37°C and 10% CO2 for 24–48 hours. Cells were then collected from co-incubation, washed, and stained to assess viability (using viability-fixing dye eFlour 780, Invitrogen), and stained with fluorescently labeled antibodies against the following: CD45-PerCP-Cy5.5, CD3-BV605, CD34-BV421, and CD64-PE. The stained cells were analyzed using a Fortessa X-20 flow cytometer (BD Biosciences). Analysis of live AML cells (CD45-PerCP-Cy5.5) was performed. 弱 / CD3 - ) and T cells (CD45) 亮 / CD3 + The ratio of α to α was calculated and normalized to simulated AML cells at each E:T ratio. The expression of CD34 and CD64 in AML cells was further analyzed. Statistical comparisons between the simulated and CAR treatments (at 24 and / or 48 hours) were performed using a two-way ANOVA with Tukey's multiple comparison test.

[0326] Ex vivo clearance of leukemia stem cells from patient-derived AML samples by CD64 CAR T cells: Cryopreserved patient-derived AML samples obtained from the University of Colorado Biobank were thawed and cultured in a tissue culture incubator at 37°C and 8% CO2.

[0327] Cell viability and concentration were determined using the trypan blue exclusion assay. Patient-derived AML cells and CD64 CAR T cells at a 1:2 E:T ratio or an equal volume of simulated T cells were co-incubated in a tissue culture incubator at 37°C and 10% CO2 for 24 hours. T cells were removed by antibody-mediated magnetic separation, and 10 6 AML cells were injected into NSG-S mice via tail vein injection. The mice were euthanized 6 weeks later. Bone marrow was collected from the femur, processed into a single-cell suspension, erythrocytes were lysed, and bone marrow cells were stained to assess viability (using the viability-fixing dye eFlour780, Invitrogen) and stained with fluorescently labeled antibodies against the following: mouse CD45-BV786, human CD45-APC-R700, CD3-FITC, CD33-PE, and CD64-BV510. Human AML cells are defined as (mCD45... - / hCD45+ / CD3 - / CD33 + / CD64 + / - The Mann-Whitney analysis was used to statistically compare the simulated treatment with CAR T cell treatment.

[0328] Example 1 – CD64 CAR T cells with a CD28 co-stimulatory domain (CD64-28z) exhibit antigen-dependent effector activity against AML cell lines.

[0329] CD64-28z-m22 CAR T cells generated from healthy donor T cells demonstrated the ability to target CD64+ AML cell lines in vitro after 24 hours of co-incubation. The cytotoxicity of CD64-28z-m22 CAR T cells was assessed by measuring residual AML cells using flow cytometry after co-incubation with CAR T cells or T cell mimics. CD64-28z-m22 CAR T cells significantly eliminated CD64+ cell lines MOLM14 and THP1. Figure 1A -B). Cytotoxicity is entirely dependent on CD64 antigen expression, as the CD64-negative cell line K562 is not targeted by CD64-28z-m22 CAR T cells. This is manifested in the fact that the number of residual K562 cells remains unchanged relative to simulated T cells after co-incubation with CAR T cells. Figure 1A -B). Compared to THP1 cells with high CD64 expression, CD64-28z-m22 CAR T cells showed a slight decrease in cytotoxicity against MOLM14 cells with low CD64 expression, which further supports the view that the activity of CD64-28z-m22 CAR T cells depends on antigen expression on target cells. Figure 1A -B). CD64-28z-m22 CAR T cells also respond to CD64+ cells (THP1) by secreting effector cytokines interferon-γ (IFNG) and interleukin-2 (IL-2), but do not secrete them spontaneously (only T cells) or respond to CD64-negative (K562) cells by secreting ( Figure 1C Unintentionally limited by theory, these results indicate that the CD64-28z-m22 CAR can recognize CD64+ AML cells and respond through cytotoxicity and cytokine production.

[0330] Example 2 – CD64-28z-m22 CAR T cells demonstrate in vivo efficacy against CD64+ AML cell lines in a xenograft model.

[0331] CD64-28z-m22 CAR T cells generated from healthy donor T cells demonstrated the ability to control AML progression in a xenograft model. Administration of CD64-28z-m22 CAR T cells to NSG mice inoculated with the CD64+ THP1 cell line significantly delayed leukemia progression and remission, ultimately significantly prolonging the survival of CAR-treated mice compared to untreated mice. Figure 2A Similarly, CD64-28z-m22 CAR T cells were able to eradicate CD64+ MOLM14 leukemia to undetectable levels and significantly prolong the survival of mice, exceeding that of untreated mice. Figure 2B ).

[0332] Example 3 – CD64-28z-m22 CAR T cells exhibit in vitro cytotoxicity against patient-derived AML.

[0333] The ability of CD64-28z-m22 CAR T cells to kill patient-derived AML cells that primarily express CD64 was tested. Figure 3A After 24 hours of co-incubation, CD64-28z-m22 CAR T cells significantly reduced the number of viable AML cells compared to simulated T cells. Figure 3B Specifically, CD64-28z-m22 CAR T cells eliminated CD64+ cells in AML, resulting in virtually undetectable CD64+ at 24 hours at an E:T ratio of 1:1 and at 48 hours at a lower E:T ratio of 1:4. Figure 3C Even at E:T ratios as low as 1:16, significant reductions were observed in both total AML and CD64+ AML at 24 and 48 hours. Figure 3B -C). Similar results were obtained when CD64-28z-m22 CAR T cells were co-incubated with patient-derived AML containing both CD64+ and CD64- subsets. Figure 4A These subpopulations originate from primitive (CD64-) and monocyte (CD64+) leukemia stem cell (LSC) populations, as described by Pei et al. in Cancer Discovery, 2020;10(4):536-551. CD64-28z-m22 CAR T cells significantly reduced the number of live AML cells within 24 hours, compared to simulated T cells ( Figure 4B CD64-28z-m22 CAR T cells were also found to significantly eliminate CD64+ AML cells, almost completely eliminating CD64+ AML at E:T ratios of 1:1 and 1:4 for 24 hours, and at E:T ratios as low as 1:16 for 48 hours. Figure 4CUnintentionally limited by theory, these results indicate that CD64-28z-m22 CAR is effective not only against AML cell lines (such as...). Figure 1A (as shown in -B), and also showed effective cytotoxicity against patient-derived AML.

[0334] Example 4 – CD64-28z-m22 CAR T cells deplete monocytic leukemia stem cells (mLSC) from patient-derived AML samples

[0335] Since monocytic leukemia stem cells (mLSCs) have been reported to drive resistance to common AML treatments such as venetoclax and azacytidine in patients (Pei et al., Cancer Discovery, 2020;10(4):536-551), the ability of CD64-28z-m22 CAR to specifically clear mLSCs from patient-derived AML samples was tested. Since in vivo engraftment is the primary assay for LSC activity, patient-derived AML samples were first co-incubated with CD64-28z-m22 CAR T cells or T cell mimics at a 1:2 E:T ratio for 24 hours to allow CAR T cells to kill the mLSC population. After 24 hours, the T cells were cleared, and the remaining AML cells were inoculated into immunocompromised NSG-S mice. Engraftment of leukemia was assessed after 6 weeks, and engraftment following CAR T cell co-incubation was found to be significantly impaired compared to co-incubation with T cell mimics. Figure 5A -D). Pretreatment of AML with CD64-28z-m22 CAR T cells resulted in reduced engraftment of the total AML population and the CD64+ AML population, which was predominantly CD64+ in patients. Figure 5A -B), indicating that mLSCs were cleared before being inoculated into mice. Similarly, engraftment of the total AML population and CD64+ subset from AML patients with mixed primitive / monocytic AML was also reduced after co-incubation with CD64-28z-m22 CAR T cells. Figure 5C -D). Unintentionally limited by theory, these results indicate that CD64 CAR T cells can specifically eliminate monocytic leukemia stem cell populations associated with patient relapse, especially after treatment with venetoclax and azacitidine.

[0336] Example 5 – CD64-BBz-m22 and CD64-28z-m22 CAR T cells demonstrate in vivo efficacy against CD64+ AML cell lines in a xenograft model.

[0337] CD64 CARs containing either a CD28 co-stimulatory domain (CD64-28z-m22) or a 4-1BB co-stimulatory domain (CD64-BBz-m22) were generated from T cells from two independent healthy donors, and their ability to control the CD64+ MOLM14 AML cell line in a xenograft model was tested. Compared with simulated T cell treatment, both CD64-28z-m22 and CD64-BBz-m22 CAR T cells significantly slowed leukemia progression and / or eliminated leukemia ( ). Figure 6A The efficacy of CD64 CAR T cells was observed when CAR T cells were generated from different healthy T cell donors. Figure 6A Furthermore, both CD64-28z-m22 and CD64-BBz-m22 CAR T cells significantly prolonged the survival of mice with AML. Figure 6B Quantitative analysis of total bioluminescence flux per mouse showed that, compared with simulated mice, mice treated with CD64-28z-m22 and CD64-BBz-m22 CAR T cells had a reduced leukemia burden. Figure 6C Unintentionally limited by theory, these results indicate that CD64-BBz-m22 CAR T cells have a significant survival advantage over CD64-28z-m22 CAR T cells. Figure 6B ).

[0338] Example 6 – CD64 CAR T cells demonstrate efficacy against monocytic and mixed monocytic / primitive AML.

[0339] The results in this embodiment restate and expand upon at least some of the data provided in other embodiments.

[0340] CD64 expression in AML models

[0341] To evaluate the preclinical efficacy of CD64 CAR, several target leukemia models with different CD64 expression levels were used. AML cell lines with high, intermediate, and negative CD64 expression were employed to test the efficacy of CD64 CAR. CD64 expression in the three model cell lines was compared by flow cytometry staining. Figure 7A The CD64+ cell line MOLM14 expresses low to moderate levels of CD64. The mononuclear AML cell line THP-1 expresses high levels of CD64. The K562 cell line was used as a negative control because it does not express CD64.

[0342] In addition, primary AML samples with different phenotypes were used, including monocytic (FAB M5) patient samples (AML04 and AML06) with uniformly high CD64 expression (CD64). 亮 CD34 暗 / - )) and mixed monocytic and primitive AML patient samples (AML03 and AML07, (CD64) 暗 / - CD34 亮 ))( Figure 7B ).

[0343] CD64 CAR expression in donor T cells

[0344] After expanding Dynabeads™ (Invitrogen) activated T cells with anti-CD3 / CD28 T cells in the presence of IL-2 (R&D, 40 IU / mL), the CD64 CAR transgene was transduced into human T cells via lentiviral transduction. Following transduction, anti-CD3 / CD28 Dynabeads™ was removed, and T cells were expanded for 5 days in the presence of IL-2 (100 IU / mL). Three different CAR constructs were tested. CD64-28-m22 contains an scFv derived from a mouse M22 antibody (targeting human CD64), a portion of which is linked to the human CD28 extracellular domain, the human CD28 transmembrane domain, and the human CD28 intracellular signal transduction domain tandem with the human CD3-ζ signal transduction domain (all three ITAMs intact). Figure 8A CD64-BB-m22 contains the same M22scFv, linked to a portion of the human CD8α extracellular domain and the human CD8α transmembrane domain. This transmembrane domain is tandemly linked to the intracellular signaling domain of human 4-1BB and human CD3-ζ (all three ITAMs intact). CD64-BB-611 binds to the scFv derived from humanized anti-CD64 antibody 611, linked to a portion of the human CD8α extracellular domain and the human CD8α transmembrane domain. This transmembrane domain is tandemly linked to the intracellular signaling domain of human 4-1BB and human CD3-ζ (all three ITAMs intact).

[0345] Healthy donor T cells were successfully transduced using all CD64 CAR constructs. Flow cytometry measurements using an antibody recognizing the G4S linker of CARscFv showed consistent expression of the CAR molecule in T cells from multiple healthy donors. Figure 8BIn CAR+ populations transduced with CAR constructs containing the 4-1BB signaling domain, the ratio of CD4 to CD8 T cells was skewed higher. In CAR+ populations transduced with CARs containing the CD28 co-stimulatory domain, a more balanced ratio of CD4 to CD8 was observed. Figure 8C ).

[0346] Studies have found that T cells transduced with CD64-28-m22 CAR mainly exhibit a phenotype consistent with T stem cell memory (TSCM) cells (CD62L+ / CD45RA+ / CD95+), while T cells transduced with CD64-BB-m22 or CD64-BB-611 exhibit a mixed phenotype consistent with TSCM, T central memory (TCM, CD62L+ / CD45RA- / CD95+), and T effector memory (TEM, CD62L- / CD45RA- / CD95+). Figure 8D These phenotypic trends were consistent in T cells from multiple healthy donors. Statistical analysis was performed on three independent donors using two-way ANOVA and Tukey's multiple comparison test.

[0347] In vitro cytotoxicity of CD64 CAR T cells

[0348] The expected effective CAR molecule elicits a cytotoxic response in CAR T cells upon contact with antigen-positive cells. To test the ability of the CD64 CAR construct to induce this effector function, CD64 CAR T cells were inoculated with 1x10 5 One from MOLM14 (CD64) 低 THP-1 (CD64) 高 Cells from the CD64+ AML cell line and the K562 (CD64-negative) AML cell line were co-cultured. Cells were co-cultured overnight at an E:T ratio of 1:1 or 1:4. The following day, viable AML cells were quantified by flow cytometry. Untransduced T cells (mock T cells) from the same T cell donor were used as a negative control. A decrease in the viable AML cell population was observed when CD64+ AML cells were cultured with CD64-28-m22, CD64-BB-m22, and CD64-BB-611 CAR T cells. Figure 9A The killing effect on target cells in three replicate experiments was quantified. Figure 9B ).

[0349] To further characterize the cytotoxicity of CD64 CAR T cells, a luciferase-based killing assay was used to test the ability of each CD64 CAR construct to kill MOLM14, THP-1, and K562 cells. Cells were co-cultured overnight with CD64-28-m22, CD64-BB-m22, or CD64-BB-611 CAR T cells from three independent donors at various E:T ratios. Figure 9C CD64-28-m22 CAR T cells showed significantly higher cytotoxicity against THP-1 cells in all donors, and higher cytotoxicity against MOLM14 cells in two of the three donors. Both CD64-BB-m22 and CD64-BB-611 showed effective killing of both THP-1 and MOLM14 cells. Figure 9C This is consistent with previous conclusions. Figures 9A-9B Importantly, no significant killing effect of any CD64 CAR constructs on CD64-negative K562 cells was observed, which supports the antigen-dependent and specific nature of these CARs.

[0350] To test CD64 CAR T cells in primary AML, CD64 CAR T cells were mixed with 1x10 5 Patient-derived AML cells were co-cultured. Cells were co-cultured for 24 hours at different E:T ratios, and residual viable AML cells were assessed by flow cytometry. Patient-derived AML cells co-cultured with T-cell mimics showed a persistent CD64+ population, while each CD64 CAR T-cell product reduced CD64+ AML cells in a dose-dependent manner. Figure 9D-9E CAR T cells from multiple donors, including CD64-28-m22, CD64-BB-m22, and CD64-BB-611, were able to eliminate CD64+ AML cells and showed cytotoxicity against cells from monocytic and mixed monocytic / primitive AML patients. Figure 9D-9E In summary, these data demonstrate the cytotoxic function of CD64 CAR T cells against CD64-expressing AML cells.

[0351] Secretion of T cell effector cytokines

[0352] Upon antigen stimulation, CAR T cells initiate effector functions, such as secreting cytokines and releasing cytotoxic granules targeting antigen-positive cells. To test the ability of CD64 CAR molecules to induce CAR-transduced T cells to produce cytokines, 1x10 5 1 x 10 CD64 CAR T cells (CD64-28-m22, CD64-BB-m22, or CD64-BB-611)5 One from CD64-expressing cell lines (MOLM14 and THP-1) or 1x10 5 CD64+ cells (monocytes and mixed monocytes / primitive primary AML) from patient samples were co-cultured. After 24 hours of co-culture, the production of T cell effector cytokines (IL-2, IFNγ, and TNFα) was measured using the LegendPlex® assay (Biolegend). Figures 10A-10F In THP-1 cells (CD64) 高 Following stimulation, it was observed that compared to MOLM14 (CD64) 低 Higher production of classical T cell effector cytokines IL-2, IFNγ, and TNFα. Figures 10A-10C No cytokine production was observed in T cell-mimicking cells (untransduced T cells) co-cultured with THP-1 or MOLM14 cells. These results indicate that AML cell lines themselves do not produce these cytokines, and that T cell release of these cytokines requires CD64 CAR transduction. Figures 10A-10C Furthermore, CD64 CAR T cells did not exhibit antigen-independent cytokine production when co-cultured with the CD64-negative cell line K562, as no cytokines were produced. Figures 10A-10C Despite the common expected differences in cytokine production between donors in CAR-T cell products, CD64-28-m22 CAR T cells were observed to secrete more IL-2. Figure 10A High levels of IL-2 secretion were also observed in CD64-BB-611 CAR T cells when co-cultured with CD64+ cell lines. Figure 10A Conversely, CD64-BB-m22 CAR T cells were observed to secrete more TNFα. Figure 10C High levels of TNFα secretion were also observed in CD64-BB-611 CAR-T cells when co-cultured with CD64+ cell lines. Figure 10C Studies have found that CD64-28-m22 and CD64-BB-m22 CAR T cell products secrete similar amounts of IFNγ in response to THP-1 cells, while the secretion level of CD64-BB-m22 CAR T cells is slightly higher in response to MOLM14. Figure 10B When co-cultured with CD64+ cell lines, CD64-BB-611 CAR-T cells also exhibited similarly high levels of IFNγ secretion. Figure 10B ).

[0353] When co-cultured with patient samples, CD64-28-m22 CAR T cells demonstrated the ability to produce large amounts of IL-2, IFNγ, and TNFα in response to monocytic and mixed primitive / monocytic patient-derived AML samples. Figure 10D-10F Similarly, CD64-BB-m22 CAR T cells secrete large amounts of IFNγ and TNFα in response to monocyte- and mixed primitive / monocyte-derived AML cells. Figure 10E-10F CD64-BB-611 CAR-T cells secrete particularly high levels of IL-2 and TNFα, as well as fairly high levels of IFNγ. Figure 10D-10F All cytokine data were analyzed in triplicate using three independent T cell donors. Statistical analyses were performed using two-way ANOVA and Tukey's multiple comparison test. Overall, these data demonstrate that CD64-28-m22, CD64-BB-m22, and CD64-BB-611 CARs induce antigen-dependent cytokine production in transduced T cells in response to AML cells, supporting their effector function against leukemia cells expressing target antigens.

[0354] Production of CD64 CAR T cells

[0355] One concern with CAR T therapy for AML is the health or ability to produce CAR T cells derived from patient-derived T cells. To address this concern, T cells from an AML patient sample biobank were transduced with CD64-BB-m22 CAR in parallel with healthy donor T cells. Patient-derived T cells proliferated similarly to healthy donor T cells during CAR T cell production. Figure 11A The efficiency of CAR transduction using patient-derived T cells and healthy donor T cells is comparable in one donor but decreases in another. Figure 11B No significant difference in T cell proliferation was observed between patient-derived CD64-BB-m22 CAR T cells and healthy donor CD64-BB-m22 CAR T cells when co-cultured with MOLM14 target leukemia cells. Figure 11C ).

[0356] In the luciferase killing assay, patient-derived CD64-BB-m22 CAR T cells showed that they killed MOLM14 cells in a dose-dependent manner, which is consistent with... Figure 9C The CD64-BB-m22 CAR T cells from healthy donors shown are similar ( Figure 11DNext, patient-derived CD64-BB-m22 CAR T cells were cultured overnight with their respective monocytic and mixed AML patient samples. The CD64-BB-m22 CAR T cells eliminated CD64+ AML cells in these patient samples. Figure 11E This data indicates that CD64 CAR T cells can be produced from patient-derived T cells and are effective against AML expressing CD64.

[0357] CAR T cells in a CD64 leukemia xenotransplantation model

[0358] The in vivo efficacy of CD64 CAR T cells was also demonstrated after THP-1 (CD64) implantation. 高 The study evaluated leukemia cells in mice, and in a xenograft model, this resulted in more resistant disease than MOLM14. Mice were injected with 1x10 cells via tail vein on day -4. 6 5 x 10 luciferase-positive THP1 cells. Engraftment was confirmed on day 0 using luciferase-based bioluminescent in vivo imaging on the Xenogen IVIS® platform. Mice were infused with 5 x 10 luciferase-positive THP1 cells on day 0. 6 One CD64 CAR+ T cell or an equivalent number of simulated T cells, and monitored weekly via BLI as described in Figure 6 above. Figure 12A This experiment was repeated three times using CAR T cells manufactured from three independent human T cell donors. All mice treated with the simulated T cells died by day 70 due to leukemia progression, and none died from the anticipated complication of xenograft-versus-host disease (xGVHD), which resulted in the mice being euthanized at a predefined, predetermined humanitarian endpoint without detectable leukemia. This is a well-known complication of infusing human T cells into immunodeficient mice and is unrelated to the function of the CAR or CAR T cells. CD64-BB-611 CAR T cells resulted in complete leukemia clearance in all eight mice within 14 days post-treatment. None of these mice died from leukemia; all deaths were caused by xGVHD. Eight out of nine mice treated with CD64-28-m22 and three out of nine mice treated with CD64-BB-m22 CAR T cells developed progressive leukemia. These data demonstrate that each CD64 CAR construct possesses in vivo efficacy against established AML in a xenograft model. Furthermore, this data demonstrates that CD64 CAR T cells using the 4-1BB co-stimulatory domain exhibit excellent survival and leukemia clearance rates, with CD64-BB-611 CAR T cells showing the best leukemia control effect. Figure 12B-12C ).

[0359] To evaluate the in vivo efficacy of CD64-BB-611 CAR T cells, NSG mice were implanted with 1x10⁻⁶ CAR T cells on day -4. 6 MOLM14 cells expressing luciferase. Leukemia engraftment was confirmed on day -1. On day 0, 3 x 10⁻⁶ cells were used. 6 Mice were treated with one CD64 CAR+ T cell or an equivalent dose of mimic T cells. The mice were then imaged weekly via BLI to track leukemia burden. Figure 12D The experiment is as follows: Figure 6A The study was performed in triplicate, using T cells derived from a third healthy donor to include CD64-BB-611 CAR T cells in the comparison. By day 30, all five mice treated with the untransduced control T cells had died of leukemia. By day 91, all mice treated with CD64-28-m22, CD64-BB-m22, and CD64-BB-611 CAR T cells achieved leukemia-free status. All five mice treated with CD64-BB-611 completely cleared leukemia within 35 days of CAR T cell administration, and no mice in this group died from leukemia progression. The leukemia-free survival rate was 100% in all CD64 CAR-T cell-treated mice. Figure 12E Quantitative analysis of total bioluminescence flux per mouse showed that, compared with simulated mice, mice treated with CD64-28-m22, CD64-BB-m22, and CD64-BB-611 CAR-T cells had a reduced leukemia burden. Figure 12F ).

[0360] Durability of CD64 CAR T cells in xenograft models

[0361] Since CAR T cell persistence has been shown to affect the prognosis of lymphocytic leukemia, the persistence of CD64 CAR T cells after clearance of MOLM14 leukemia was evaluated. NSG mice were engrafted with 1x10⁻⁶ CAR T cells on day -4. 6 MOLM14 cells. Then use 5x10 6 Mice were treated with either CD64-28-m22 or CD64-BB-m22 CAR+ T cells. On day 35, mice were euthanized, and bone marrow was assessed by flow cytometry to evaluate persistent CAR T cells. The percentage and absolute number of CD64-BB-m22 CAR T cells in the bone marrow after leukemia clearance were significantly higher (mean 335,811 cells / tibia) compared to CD64-28-m22 CAR T cells (15,148 cells / tibia; p < 0.0001, Mann-Whitney test). Figure 13ASimilar patterns were observed in the CD4+ and CD8+ CAR T cell compartments. Figure 13A Phenotypically, most persistent CAR-T cells were T effector memory cells (TEMs), and no differences were observed between CAR co-stimulatory domains (CD64-28-m22 69%, CD64-BB-m22 73%; p=0.95). Figure 13B The remaining cells mainly possess T-effect memory and re-express the CD45RA (TEMRA) phenotype. Figure 13B Most persistent CAR T cells do not express exhaustion markers. Figure 13C These data indicate that CD64 CAR T cells with a 4-1BB co-stimulatory domain exhibit improved in vivo efficacy against monocytic AML and prolonged CAR T cell persistence. Furthermore, CD64-BB-611 CAR T cells conjugated with the humanized conjugate showed the strongest in vivo efficacy against the monocytic THP-1 cell line.

[0362] Previous experiments were repeated to incorporate the CD64-BB-611 CAR and investigate CAR T cell persistence and phenotype at later time points after leukemia clearance. Mice were cleared of MOLM14 leukemia and euthanized after 105 days. An average of 62,113 CD64-BB-611 CAR+ cells were detected per femur, compared to 7,992 CD64-28-m22 CAR+ cells and 21,655 CD64-BB-m22 CAR+ cells per femur, indicating further improved persistence of the CD64-BB-611 CAR construct compared to CD64-BB-m22. Figure 13D CD64-BB-611 also produced TEM cells, and most CD64 CAR T cells remained negative for the exhaustion markers PD1 and TIM3 at this later time point. Figure 13E-13F ).

[0363] CD64 CAR T-cell cytotoxicity against monocytes

[0364] Given the known expression patterns of CD64 on monocytes and macrophages, CD64 CAR T-cell therapy may lead to the elimination of these cell populations as a targeted / non-tumor toxicity. To test this, CD64 CAR-T cells from three different T-cell donors were co-cultured with normal monocytes from two different healthy blood donors. All CD64 CAR-T cells reduced the viable cell population of both classical and non-classical CD14+ monocytes. At an E:T ratio of 1:2, CD64-28-m22 CAR T cells produced the strongest monocyte-killing effect after 24 hours. Figures 14A-14B CD64 CAR-T cells produced IL-2, IFNγ, and TNFα effector cytokines after being co-cultured with normal monocytes for 24 hours. Figure 14C ), and showed a similar cytokine expression pattern to that observed after co-culturing with AML cells. Figures 10A-10F Statistical analysis was performed using two-way ANOVA and Tukey's multiple comparison test. Therefore, reducing normal monocytes is an expected targeted / non-tumor toxicity of CD64 CAR T-cell therapy. This may have further therapeutic potential for clearing macrophages from the tumor microenvironment in other malignancies. Tumor-associated macrophages (TAMs) have been shown to negatively impact cancer progression and prognosis by infiltrating the tumor microenvironment and driving promotion of cancer cell division and metastasis, accelerating angiogenesis, supporting the cytokine and growth factor environment maintained by cancer stem cells, and producing immunosuppressive effects through cytokine secretion and recruitment of inhibitory regulatory T cells. (Li et al., J. Immunother. Cancer., 9(1): e001341 (2021) doi:10.1136 / jitc-2020-001341. PMID: 33504575; DeNardo et al., Nat. Rev. Immunol., 19(6): 369-382 (2019). doi: 10.1038 / s41577-019-0127-6. PMID: 30718830; PMCID:PMC7339861). Methods to reduce tumor microenvironmental endothelial growth (TAM) in the tumor microenvironment are an active area of ​​research in clinical trials targeting a wide range of hematologic and solid tumors, including AML, multiple myeloma, lymphoma, ovarian cancer, lung cancer, pancreatic cancer, sarcoma, colorectal cancer, and advanced / metastatic cancers of any origin. In addition to the main topic discussed in this article—the intended use of venetoclax in relapsed / refractory AML—there is potential to eliminate these TAMs in various tumor types by leveraging the significant antimonocyte / macrophage, targeted, non-tumor effects of CD64 CAR-T cell therapy.

[0365] CD64 CAR T-cell therapy combined with ven / aza

[0366] The effects of CD64 CAR-T cell therapy in tandem with ven / aza therapy were evaluated in patients with monocytic and mixed monocytic / primitive primary AML. Venetoclax combined with a hypomethylating agent (usually azacitidine) is frequently used as standard therapy for AML. Unfortunately, patients with monocytic AML are more likely to relapse or initially develop resistance to this treatment after receiving this combination therapy. Samples from monocytic or mixed AML patients (1 x 10⁻⁶ replicates) were used.5 Cells were thawed and cultured in vitro in IMDM medium supplemented with BIT, FBS, BME, and LDL, as well as 10 ng / mL of supportive myeloid cytokines FLT3, IL-3, and SCF. Cells were treated with venetoclax in 500 nM DMSO and azacitidine in 1.5 μM PBS for 48 hours, or with 500 nM DMSO alone as a control. Then, 50,000 CD64-BB-m22CAR-T cells or simulated transduced T cells were added, and cultured for another 48 hours. After 4 days of culture, the number of viable AML cells and CD64 expression were assessed by flow cytometry. Mixed monocytic / primitive AML was sensitive to ven / aza monotherapy (21-fold reduction in viable AML cells compared to control, p < 0.0001), but the remaining cells at the end of treatment were highly CD64+. Figures 15A-15C This is consistent with previously described results (Pei et al., Cancer Discovery (2020)). Treatment with CD64-BB-m22 CAR-T cells alone reduced the number of viable AML cells (4.2-fold reduction in all AMLs) and nearly eliminated CD64+ AMLs. Sequential treatment with ven / aza and CD64-BB-m22 CAR-T cells reduced viable AMLs by 34.25-fold and CD64+ AMLs by 353.5-fold (p<0.0001). Treatment with ven / aza in samples from monocytic AML patients did not significantly alter AML cell viability (p=0.998). Figure 15D or significantly alter CD64 expression ( Figure 15E-15F CD64 CAR-T therapy (with or without combination with ven / aza) significantly reduced CD64+ AML by >40-fold (p<0.0001). Figure 15F This data supports the efficacy of CD64 CAR-T therapy in primary monocytic AML and demonstrates the ability of the combination therapy of ven / aza and CD64 CAR-T to effectively target ven / aza-resistant AML.

[0367] Example 7 – Exemplary CD64 Binding Molecule

[0368] This embodiment provides the amino acid sequence of an exemplary scFv having the ability to bind CD64. The amino acid sequences of each scFv are also provided and described. , connector and frame sequence.

[0369] Anti-CD64 Cloning #1

[0370] Full length scFV

[0371] VH structural domain:

[0372] VH structural domain frame region 1

[0373] QVQLVEAGGGVVQPGRSLRLSCAA (SEQ ID NO:27)

[0374] CDR1 of the VH domain

[0375] SGFIFSGYG (SEQ ID NO:24)

[0376] Frame region 2 of the VH structural domain

[0377] MHWVRQAPGKGLEWVTV (SEQ ID NO:28)

[0378] CDR2 of the VH domain: IWYDGSNK (SEQ ID NO:25)

[0379] VH structural domain frame region 3

[0380] YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO:29)

[0381] CDR3 of the VH domain: ARDTGDRFFDY (SEQ ID NO:26)

[0382] VH structural domain frame region 4

[0383] WGQGTTVTVSS (SEQ ID NO:30)

[0384] In some cases, the frame region 4 of the VH domain may be WGQGTLVTV (SEQ ID NO: 381), and / or an “SS” sequence may be added to the 5' end of the connector shown in SEQ ID NO: 420.

[0385] Connector: GGGGSGGGGSGGGGS (SEQ ID NO: 420)

[0386] VL domain:

[0387] VL structural domain frame area 1: EIVLTQSPATLSLSPGERATLSCRAS (SEQ ID NO:35)

[0388] CDR1 of the VL domain: QSVSSY (SEQ ID NO:32)

[0389] VL structural domain frame area 2: LAWYQQKPGQAPRLLIY (SEQ ID NO:36)

[0390] CDR2 of VL domain: DAS (SEQ ID NO:33)

[0391] VL structural domain frame area 3: SRATGIPARFGGSGSGTTDFTLTISSLEPEDFAVYYC (SEQ ID NO:37)

[0392] CDR3 of VL domain: QLRSNWPPYT (SEQ ID NO:34)

[0393] VL structural domain frame area 4: FGQGTKLEIKTS (SEQ ID NO:38) In some cases, the frame region 4 of the VL domain may be missing the 3' "TS" sequence: FGQGTKLEIK (SEQ ID NO:382)

[0394] Anti-CD64 clone #2

[0395] Full length scFV

[0396] VH structural domain:

[0397] VH structural domain frame region 1

[0398] QVQLVEAGGGVVQPGRSLRLSCAASGFIFS (SEQ ID NO:43)

[0399] CDR1 of the VH domain

[0400] GYGMH (SEQ ID NO:40)

[0401] Frame region 2 of the VH structural domain

[0402] WVRQAPGKGLEWVT (SEQ ID NO:44)

[0403] CDR2 of the VH domain: VIWYDGSNKYYADSVKG (SEQ ID NO:41)

[0404] VH structural domain frame region 3

[0405] FTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:45)

[0406] CDR3 of the VH domain: DTGDRFFDY (SEQ ID NO:42)

[0407] VH structural domain frame region 4

[0408] WGQGTLVTVSS (SEQ ID NO:46)

[0409] In some cases, the frame region 4 of the VH domain may be WGQGTLVTV (SEQ ID NO: 383), and / or an “SS” sequence may be added to the 5' end of the connector shown in SEQ ID NO: 420.

[0410] Connector: GGGGSGGGGSGGGGS (SEQ ID NO: 420)

[0411] VL domain:

[0412] VL structural domain frame area 1: EIVLTQSPATLSLSPGERATLSC (SEQ ID NO:51)

[0413] CDR1 of the VL domain: RASQSVSSYLA (SEQ ID NO:48)

[0414] VL structural domain frame area 2: WYQQKPGQAPRLLIY (SEQ ID NO:52)

[0415] CDR2 of VL domain: DASSRAT (SEQ ID NO:49)

[0416] VL structural domain frame area 3: GIPARFGGSGSGTTDFTLTISSLEPEDFAVYYC (SEQ ID NO:53)

[0417] CDR3 of VL domain: QLRSNWPPYT (SEQ ID NO:50)

[0418] VL structural domain frame area 4: FGQGTKLEIKTS (SEQ ID NO:54) In some cases, the frame region 4 of the VL domain may be missing the 3' "TS" sequence: FGQGTKLEIK (SEQ ID NO:384)

[0419] Anti-CD64 clone #3

[0420] Full length scFV

[0421] VH structural domain:

[0422] VH structural domain frame region 1

[0423] EVQLVESGGGLVKPGGSLRLSCVAS (SEQ ID NO:386)

[0424] CDR1 of the VH domain

[0425] GFIFSDNY (SEQ ID NO:387)

[0426] Frame region 2 of the VH structural domain

[0427] MYWVRQTPEKRLEWVAT (SEQ ID NO:388)

[0428] CDR2 of the VH domain: ISDGGSYT (SEQ ID NO:389)

[0429] VH structural domain frame region 3

[0430] YYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAIYYC (SEQ ID NO:390)

[0431] CDR3 of the VH domain: ARGYYRYEGAMDY (SEQ ID NO:391)

[0432] VH structural domain frame region 4

[0433] WGQGTSVTVSS (SEQ ID NO:392)

[0434] In some cases, the frame region 4 of the VH structural domain may be WGQGTSVTV (SEQ ID NO:393), and / or an “SS” sequence may be added to the 5' end of the connector shown in SEQ ID NO:420.

[0435] Connector: GGGGSGGGGSGGGGS (SEQ ID NO: 420)

[0436] VL domain:

[0437] VL structural domain frame area 1: NIVMTQSPSSLAVSAGEKVTMSCKSS (SEQ ID NO:395)

[0438] CDR1 of the VL domain: QSVLYSSNQKNY (SEQ ID NO:396)

[0439] VL structural domain frame area 2: LAWYQQKPGQSPKLLIY (SEQ ID NO:397)

[0440] CDR2 of VL domain: WAS (SEQ ID NO:398)

[0441] VL structural domain frame area 3: TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC (SEQ ID NO:399)

[0442] CDR3 of VL domain: HQYLSSWT (SEQ ID NO:400)

[0443] VL structural domain frame area 4: FGGGTKLEIKTS (SEQ ID NO:401) In some cases, the frame region 4 of the VL domain may be missing the 3' "TS" sequence: FGGGTKLEIK (SEQ ID NO:402)

[0444] Anti-CD64 Cloning #4

[0445] Full length scFV

[0446] VH structural domain:

[0447] VH structural domain frame region 1

[0448] EVQLVESGGGLVKPGGSLRLSCVASGFIFS (SEQ ID NO:404)

[0449] CDR1 of the VH domain

[0450] DNYMY (SEQ ID NO:405)

[0451] Frame region 2 of the VH structural domain

[0452] WVRQTPEKRLEWVA (SEQ ID NO:406)

[0453] CDR2 of the VH domain: TISDGGSYTYYPDSVKG (SEQ ID NO:407)

[0454] VH structural domain frame region 3

[0455] RFTISRDNAKNNLYLQMSSLKSEDTAIYYCAR (SEQ ID NO:408)

[0456] CDR3 of the VH domain: GYYRYEGAMDY (SEQ ID NO:409)

[0457] VH structural domain frame region 4

[0458] WGQGTSVTVSS (SEQ ID NO:410)

[0459] In some cases, the frame region 4 of the VH domain may be WGQGTSVTV (SEQ ID NO:411), and / or an “SS” sequence may be added to the 5' end of the connector shown in SEQ ID NO:420.

[0460] Connector: GGGGSGGGGSGGGGS (SEQ ID NO: 420)

[0461] VL domain:

[0462] VL structural domain frame area 1: NIVMTQSPSSLAVSAGEKVTMSC (SEQ ID NO:413)

[0463] CDR1 of the VL domain: KSSQSVLYSSNQKNYLA (SEQ ID NO:413)

[0464] VL structural domain frame area 2: WYQQKPGQSPKLLIY (SEQ ID NO:414)

[0465] CDR2 of VL domain: WASTRES (SEQ ID NO:415)

[0466] VL structural domain frame area 3: GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC (SEQ ID NO:416)

[0467] CDR3 of VL domain: HQYLSSWT (SEQ ID NO:417)

[0468] VL structural domain frame area 4: FGGGTKLEIKTS (SEQ ID NO:418) In some cases, the frame region 4 of the VL domain may be missing the 3' "TS" sequence: FGGGTKLEIK (SEQ ID NO:419)

[0469] Anti-CD64 clone #5

[0470] Full length scFV

[0471] QVQLVESGGGWQPGRSLRLSCSSSGFIFSDNYMYWVRQAPGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGYYRYEGAMDYWGQGTPVTVSS GGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCKSSQSVLYSSNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSSWTFGQGTKLEIKTS (SEQ ID NO:2)

Claims

1. A polypeptide comprising: a) A first domain comprising an amino acid sequence of one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; b) A second domain containing the amino acid sequence of SEQ ID NO: 4; c) A third domain containing the amino acid sequence of SEQ ID NO: 5; d) A fourth domain comprising the amino acid sequence of SEQ ID NO: 6; and e) The fifth domain, which contains the amino acid sequence of SEQ ID NO:

7.

2. The polypeptide according to claim 1, wherein the first domain further comprises the amino acid sequence of SEQ ID NO:

23.

3. The polypeptide according to claim 2, wherein the polypeptide comprises an amino acid sequence of one of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO:

13.

4. A polypeptide comprising: a) A first domain comprising an amino acid sequence of one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; b) A second domain containing the amino acid sequence of SEQ ID NO: 8; c) A third domain containing the amino acid sequence of SEQ ID NO: 9; d) A fourth domain comprising the amino acid sequence of SEQ ID NO: 10; and e) The fifth domain, which contains the amino acid sequence of SEQ ID NO:

7.

5. The polypeptide according to claim 4, wherein the first domain further comprises the amino acid sequence of SEQ ID NO:

23.

6. The polypeptide of claim 5, wherein the polypeptide comprises an amino acid sequence of one of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO:

16.

7. A nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide as described in any one of claims 1-6.

8. The nucleic acid molecule according to claim 7, wherein the nucleic acid molecule comprises a nucleic acid sequence of one of SEQ ID NO: 17-22.

9. A vector comprising a nucleic acid molecule as claimed in claim 7 or claim 8, preferably wherein the vector is a viral vector, and preferably wherein the viral vector is a lentiviral vector.

10. A cell comprising a polypeptide as described in any one of claims 1-6, a nucleic acid molecule as described in any one of claims 7-8, and / or a carrier as described in claim 9.

11. The cell of claim 10, wherein the cell is an immune cell, preferably wherein the immune cell is a T cell or a natural killer (NK) cell.

12. A cell population of the cells as described in claim 10 or claim 11.

13. A method of treating acute myeloid leukemia (AML) in a subject, the method comprising administering the cell population of claim 12 to the subject.

14. The method of claim 13, wherein the subject suffering from AML has a CD64+ AML cell population.

15. The method of claim 13 or claim 14, wherein the subject suffering from AML has a monocytic leukemia stem cell (mLSC) population.

16. The method according to any one of claims 13-15, wherein the subject has previously been given at least one therapy targeting AML, preferably wherein the therapy targeting AML comprises administration of a combination of venetoclax and azacitidine.

17. The method of claim 16, wherein the subject relapses after receiving treatment with the at least one targeted AML therapy, and / or the subject is resistant to the at least one targeted AML therapy.

18. A chimeric antigen receptor (CAR) comprising: (a) An antigen recognition domain, wherein the antigen recognition domain contains: (i) A heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence as shown in SEQ ID NO: 24 (or SEQ ID NO: 24 having one, two, or three amino acid additions, deletions, or substitutions), an amino acid sequence as shown in SEQ ID NO: 25 (or SEQ ID NO: 25 having one, two, or three amino acid additions, deletions, or substitutions), and an amino acid sequence as shown in SEQ ID NO: 26 (or SEQ ID NO: 26 having one, two, or three amino acid additions, deletions, or substitutions), and the light chain variable domain comprises an amino acid sequence as shown in SEQ ID NO: 32 (or SEQ ID NO: 32 having one, two, or three amino acid additions, deletions, or substitutions), an amino acid sequence as shown in SEQ ID NO: 33 (or SEQ ID NO: 33 having one, two, or three amino acid additions, deletions, or substitutions), and an amino acid sequence as shown in SEQ ID NO: 34 (or SEQ ID NO: 34 having one, two, or three amino acid additions, deletions, or substitutions); or (ii) A heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 40 (or SEQ ID NO: 40 having one, two, or three amino acid additions, deletions, or substitutions), the amino acid sequence shown in SEQ ID NO: 41 (or SEQ ID NO: 41 having one, two, or three amino acid additions, deletions, or substitutions), and the amino acid sequence shown in SEQ ID NO: 42 (or SEQ ID NO: 42 having one, two, or three amino acid additions, deletions, or substitutions), the light chain variable domain being shown in SEQ ID NO: 48 (or SEQ ID NO: 48 having one, two, or three amino acid additions, deletions, or substitutions), the light chain variable domain being shown in SEQ ID NO: 49 (or SEQ ID NO: 49 having one, two, or three amino acid additions, deletions, or substitutions), and the light chain variable domain being shown in SEQ ID NO: 50 (or SEQ ID NO: 50 having one, two, or three amino acid additions, deletions, or substitutions). (b) Hinge area; (c) Transmembrane domains; and (d) One or more signal transduction domains.

19. The CAR of claim 18, wherein the antigen recognition domain comprises the heavy chain variable domain and the light chain variable domain as shown in (i).

20. The CAR of claim 18, wherein the antigen recognition domain comprises the heavy chain variable domain and the light chain variable domain as shown in (ii).

21. The CAR according to any one of claims 19-20, wherein the antigen recognition domain comprises the amino acid sequence shown in SEQ ID NO:

1.

22. The CAR according to any one of claims 18-21, wherein the antigen recognition domain has the ability to bind differentiation cluster 64 (CD64) antigen.

23. The CAR according to any one of claims 18-22, wherein the hinge region is a CD8α hinge region.

24. The CAR of claim 23, wherein the CD8α hinge region comprises an amino acid sequence as shown in SEQ ID NO:

4.

25. The CAR according to any one of claims 18-24, wherein the transmembrane domain is a CD8α transmembrane domain.

26. The CAR of claim 25, wherein the CD8α transmembrane domain comprises an amino acid sequence as shown in SEQ ID NO:

5.

27. The CAR according to any one of claims 18-26, wherein the one or more signal transduction domains comprise a co-stimulatory domain and an activation domain.

28. The CAR of claim 27, wherein the co-stimulatory domain is a 4-1BB co-stimulatory domain.

29. The CAR of claim 28, wherein the 4-1BB co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:

6.

30. The CAR of claim 27, wherein the activation domain is a CD3ζ (CD3z) intracellular signal transduction domain.

31. The CAR of claim 30, wherein the CD3z intracellular signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:

7.

32. The CAR according to any one of claims 18-31, wherein the CAR further comprises a CD8α signal peptide.

33. The CAR of claim 32, wherein the CD8α signal peptide comprises the amino acid sequence shown in SEQ ID NO:

23.

34. The CAR according to any one of claims 18-33, wherein the CAR comprises the amino acid sequence shown in SEQ ID NO:

11.

35. A nucleic acid comprising a nucleic acid sequence encoding a CAR as described in any one of claims 18-34.

36. The nucleic acid of claim 35, wherein the nucleic acid comprises a nucleic acid sequence as shown in SEQ ID NO:

17.

37. The nucleic acid according to any one of claims 35-36, wherein the nucleic acid is a viral vector.

38. A cell comprising the nucleic acid as described in any one of claims 35-37.

39. A cell expressing the CAR as described in any one of claims 18-34.

40. The cell according to any one of claims 38-39, wherein the cell is a T cell, a stem cell, or a natural killer (NK) cell.

41. A composition comprising the cells as described in claim 40.

42. A method of treating a mammal with cancer, wherein the method comprises administering the composition of claim 41 to the mammal.

43. The method of claim 42, wherein the mammal is a human.

44. The method according to any one of claims 42-43, wherein the cancer is a CD64-positive cancer.

45. The method according to any one of claims 42-44, wherein the cancer is acute myeloid leukemia (AML).

46. ​​The method of claim 45, wherein the AML is a mononuclear cell type AML.

47. The method of claim 45, wherein the AML is a hybrid mononuclear / primitive cell type primary AML.

48. The method according to any one of claims 45-47, wherein the AML has a population of CD64-positive AML cells.

49. The method according to any one of claims 45-47, wherein the AML has a monocytic leukemia stem cell (mLSC) population.

50. The method according to any one of claims 42-49, wherein the number of cancer cells in the mammal is reduced after the administration step.

51. The method according to any one of claims 42-50, wherein the mammal has previously been given at least one therapy targeting AML.

52. The method of claim 51, wherein the targeted AML therapy comprises administering a combination of venetoclax and azacitidine.

53. The method according to any one of claims 51-52, wherein the mammal relapses after receiving treatment with at least one therapy targeting AML.

54. The method according to any one of claims 51-52, wherein the mammal is resistant to at least one therapy targeting AML.

55. The method according to any one of claims 51-54, wherein the AML lacks a population of CD64-positive AML cells prior to administering at least one therapy targeting AML to the mammal.

56. The method of claim 55, wherein after administering at least one therapy targeting AML to the mammal, the AML has a population of CD64-positive AML cells.

57. A chimeric antigen receptor (CAR) comprising: (a) An antigen recognition domain comprising an scFv containing the amino acid sequence shown in SEQ ID NO: 1; (b) CD8α hinge region, which contains the amino acid sequence shown in SEQ ID NO: 4; (c) CD8α transmembrane domain containing the amino acid sequence shown in SEQ ID NO: 5; (d) Human 4-1BB costimulatory domain, comprising the amino acid sequence shown in SEQ ID NO: 6; and (e) Human CD3z intracellular signal transduction domain, which contains the amino acid sequence shown in SEQ ID NO: 7.

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