Engineering NK cells with CAR constructs with optimal signaling

Abstract

Embodiments of the present disclosure encompass certain chimeric antigen receptor constructs that optionally include a hinge, one of the CD28 transmembrane domain or the DAP10 transmembrane domain, a DAP10 costimulatory domain, and CD3 zeta. In certain embodiments, the chimeric antigen receptor is expressed by natural killer (NK) cells, and in some cases, the NK cells are further modified to express, for example, one or more cytokines and optionally a suicide gene.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 257,608, filed October 20, 2021.

[0002] This application contains a Sequence Listing that has been submitted in XML format, which is incorporated by reference in its entirety. The XML copy, created on October 11, 2022, is named MDAC_1299WO_Sequence_Listing_ST26.xml and is 13,926 bytes in size. I.Technical field

[0003] Embodiments of the present disclosure include the fields of medicine including at least cell biology, molecular biology, immunology, and cancer medicine. II. Background

[0004] In recent years, adoptive cell therapy using autologous T cells transduced with chimeric antigen receptors (CARs) has proven to be a very powerful approach for the treatment of cancer and has gained FDA approval in B-cell leukemia / lymphoma. 1~3 However, challenges remain, such as uncoupling cytotoxicity against tumor cells from systemic toxicity, finding a solution to target antigen-negative relapse, and developing a universal off-the-shelf cell therapy product to avoid the logistical barriers to generating an autologous product while managing the issues that arise with allogeneic T cell products. 4 Natural killer (NK) cells are attractive candidates for CAR engineering because they mediate effective cytotoxicity against tumor cells and, unlike T cells, do not have the potential to cause graft-versus-host disease (GVHD) in the allogeneic setting. 5 Thus, NK cells can be utilized as an off-the-shelf cell therapy product for immediate clinical use. CAR-NK cells also retain the intrinsic ability to recognize and target tumor cells via their native receptors, so in principle disease escape by downregulation of CAR target antigens is less likely than observed with CAR-T cells.6 Umbilical cord blood (CB) is a readily available, "off-the-shelf" source of allogeneic NK cells (as an example) that can be expanded to large and highly functional doses using GMP-compliant universal antigen-presenting cells (uAPCs), which are K562 cells engineered to express CD48, 4-1BBL, and membrane-bound IL-21 (mbIL21). 7 . Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure fulfills a long-standing need in the art of adoptive cell therapy to provide highly effective NK cells that can be targeted to desired antigens with specific CAR configurations. [Means for solving the problem]

[0006] Embodiments of the present disclosure include methods and compositions relating to adoptive cell therapy for an individual in need thereof, including cell therapy in which the cells are modified NK cells that express certain synthetic proteins that are particularly effective in treating certain disease conditions, for example, allowing for enhanced effectiveness of the NK cells against cells that target the synthetic proteins.

[0007] In certain embodiments, the present disclosure relates to chimeric antigen receptor constructs that include a DAP10 costimulatory domain (which is more relevant to NK cell biology compared to T cell biology) in combination with either a DAP10 transmembrane domain or a CD28 transmembrane domain, and / or optionally in combination with a CD28 hinge. Such DAP10-containing CAR constructs result in enhanced CAR-NK cell anti-tumor activity compared to other costimulatory domains, such as the CD28 costimulatory domain, which are more specific to T cell biology. In specific embodiments, such constructs are utilized to improve adoptive CAR-NK cell therapy, enhancing efficacy and thereby allowing the use of fewer CAR-NK (or alternatively, CAR-T) cells for individuals in need thereof to reduce the risk of toxicity.

[0008] Certain embodiments of the present disclosure encompass adoptive cell therapy using CAR-NK cells (or other alternative CAR vehicles) to treat patients with any type of hematological malignancy, solid cancer, and / or infectious disease.

[0009] An embodiment of the present disclosure includes a polynucleotide encoding a fusion protein, the fusion protein comprising (a) optionally a CD28 hinge; and (b1) a CD28 transmembrane domain or (b2) a DAP10 transmembrane domain; and (c) a DAP10 costimulatory domain. In a specific embodiment, the fusion protein is further defined as a chimeric antigen receptor (CAR). In some cases, the CAR further comprises one or more antigen binding domains, including those targeting a cancer antigen (solid tumor or hematological malignancy) or an infectious agent. In a specific case, the CAR further comprises CD3 zeta, such as one comprising SEQ ID NO: 3. The polynucleotide may encode the CD28 transmembrane domain comprised in SEQ ID NO: 1. In certain embodiments, the CAR further comprises one or more additional costimulatory domains, such as one or more additional costimulatory domains selected from the group consisting of CD28, DAP12, 4-1BB, NKG2D, 2B4, and combinations thereof. The CAR may or may not further comprise a signal peptide, such as, by way of example, a signal peptide from CD8, CD27, granulocyte-macrophage colony-stimulating factor receptor (GMSCF-R), Ig heavy chain (IgH), CD3, or CD4.

[0010] In certain embodiments, the polynucleotide of the present disclosure further comprises encoding one or more additional polypeptides of interest. The sequence encoding the one or more additional polypeptides of interest and the sequence encoding the CAR may be separated on the polynucleotide by a 2A element or an IRES. In certain cases, the additional polypeptide of interest is one or more therapeutic proteins and / or a protein that enhances cell activity, expansion, cytotoxicity, and / or persistence. In some cases, the additional polypeptide of interest is a suicide gene product, one or more cytokines (e.g., IL-15, IL-2, IL-12, IL-18, IL-21, IL-23, and / or IL-7), and / or one or more human or viral proteins that enhance proliferation, expansion, and / or metabolic fitness. When the cytokine is IL-15, the IL-15 sequence may include SEQ ID NO:8.

[0011] In certain embodiments, any type of vector comprises any of the polynucleotides of the present disclosure, including viral vectors such as adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, or retroviral vectors, or non-viral vectors such as plasmids. Also encompassed in the present disclosure are any type of cells comprising any of the polynucleotides encompassed herein and / or any of the vectors encompassed herein. The cells may be stem cells or immune cells, or a mixture thereof. Specific immune cells include: natural killer (NK) cells, T cells, gamma delta T cells, alpha beta T cells, invariant NKT (iNKT) cells, B cells, macrophages, mesenchymal stromal cells, dendritic cells, or a mixture thereof. When the immune cells are NK cells, the NK cells may be derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, hematopoietic stem cells, bone marrow, or from a cell line, such as NK cells from the NK-92 cell line. The NK cells may be derived from umbilical cord blood mononuclear cells. The NK cells may be CD56+ NK cells. In a specific embodiment, the NK cells express recombinant cytokines such as IL-15, IL-2, IL-12, IL-18, IL-21, IL-7, and / or IL-23. Also included herein is a population of immune cells or stem cells expressing one or more CAR molecules of the present disclosure. When two or more CAR molecules are expressed by a cell, the CAR can target different antigens, enhancing the ability to specifically bind to the intended cell. The population may or may not be composed of a mixture of all types of cells.

[0012] An embodiment of the present disclosure includes a method of killing cancer cells in an individual, comprising administering to the individual an effective amount of any cell carrying any polynucleotide encompassed herein and / or any cell carrying any vector encompassed herein. In a specific embodiment, the polynucleotide-carrying cell is an immune cell, e.g., a NK cell, a T cell, a gamma delta T cell, an alpha beta T cell, an iNKT cell, a B cell, a macrophage, a dendritic cell, or a mixture thereof. The immune cell may comprise a NK cell, the NK cell being derived from umbilical cord blood (including CB mononuclear cells), peripheral blood, induced pluripotent stem cells, hematopoietic stem cells, bone marrow derived, a cell line, or a mixture thereof. The immune cell may be autologous or allogeneic with respect to the individual. In certain embodiments of the method, the polynucleotide-carrying cell and / or the vector-carrying cell are administered to the individual once or more than once, and the period between administration of the polynucleotide-carrying cell to the individual may be 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, or more than a year. The method may further comprise providing to the individual an effective amount of an additional therapy, such as surgery, radiation, gene therapy, immunotherapy, or hormonal therapy. The polynucleotide-carrying cells and / or vector-carrying cells may be administered to the individual by infusion, injection, intravenous, intraarterial, intraperitoneal, intratracheal, intratumoral, intramuscular, endoscopic, intralesional, intracranial, percutaneous, subcutaneous, topical, perfusion, into the tumor microenvironment, or combinations thereof.

[0013] The foregoing has outlined, rather broadly, the features and technical advantages of the present disclosure in order that the following detailed description may be better understood. Additional features and advantages will now be described which form the subject matter of the claims herein. It should be appreciated by those skilled in the art that the ideas and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present design. It should also be appreciated by those skilled in the art that such equivalent constructions do not depart from the spirit and scope as defined by the appended claims. The novel features believed characteristic of the design disclosed herein, both as to organization and method of operation, together with further objects and advantages, will be better understood from the following description when considered in conjunction with the accompanying figures. It is to be expressly understood, however, that each figure is provided for the purpose of illustration and description only, and is not intended as a definition of the limits of the present disclosure.

[0014] For a more complete understanding of this disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0015] [Figure 1A] Figure 1A-B. Phenotypic analysis using a mass cytometry panel. 1A. TSNE phonograph plot showing distinct clusters in non-transduced NT (left) and CD5CAR-NK CD28TMDAP10CD3z transduced NK cells. Clusters are numbered with a color code legend as shown below the phenograph. Two new clusters (#8 and #11) expressed in CARCD5 NK cells are highlighted by circles and squares. [Figure 1B] 1B. Heatmap showing normalized expression of various markers (shown on the X-axis) in each cluster (shown on the Y-axis). Activation, cytotoxicity, and maturation markers highly expressed in clusters #8 and #11 are highlighted with rectangles.

[0016] [Figure 2A]Figure 2A-Figure 2C. Isoplexis single cell secretome data showing polyfunctionality of CD5 CAR-NK cells with DAP10 costimulatory domain. 2A. Bar graph showing the percentage of polyfunctionality of different CD5 CAR-NK cells compared to non-transduced (NT) NK cells. [Figure 2B] 2B. Bar graph showing the polyfunctional strength index between different CD5 CAR-NK cells compared to non-transduced (NT) NK cells. [Figure 2C] 2C Multifunctional heatmap showing which constructs are most capable of secreting various permutations of cytokines at the single cell level.

[0017] [Figure 3A] 3A-C. Incucyte killing assay experiment with multiple tumor re-challenge. 3A. Schematic showing one embodiment of the experimental design and methodology for the Incucyte killing assay re-challenge experiment. [Figure 3B] 3B. Graph showing the number of red cells (y-axis; a measure of viable tumor numbers) after each tumor rechallenge (indicated by arrows) between various CD5 CAR-NK cell conditions. [Figure 3C] 3C. Graph showing percent confluence (a measure of tumor burden) after each tumor rechallenge (indicated by arrows).

[0018] [Figure 4A] Figure 4A-Figure 4B. Seahorse metabolic assay measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of various CD5 CAR-NK cells. 4A. Graph showing OCR between various CD5 CAR-NK cell designs compared to non-transduced (NT) NK cells. [Figure 4B] 4B. Graph showing ECAR between various CD5 CAR-NK cell designs compared to non-transduced (NT) NK cells.

[0019] [Figure 5A-C]Figure 5A-C. CD5 CAR-NK cells with DAP10 costimulatory domain show good activity in PDX mouse models of mantle cell lymphoma. 5A. Bar graph showing absolute number of CD45+CD5+ cells in subcutaneous tumors of mice receiving tumor alone (left) and tumor + CD5CAR-NK (right) in the subcutaneous tumor. 5B. Bar graph showing absolute number of CD45+CD5+ cells in subcutaneous tumors of mice receiving tumor alone (left) and tumor + CD5CAR-NK (right) in the spleen. 5C. Bar graph showing absolute number of CD45+CD5+ cells in subcutaneous tumors of mice receiving tumor alone (left) and tumor + CD5CAR-NK (right) in the bone marrow.

[0020] [Figure 6A] Figure 6A-6B. CD27 CAR-NK cells with DAP10 costimulatory domain improve tumor control and survival in the NSG mouse model of acute myeloid leukemia (THP-1 transduced with firefly luciferase (FFLuc)). 6A. A series of bioluminescent imaging (BLI) shows tumor burden as THP-1 FFLuc luminescence among various mouse groups. [Figure 6B] 6B. Survival curves showing the survival rate of mice in each group over time.

[0021] [Figure 7A-B] Figures 7A-7B. Figure 7A illustrates the identification of various constructs and the corresponding transduction efficiency (Figure 7B). CB-NK cells were transduced with various CD5 CAR constructs as shown in Figure 7A, and transduction efficiency was measured by flow cytometry. Transduction efficiency is based on the percentage of positive cells (Figure 7B). In Figure 7B, the bars from left to right of the bar graph correspond to the bars in the legend when read from top to bottom.

[0022] [Figure 8]An example of an experimental design is provided in which different CD5 constructs are injected into mice, with the corresponding timeline.The schematic shows the testing of the in vivo antitumor activity of different CD5 CAR NK cells against the T lymphoblastoid cell line CCRF-CEM as a target.

[0023] [Figure 9A] Figure 9A-Figure 9B. Figure 9A and Figure 9B show that mice treated with anti-CD5 CAR NK with IgG1 hinge cells survive significantly longer than NT NK cells and tumor alone. Bioluminescence images of mice from each group are shown (Figure 9A). [Figure 9B] Quantification of the luciferase signal is shown in FIG. 9B.

[0024] [Figure 10A] Figure 10A-B. Figure 10A and Figure 10B demonstrate that mice treated with anti-CD5 CAR NK with a CD28 hinge have significantly reduced tumor burden compared to tumor alone, NT NK cells, and CD5 CAR NK cells with an IgG1 hinge. Bioluminescence images of mice from each group are provided in Figure 10A. [Figure 10B] Quantification of the luciferase signal is shown in FIG. 10B.

[0025] [Figure 11] Figure 11. CD5 CAR-NK cells with DAP10 signaling show evidence of enhanced proliferative and metabolic advantage at the single cell transcriptome level. Heatmap showing pathway enrichment analysis of scRNAseq data comparing CD5CAR-DAP10-CD3z, CD5CAR-CD3z vs NT NK cells. N=2.

[0026] [Figure 12]Figure 12. CD5 CAR-NK cells with DAP10 signaling show enrichment of TFs associated with the AP1 complex and BATF family at the single cell epigenetic level. Volcano plot showing TF enrichment analysis of scATACseq data comparing CD5CAR-DAP10-CD3z vs. CD5CAR-CD3z. N=2.

[0027] [Figure 13A] Figure 13A-B. CD5 CAR-NK cells with DAP10 signaling show enhanced activation at the proteomic level by RPPA. 13A. Heatmap of RPPA analysis showing Log2 protein expression data of CD5CAR-DAP10-CD3z and CD5CAR-CD3z normalized to NT NK cells unstimulated and after 2 and 15 min stimulation with CD5 target antigen. [Figure 13B] 13B. Pathway network analysis showing interactions between different protein pathways related to proliferation, stemness, metabolic activity, immune synapse formation and memory features. N=2.

[0028] [Figure 14] Figure 14A-B. CD5 CAR-NK cells with DAP10 signaling have the ability to persist and mount recall responses after tumor rechallenge in vivo. 14A. Schematic detailing the experimental design of the in vivo mouse model showing the timing of irradiation, timing of injection of CD5+ CCRF tumors, timing of injection of CD5 CAR-NK cells, and timing of rechallenge of CD5+ CCRF tumors (transduced with FFLuc-GFP). 14B. FACS plots showing flow cytometry data before (left panel) and after rechallenge (right panel) show a human CD45+ gate followed by an NK cell gate (CD56+ and GFP-). This indicates that CD5 CAR-NK cells can expand after tumor rechallenge and mount recall responses against CD5+ CCRF tumors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description Definition Examples Following long-standing patent law practice, the words "a" and "an" when used herein in conjunction with the word comprising, including in the claims, refer to "one or more." Some embodiments of the present disclosure may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, and that different embodiments may be combined.

[0030] Throughout this specification, unless the context dictates otherwise, the words "comprise", "comprises" and "comprising" are understood to mean the inclusion of the recited step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and other elements may not be present. "Consisting essentially of" means including any elements recited after the phrase, and is limited to those that do not interfere with or contribute to the activity or action specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or mandatory, but other elements are optional and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0031] Throughout this specification, reference to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, means that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0032] As used herein, the terms "or" and "and / or" are utilized to describe multiple components in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0033] Throughout this application, the term "about" is used according to its plain and ordinary meaning within the art of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0034] The term "engineered," as used herein, refers to entities made by the hand of man, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, engineered entities are synthetic and contain elements that do not occur in nature or are not constructed by the methods utilized in the present disclosure.

[0035] The term "isolated" as used herein refers to a molecule or biological or cellular material that is substantially free of other materials. In one aspect, the term "isolated" refers to a nucleic acid such as DNA or RNA, or a protein or polypeptide, or a cell or organelle, or a tissue or organ, that is separated from other DNA or RNA, or proteins or polypeptides, or cells or organelles, or tissues or organs, respectively, as they are present in a natural source. The term "isolated" refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, culture medium, if produced by recombinant DNA technology, or chemical precursors or other chemicals, if chemically synthesized. Furthermore, "isolated nucleic acid" is meant to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to a polypeptide that is isolated from other cellular proteins, and is meant to encompass both purified and recombinant polypeptides. The term "isolated" is also used herein to refer to a cell or tissue that is isolated from other cells or tissues, and is meant to encompass both cultured and engineered cells or tissues.

[0036] As used herein, "prevent" and similar terms such as "prevented", "preventing" refer to an approach to prevent, inhibit, or reduce the likelihood of occurrence or recurrence of a disease or condition, such as cancer. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, impact, symptoms and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0037] The term "sample" as used herein generally refers to a biological sample. A sample can be taken from tissue or cells from an individual. In some cases, a sample can include or be derived from a tissue biopsy, blood (e.g., whole blood), plasma, extracellular fluid, dried blood spot, cultured cells, discarded tissue. A sample may be isolated from a source prior to collection. Non-limiting examples include blood, cerebrospinal fluid, pleural fluid, amniotic fluid, lymphatic fluid, saliva, urine, stool, tears, sweat, or mucosal excretions, and other bodily fluids isolated from a primary source prior to collection. In some cases, a sample is isolated from its primary source (cells, tissues, bodily fluids such as blood, environmental samples, etc.) during sample preparation. A sample may or may not be purified or concentrated from its primary source. In some cases, the primary source is homogenized prior to further processing. A sample may be filtered or centrifuged to remove buffy coat, lipids, or particulate matter. The sample may also be purified or enriched for nucleic acids or treated with RNase. The sample may contain intact tissues or cells, fragmented tissues or cells, or partially degraded tissues or cells.

[0038] The term "subject" as used herein generally refers to an individual having a biological sample being processed or analyzed, and in certain cases, an individual having or suspected of having cancer. A subject can be any living organism or animal subject that is the subject of a method or material, including mammals, such as humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. A subject can be, for example, a patient having or suspected of having a disease (sometimes referred to as a pathology), such as a benign or malignant neoplasm, or cancer. A subject can be undergoing or having been undergoing treatment. A subject can be asymptomatic. A subject can be an individual who is healthy but desires to prevent cancer. The term "individual" can be used interchangeably in at least some cases. A "subject" or "individual" as used herein may or may not be housed in a medical facility, or may be treated as an outpatient in a medical facility. An individual may receive one or more medical compositions via the Internet. An individual may include humans or non-human animals of any age, and thus includes both adults and juveniles (i.e., children) and infants, including individuals in utero. The term is not intended to imply a need for medical treatment, and an individual may voluntarily or involuntarily participate in an experiment, whether clinical or in support of basic science research.

[0039] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated, such as cancer. Treatment may optionally include either a reduction or amelioration of one or more symptoms of the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily indicate a complete eradication or cure of the disease or condition, or the symptoms associated therewith.

[0040] In the context of a therapeutic, diagnostic, or physiological purpose or effect, any method may also be described in "use" claim language, such as the "use" of any compound, composition, or agent discussed herein to achieve or carry out the described therapeutic, diagnostic, or physiological purpose or effect.

[0041] The present disclosure relates to methods and compositions in which chimeric antigen receptor constructs have one or more components that are more relevant to NK cells, in contrast to the biology that is suitable for other immune cells, including T cells, and therefore are more suitable for use with NK cells, at least in some cases.In one example, considering that CD28 is a costimulatory molecule related to T cell biology and is not present in NK cells, the inventors have developed other CAR vectors with alternative costimulatory molecules that are more relevant to NK cell biology, such as, for example, DAP10, DAP12, NKG2D and / or 4-1BB.As encompassed herein, different CAR designs with various costimulatory molecules have been characterized using either CD5 scFv or CD27 extracellular domain as antigen binding domain, but these are merely examples. The DAP10 costimulatory domain confers a more activated phenotype to CAR-NK cells, with specific clusters identified by mass cytometry showing increased expression of activation markers such as DNAM, NKG2D, CD3z and ZAP70, cytotoxic markers such as TRAIL, granzyme B, and perforin, as well as maturation markers such as Eomes and T-bet (Figure 1). Furthermore, CAR-NK cells bearing the DAP10 costimulatory domain and either the DAP10 or CD28 transmembrane domains exhibited high polyfunctionality compared to CAR-NK cells bearing other costimulatory domains (Figure 2). Importantly, CD5-directed CAR-NK cells bearing the DAP10 costimulatory domain retained the ability to kill CCRF T-ALL cell lines even after multiple tumor rechallenges, whereas CAR-NK cells bearing other costimulatory molecules lost the ability to kill CCRF upon later tumor rechallenge, likely due to functional exhaustion (Figure 3). From a metabolic standpoint, CAR-NK cells bearing the DAP10 costimulatory domain exhibited increased metabolic fitness with higher oxidative phosphorylation as evidenced by higher oxygen consumption rate (OCR) and glycolytic capacity, as well as higher extracellular acidification rate (ECAR) (Figure 4), which may, in certain embodiments, correlate with their enhanced efficacy.This translated into enhanced antitumor activity of DAP10 constructs in a PDX mouse model of mantle cell lymphoma testing the efficacy of CD5 CAR-NK cells (Figure 5) and in an NSG mouse model of acute myeloid leukemia (THP-1) testing the efficacy of CD70 CAR-NK cells (Figure 6). As shown herein, DAP10, a key adaptor molecule downstream of the NK activating receptor NKG2D, can act as a potent co-stimulatory domain of CAR-NK cells, enhancing their metabolic fitness and antitumor activity in vitro and in vivo. I. Engineered Receptors

[0042] The present disclosure relates to engineered receptors that utilize certain components, which have enhanced potency over other engineered receptors that lack certain components. In certain embodiments, the receptor optionally includes a hinge. If the receptor includes an scFv as at least a portion of the extracellular domain, the receptor may include a hinge between the scFv and the transmembrane domain. In specific examples, if the extracellular domain of the receptor lacks an scFv, e.g., includes at least a portion of the extracellular domain of an endogenous receptor or another receptor, the receptor may or may not include a hinge. In certain cases, the receptor includes at least a CD28 hinge. In some cases, the receptor includes at least a CD28 transmembrane domain. In certain cases, the receptor includes at least a DAP10 transmembrane domain. In certain cases, the receptor includes at least a DAP10 costimulatory domain.

[0043] In specific embodiments, the enhanced receptor comprises (including in the form of a fusion protein):

[0044] (a) optionally a hinge; and

[0045] (b1) CD28 transmembrane domain or

[0046] (b2) the transmembrane domain of DAP10;

[0047] (c) the DAP10 costimulatory domain; and

[0048] (d) CD3 zeta.

[0049] Thus, in certain cases, the receptor comprises a CD28 hinge (or, for example, a CD8 alpha hinge, an IgG1 hinge), a CD28 transmembrane domain, and a DAP10 costimulatory domain. In other cases, the receptor comprises a CD28 hinge, a DAP10 transmembrane domain, and a DAP10 costimulatory domain. In a specific embodiment, the components (a) (optional), (b1) or (b2), (c) and (d) are in the form of a fusion protein, and are in the following order from N-terminus to C-terminus: (a) (optional), (b1) or (b2), (c) and (d). In certain cases, the fusion protein lacks (a).

[0050] In a specific embodiment, the components (a) (optional), (b1) or (b2), (c) and (d) in the form of a fusion protein further comprise one or more antigen binding domains, including as the configuration of CAR. In a specific embodiment, the one or more antigen binding domains in the N-terminal to C-terminal direction are on the N-terminal side of (a) (optional), (b1) or (b2), (c) and (d) (in this order). In a specific embodiment, the fusion protein as an engineered receptor essentially consists of one or more antigen binding domains, (a) (optional), (b1) or (b2), (c) and (d). That is, in a specific case, the engineered receptor lacks any other costimulatory domains other than DAP10, while in an alternative case, the engineered receptor comprises one or more costimulatory domains other than DAP10. In such alternative cases where the engineered receptor includes one or more costimulatory domains other than DAP10, the other costimulatory domains may or may not be costimulatory domains present in proteins naturally found in NK cells but not in T cells, examples include CD28, DAP12, 4-1BB, NKG2D, 2B4, combinations thereof, etc.

[0051] The components of (a) (optional), (b1) or (b2), (c) and (d) may be expressed on a single polynucleotide as a fusion protein, and in certain embodiments, the fusion protein comprises one or more antigen binding domains. The polynucleotide may be isolated or may be included in any type of vector, including viral or non-viral. In specific embodiments, the vector is present in any type of cell, including immune cells such as NK cells, T cells, gamma delta T cells, alpha beta T cells, iNKT cells, B cells, macrophages, dendritic cells, or mixtures thereof. Suitable methods for cell modification are known in the art. See, for example, Sambrook and Ausubel, supra. For example, the gene transfer techniques described in Heemskerk et al., 2008 and Johnson et al., 2009 can be used to transduce cells to express engineered receptors with antigen specificity for cancer antigens or antigens of infectious agents.

[0052] In some embodiments, the cells contain one or more nucleic acids introduced via genetic engineering that code for one or more engineered receptors, and express the engineered products of such nucleic acids. In certain embodiments, the nucleic acids are heterologous, i.e., not normally present in the cell or sample obtained from the cell, such as those obtained from another organism or cell that is not normally found in the engineered cell and / or the organism from which such cell is derived. In some embodiments, the nucleic acids are non-naturally occurring nucleic acids, such as nucleic acids not found in nature (e.g., chimeras). They may be products of the hand of man.

[0053] Exemplary antigen receptors, including CARs, as well as methods for engineering and introducing receptors into cells, include those described in, for example, International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US20130149337, U.S. Patent Nos. 6,451,995, 7,446,190, 8,446,192, and 9,542,742. ,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, as well as European Patent Application No. EP2537416, and / or those described by Sadelain et al., 2013, Davila et al., 2013, Turtle et al., 2012, Wu et al., 2012. In some embodiments, engineered antigen receptors include CARs such as those described in U.S. Pat. No. 7,446,190 and those described in International Patent Application Publication No. WO / 2014055668.

[0054] In certain embodiments, the engineered receptor comprises one or more antigen binding domains and components (a), (b1) or (b2), and (c). In specific embodiments, the engineered receptor is a CAR, and in some embodiments, the antigen binding domain is an antibody or a functional fragment thereof. In other cases, the antigen binding domain of the CAR is not an antibody or a functional fragment thereof, but instead is a natural ligand for the receptor. A CAR may be a single polypeptide that is bispecific by containing two or more antigen binding domains, one of which binds to a desired antigen and the other of which binds to another non-identical antigen.

[0055] In some embodiments, the engineered antigen receptor comprises a CAR, including an activating or stimulatory CAR, or a costimulatory CAR (see WO2014 / 055668). CARs generally comprise an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some cases via components (a), (b1) or (b2), and (c). Such molecules typically mimic or approximate the signaling through a natural antigen receptor, the signaling through such receptor in combination with a costimulatory receptor, and / or the signaling through a costimulatory receptor alone.

[0056] It is contemplated that chimeric constructs can be introduced into immune cells as naked DNA or in suitable vectors.Methods for stable transfection of cells by electroporation using naked DNA are known in the art.See, for example, U.S. Patent No. 6,410,319.Naked DNA generally refers to the DNA encoding chimeric receptor contained in a plasmid expression vector in the appropriate orientation for expression.

[0057] Alternatively, a viral vector (e.g., retroviral vector, adenoviral vector, adeno-associated viral vector, or lentiviral vector) can be used to introduce chimeric CAR construct into immune cells.The vector suitable for use according to the method of the present disclosure is non-replicative in immune cells.There are many known viral vectors, such as HIV, SV40, EBV, HSV, or BPV-based vectors, whose viral copy number maintained in cells is low enough to maintain cell viability.

[0058] Certain embodiments of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding specific CAR polypeptides that include the components (a) (optional), (b1) or (b2), (c) and (d), in some cases including humanized CARs (hCARs) to reduce immunogenicity. In certain embodiments, the CAR may recognize an epitope that includes a shared space between one or more antigens. In certain embodiments, the binding region may include a complementarity determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor.

[0059] It is contemplated that the human CAR nucleic acid may be a human gene used to enhance cellular immunotherapy for human patients. In a specific embodiment, the present disclosure includes a full-length antigen-specific CAR cDNA or coding region. The antigen-binding region or domain may be the V of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody, as described in U.S. Patent No. 7,109,304, which is incorporated herein by reference. H Chain and V L The fragments may include fragments of the chains. The fragments may also be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence optimized for human codon usage for expression in human cells.

[0060] In some embodiments, antigen-specific CARs are constructed with specificity for an antigen, such as the antigen being expressed on diseased cell types (cancer cells or cells infected with infectious agents). Thus, CARs typically comprise one or more antigen-binding molecules, such as one or more antigen-binding fragments, domains, antibody variable domains, ligands, receptors, and / or any type of antibody molecule, in their extracellular portion. Those skilled in the art can generate antibodies comprising scFvs against antigens based at least on their knowledge of polypeptides and routine practice, but many anti-antigen scFvs and monoclonal antibodies may already exist in the art. In some embodiments, the antigen-specific scFv is an scFv from one or more antibody clones.

[0061] In some embodiments, the antigen-specific CAR comprises an antigen-binding portion or part of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb). In a specific embodiment, the antibody or functional fragment thereof is or is derived from a known antibody. The antibody may also be generated de novo against the antigen, and the scFv sequence may be obtained or derived from such a de novo antibody.

[0062] In certain embodiments, the CAR comprises an extracellular domain that is or includes a natural ligand or receptor for a target antigen or receptor. In some embodiments, the CAR comprises an extracellular domain that is or includes a VH and / or VL from an antibody that targets the antigen.

[0063] The sequence of the open reading frame encoding the chimeric receptor can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g., by PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, since introns have been shown to stabilize mRNA. It may also be more advantageous to use endogenous or exogenous non-coding regions to stabilize mRNA.

[0064] In some aspects, the antigen-specific binding domain is linked to the CD28 or DAP10 transmembrane domain, and in some cases also to the DAP10 costimulatory domain. In some examples, the CD28 or DAP10 transmembrane domain is modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein, so as to minimize interaction with other members of the receptor complex. The transmembrane domain is, in some embodiments, either naturally derived or synthetic. Alternatively, the transmembrane domain is, in some embodiments, synthetic. In some aspects, the synthetic transmembrane domain mainly comprises hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine may be found at each end of the synthetic CD28 or DAP10 transmembrane domain.

[0065] In some embodiments, the CAR nucleic acid comprises sequences encoding DAP10 and optionally other than CD3 zeta. In addition to primary T cell activation signals, such as those initiated by CD3ζ and / or FcεRIγ, additional stimulatory signals for immune effector cell proliferation and effector function following engagement of the chimeric receptor with the target antigen, other than DAP10, can be utilized. For example, some or all of the human costimulatory receptors can be utilized to enhance cell activation, improve in vivo persistence, and improve the success of adoptive immunotherapy treatment. Examples include costimulatory domains from molecules such as DAP12, NKG2D, 2B4, CD2, CD28, CD27, 4-1BB, OX40, ICOS, (CD278), CD30, HVEM, CD40, LFA-1 (CD11a / CD18), and / or ICAM-1, although in certain alternative embodiments, any one of these listed may be excluded from use in the CAR. II. Examples of Specific CAR Embodiments

[0066] In certain embodiments, specific CAR molecules are encompassed herein and include (a) optionally a hinge, and (b1) a CD28 transmembrane domain or (b2) a DAP10 transmembrane domain, (c) a DAP10 costimulatory domain, and (d) CD3 zeta. In some cases, the CAR further includes any kind of antigen-binding domain, which may be any kind of scFv. When an scFv is utilized in the extracellular domain of the CAR, the variable heavy chain and the variable light chain of a particular scFv may be in any order from the N-terminus to the C-terminus. For example, the variable heavy chain may be N-terminal to the variable light chain, or vice versa. The scFv that binds to the antigen in the CAR may or may not be codon-optimized. In certain embodiments, the vector encodes an antigen-specific CAR that includes (a) (optional), (b1) or (b2), (c) and (d), and also encodes one or more other molecules. For example, the vector may encode such a CAR and may also encode another protein of interest, such as one or more other engineered antigen receptors, a suicide gene, and / or one or more specific cytokines.

[0067] On the same molecule, a CAR may contain one or more antigen-specific extracellular domains that target two different antigens, and there may be a linker between the two antigen-specific extracellular domains.

[0068] In certain embodiments of specific CAR molecules, the CAR utilizes DAP10, but also CD28, DAP12, 4-1BB, NKG2D, or other costimulatory domains (sometimes referred to herein as cytoplasmic domains), including those encompassed herein.

[0069] Examples of specific sequence embodiments of CARs are shown below, in no particular order:

[0070] The amino acid sequence of the CD28 transmembrane domain: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 1)

[0071] Any polypeptide encompassed by this disclosure may comprise SEQ ID NO:1, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more percent identical to SEQ ID NO:1.

[0072] One or more intracellular domains (which may be referred to herein as signal activation domains or costimulatory domains, as appropriate) may or may not be utilized in a particular CAR of the present disclosure. Specific examples include the intracellular domain from DAP10, and in certain cases, CD3 zeta.

[0073] Examples of particular intracellular domains that can be used in the CARs of the present disclosure are shown below:

[0074] An example of the DAP10 intracellular domain amino acid sequence: LCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO: 2) Any polypeptide encompassed by this disclosure may comprise SEQ ID NO:2, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more percent identical to SEQ ID NO:2.

[0075] An example of the CD3zeta intracellular domain amino acid sequence:

[0076] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRG (SEQ ID NO: 3)

[0077] Any polypeptide encompassed by this disclosure may comprise SEQ ID NO:3, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more percent identical to SEQ ID NO:3.

[0078] In some cases, the CAR further comprises an intracellular domain other than DAP10 (and in some cases CD3 zeta), such as:

[0079] Amino acid sequence of the 4-1BB intracellular domain: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 4) Any polypeptide encompassed by this disclosure may comprise SEQ ID NO:4, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more percent identical to SEQ ID NO:4.

[0080] Amino acid sequence of the DAP12 intracellular domain: YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO: 5) Any polypeptide encompassed by this disclosure may comprise SEQ ID NO:5, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more percent identical to SEQ ID NO:5.

[0081] Amino acid sequence of the NKG2D intracellular domain: SANERCKSKVVPCRQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGM (SEQ ID NO: 6)

[0082] Amino acid sequence of the CD28 intracellular domain: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 14)

[0083] Any polypeptide encompassed by this disclosure may comprise SEQ ID NO:6, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more percent identical to SEQ ID NO:6.

[0084] In some embodiments of the CAR, a hinge region is present between one or more extracellular antigen binding domains and the transmembrane domain, in particular cases when scFvs are used in the CAR, but not when the scFv is deleted in the CAR. In particular embodiments, the hinge is of a particular length, such as 10-20, 10-15, 11-20, 11-15, 12-20, 12-15, or 15-20 amino acids in length. In particular embodiments, the hinge is a CD28 hinge. In particular cases, the identity or length of the CD28 hinge can be modified to enhance the efficacy of the CAR. See, for example, Hudecek et al. (2014) and Jonnalagadda et al. (2015). In particular embodiments, the hinge is derived from CD28, CD8 alpha, or IgG1.

[0085] Examples of CD28 hinge amino acid sequences include the following: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 7)

[0086] Any polypeptide encompassed by this disclosure may comprise SEQ ID NO:7, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more percent identical to SEQ ID NO:7.

[0087] In specific embodiments, the following examples of expression constructs for CARs can be utilized. These CARs target CD5, although other antigens may be targeted.

[0088] CD5#1 contains an IgG hinge and a DAP12 transmembrane domain, a DAP12 costimulatory domain, and CD3 zeta.

[0089] CD5#2 contains an IgG hinge and a CD28 transmembrane domain, a DAP12 costimulatory domain, and CD3 zeta.

[0090] CD5#3 contains an IgG hinge and a CD28 transmembrane domain, a 4-1BB costimulatory domain, and CD3 zeta.

[0091] CD5#4 contains an IgG hinge and a DAP10 transmembrane domain, a DAP10 costimulatory domain, and CD3 zeta.

[0092] CD5#5 contains an IgG hinge and a CD28 transmembrane domain, a DAP10 costimulatory domain, and CD3 zeta.

[0093] CD5#7 contains an IgG hinge and a CD28 transmembrane domain, an NKG2D costimulatory domain, and CD3 zeta.

[0094] CD5#8 contains the IgG hinge and CD28 transmembrane domains, and CD3 zeta.

[0095] CD5#9 contains an IgG1 hinge, a CD28 transmembrane domain, a CD28 costimulatory domain, and CD3 zeta.

[0096] CD5#10 contains the CD28 hinge, the CD28 transmembrane domain, the DAP10 costimulatory domain, and CD3 zeta.

[0097] CD5#11 contains the CD28 hinge, the DAP10 transmembrane domain, the DAP10 costimulatory domain, and CD3 zeta.

[0098] CD5#12 contains the CD28 hinge, the CD28 transmembrane domain, the DAP12 costimulatory domain, and CD3 zeta.

[0099] CD5#13 contains the CD28 hinge, the CD28 transmembrane domain, the CD28 costimulatory domain, and CD3 zeta.

[0100] In this method, any suitable antigen can be targeted. In some cases, antigen may be associated with a certain cancer cell but not associated with non-cancer cell. Exemplary antigens include, but are not limited to, antigen molecules derived from infectious agents, autologous / self-antigens, tumor / cancer-associated antigens, and tumor neo-antigens (Linnemann et al., 2015).

[0101] In certain embodiments, the antigen is associated with cancer and is selected from the group consisting of CD19, EBNA, CD123, HER2, CA-125, TRAIL / DR4, CD20, CD70, CD38, CD123, CLL1, carcinoembryonic antigen, alpha fetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4, CD33 , CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD70, TROP-2, CD99, p53, mutant p53, Ras, mutant ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, melanoma-associated antigen, GAGE-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, -31, -32, -33, -34, -35, -36, -37, -38, -39, -40, -41, -42, -43, -44, -45, -46, -47, -48, -49, -51, -52, -53, -54, -55, -66, -67, -68, -79, -81, -82, -83, -84, -85, -86, -87, -89, -98, -106, -117, -121, -138, -141, -155, -162, -173, -184, -195, -196, -197, -198, -199, -199, -199, -1 4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, Gp100, PSA, PSM, tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G25 0, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signaling transduction 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (especially EGFRvIII),Platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), VEGFR2, cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), Nuclear Factor-Kappa B (NF-B), Notch receptors (e.g., Notch1-4), NY ESO 1, c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinases (ERKs) and their regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl-GM1, mesothelial, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, and LRRN1. Examples of antigen sequences are known in the art, for example in the GENBANK® database: CD19 (accession number NG_007275.1), EBNA (accession number NG_002392.2), WT1 (accession number NG_009272.1), CD123 (accession number NC_000023.11), NY-ESO (accession number: NC_000023.11), EGFRvIII (accession number: NG_007726.3), MUC1 (accession number: NG_029383.1),HER2 (accession number: NG_007503.1), CA-125 (accession number NG_055257.1), WT1 (accession number NG_009272.1), Mage-A3 (accession number NG_013244.1), Mage-A4 (accession number NG_013245.1), Mage-A10 (accession number NC_000023.11), TRAIL / DR4 (accession number NC_000003.12), and / or CEA (accession number NC_000019.10).

[0102] Tumor-associated antigens can be derived from, for example, prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, kidney cancer, mesothelioma, ovarian cancer, liver cancer, brain cancer, bone cancer, stomach cancer, spleen cancer, testicular cancer, cervical cancer, anal cancer, gallbladder cancer, thyroid cancer, or melanoma cancer. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE 1, 3, and MAGE 4 (or other MAGE antigens such as those disclosed in International Patent Publication No. WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of tumor types, such as melanoma, lung cancer, sarcoma, and bladder cancer. See, for example, U.S. Patent No. 6,544,518. Prostate cancer tumor associated antigens include, for example, prostate specific membrane antigen (PSMA), prostate specific antigen (PSA), prostatic acid phosphate, NKX3.1, and six-stage membrane epithelial antigen of the prostate (STEAP).

[0103] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto and Criptin. In addition, tumor antigens may be self-peptide hormones, such as full-length gonadotrophin-releasing hormone (GnRH), a short peptide of 10 amino acids that is useful in the treatment of many cancers.

[0104] Antigens may include epitope regions or epitope peptides derived from genes mutated or transcribed at different levels in tumor cells compared to normal cells, aberrantly expressed intronic sequences such as telomerase enzyme, survivin, mesothelin, mutant ras, bcr / abl rearrangements, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myelomas and B-cell lymphomas; tumor antigens that include epitope regions or epitope peptides derived from tumor viral processes such as human papilloma virus proteins E6 and E7; Epstein-Barr virus protein LMP2; non-mutated oncofetal proteins with tumor selective expression such as carcinoembryonic antigen and alpha-fetoprotein.

[0105] In other embodiments, antigens are obtained or derived from infectious agents, including pathogenic or opportunistic pathogenic microorganisms (also referred to herein as infectious disease microorganisms), such as viruses, fungi, parasites, protozoa, and bacteria. In certain embodiments, antigens derived from such microorganisms include full-length proteins.

[0106] Exemplary pathogenic organisms whose antigens are contemplated for use in the methods described herein include human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza A, B and C, vesicular stomatitis virus (VSV), polyomaviruses (e.g., BK virus and JC virus), adenovirus, Staphylococcus species, including methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus species, including Streptococcus pneumoniae. As will be appreciated by those of skill in the art, proteins from these and other pathogenic microorganisms, as well as nucleotide sequences encoding the proteins, for use as antigens as described herein can be identified in publications and public databases such as GENBANK®, SWISS-PROT® and TREMBL®.

[0107] Antigens from Human Immunodeficiency Virus (HIV) include any of the structural proteins of the HIV virion (e.g., gpl20, gp41, pl7, p24), protease, reverse transcriptase, or the HIV proteins encoded by tat, rev, nef, vif, vpr, and vpu.

[0108] Antigens from herpes simplex viruses (e.g., HSV1 and HSV2) include, but are not limited to, proteins expressed from HSV late genes. The late gene group primarily codes for proteins that form virion particles. Such proteins include five proteins from (UL) that form the viral capsid: UL6, UL18, UL35, UL38, and major capsid proteins UL19, UL45, and UL27, each of which can be used as an antigen as described herein. Other exemplary HSV proteins contemplated for use as antigens herein include ICP27 (HI, H2), glycoprotein B (gB), and glycoprotein D (gD) proteins. The HSV genome contains at least 74 genes, each of which codes for a protein that may be used as an antigen.

[0109] Antigens from cytomegalovirus (CMV) include CMV structural proteins, viral antigens expressed during immediate early and early vims replication, glycoproteins I and III, capsid proteins, coat proteins, lower matrix proteins pp65 (ppUL83), p52 (ppUL44), IE1 and 1E2 (UL123 and UL122), protein products from the UL128-UL150 gene cluster (Rykman et al., 2006), envelope glycoproteins B (gB), gH, gN, and ppl50. As will be appreciated by those of skill in the art, CMV proteins for use as antigens as described herein may be identified in public databases such as GENBANK®, SWISS-PROT®, and TREMBL® (see, e.g., Bennekov et al., 2004; Loewendorf et al., 2010; Marschall et al., 2009).

[0110] In certain embodiments, antigens derived from Epstein-Burn virus (EBV) contemplated for use include EBV proteins produced during the latent cycle of infection, including EBV lysis proteins gp350 and gpl lO, Epstein-Burn nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein (EBNA-LP), and latent membrane protein (LMP)-1, LMP-2A, and LMP-2B (see, e.g., Lockey et al., 2008).

[0111] Antigens from respiratory syncytial virus (RSV) contemplated for use herein include any of 11 proteins, or antigenic fragments thereof, encoded by the RSV genome: NS1, NS2, N (nucleocapsid protein), M (matrix protein) SH, G and F (viral coat proteins), M2 (second matrix protein), M2-1 (elongation factor), M2-2 (transcriptional regulator), RNA polymerase, and phosphoprotein P.

[0112] Antigens derived from vesicular stomatitis virus (VSV) contemplated for use include any one of the five major proteins encoded by the VSV genome, and antigenic fragments thereof: large protein (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and matrix protein (M) (see, e.g., Rieder et al., 1999).

[0113] In certain embodiments, antigens derived from influenza virus that are contemplated for use include hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins M1 and M2, NS 1, NS2 (NEP), PA, PB 1, PB 1-F2, and PB2.

[0114] Exemplary viral antigens include, but are not limited to, adenovirus polypeptides, alphavirus polypeptides, calici vims polypeptides (e.g., calici vims capsid antigen), coronavirus polypeptides, distemper virus polypeptides, ebolavirus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis vims (AE) polypeptides (Hepatitis B core or surface antigen, Hepatitis C vims E1 or E2 glycoproteins, core, or nonstructural proteins), herpesvirus polypeptides (including glycoproteins of herpes simplex virus or varicella zoster virus), infectious peritonitis vims polypeptides, leukemia vims polypeptides, Also included are rabies vims polypeptides, Marburg vims polypeptides, orthomyxovirus polypeptides, papilloma vims polypeptides, parainfluenza vims polypeptides (e.g., hemagglutinin and neuraminidase polypeptides), paramyxovirus polypeptides, parvovirus polypeptides, pesti vims polypeptides, picorna vims polypeptides (e.g., poliovims capsid polypeptides), pox vims polypeptides (e.g., vaccinia vims polypeptides), rabies vims polypeptides (e.g., rabies vims glycoprotein G), reovims polypeptides, retrovirus polypeptides, and rotavirus polypeptides.

[0115] In certain embodiments, the antigen may be a bacterial antigen. In certain embodiments, the bacterial antigen of interest may be a secreted polypeptide. In other certain embodiments, the bacterial antigen comprises an antigen having a portion or portions of a polypeptide exposed on the outer cell surface of the bacteria.

[0116] Antigens from Staphylococcus species, including Methicillin-resistant Staphylococcus aureus (MRSA), contemplated for use include virulence regulators such as the Agr system, Sar and Sae, the Arl system, Sar homologues (Rot, MgrA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ and TcaR), the Srr system and TRAP. Other Staphylococcus proteins that can act as antigens include Clp proteins, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA and CfvB (see, e.g., Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, edited by Jodi Lindsay). The genomes of two species of Staphylococcus aureus (N315 and Mu50) have been sequenced and are publicly available, for example, in PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, Snyder et al., 2007). As will be appreciated by those skilled in the art, staphylococcal proteins for use as antigens may also be identified in other public databases, such as GenBank®, Swiss-Prot®, and TrEMBL®.

[0117] In certain embodiments described herein, antigens from pneumococcus contemplated for use include pneumolysin, PspA, choline-binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and pilin proteins (RrgA, RrgB, RrgC). Antigenic proteins of pneumococcus are also known in the art and may be used as antigens in some embodiments (see, for example, Zysk et al., 2000). The entire genome sequence of pathogenic strains of pneumococcus has been sequenced, and as will be appreciated by those skilled in the art, pneumococcal proteins used herein may also be identified in other public databases, such as GENBANK®, SWISS-PROT®, and TREMBL®.

[0118] Proteins of particular interest as antigens according to the present disclosure include virulence factors and proteins predicted to be exposed on the surface of pneumococcus (see, e.g., Frolet et al., 2010).

[0119] Examples of bacterial antigens that can be used as antigens include, but are not limited to, Actinomyces polypeptides, Bacillus polypeptides, Bacteroides polypeptides, Bordetella polypeptides, Bartonella polypeptides, Borrelia polypeptides (e.g., OspA of B. burgdorferi), Brucella polypeptides, Campylobacter polypeptides, Capnocytophaga polypeptides, Chlamydia polypeptides, Corynebacterium polypeptides, Coxiella polypeptides, Dermatophilus polypeptides, Enterococcus polypeptides, Ehrlichia polypeptides, Escherichia polypeptides, Francisella polypeptides, Fusobacterium polypeptides, Haemobartonella polypeptides, Haemophilus polypeptides (e.g., H. influenzae polypeptides, b type outer membrane proteins), Helicobacter polypeptides, Klebsiella polypeptides, L-type bacteria polypeptides, Leptospira polypeptides, Listeria polypeptides, Mycobacteria polypeptides, Mycoplasma polypeptides, Neisseria polypeptides, Neorickettsia polypeptides, Nocardia polypeptides, Pasteurella polypeptides, Peptococcus polypeptides, Peptostreptococcus polypeptides, Pneumococcus polypeptides (i.e. S. pneumoniae polypeptides) (see description herein), Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Rochalimaea polypeptides, Salmonella polypeptides, Shigella polypeptides, Staphylococcus polypeptides, streptococcus polypeptides of group A (e.g. S.pyogenes M protein), group B streptococcus (S. agalactiae) polypeptides, Treponema polypeptides, and Yersinia polypeptides (e.g., Y pestis FI and V antigens).

[0120] Examples of fungal antigens include, but are not limited to, Absidia polypeptides, Acremonium polypeptides, Alternaria polypeptides, Aspergillus polypeptides, Basidiobolus polypeptides, Bipolaris polypeptides, Blastomyces polypeptides, Candida polypeptides, Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum polypeptides, Histoplasma polypeptides, Madurella polypeptides, Malassezia polypeptides, Microsporum polypeptides, Examples of polypeptides include polypeptides of the genus Moniliella, Mortierella, Mucor, Paecilomyces, Penicillium, Phialemonium, Phialophora, Prototheca, Pseudallescheria, Pseudomicrodochium, Pythium, Rhinosporidium, Rhizopus, Scolecobasidium, Sporothrix, Stemphylium, Trichophyton, Trichosporon, and Xylohypha.

[0121] Examples of protozoan parasite antigens include, but are not limited to, Babesia genus polypeptides, Balantidium genus polypeptides, Besnoitia genus polypeptides, Cryptosporidium genus polypeptides, Eimeria genus polypeptides, Encephalitozoon genus polypeptides, Entamoeba genus polypeptides, Giardia genus polypeptides, Hammondia genus polypeptides, Hepatozoon genus polypeptides, Isospora genus polypeptides, Leishmania genus polypeptides, Microsporidia genus polypeptides, Neospora genus polypeptides, Nosema genus polypeptides, Pentatrichomonas genus polypeptides, and Plasmodium genus polypeptides. Examples of helminth parasite antigens include, but are not limited to, Acanthocheilonema spp. polypeptides, Aelurostrongylus spp. polypeptides, Ancylostoma spp. polypeptides, Angiostrongylus spp. polypeptides, Ascaris spp. polypeptides, Brugia spp. polypeptides, Bunostomum spp. polypeptides, Capillaria spp. polypeptides, Chabertia spp. polypeptides, Cooperia spp. polypeptides, Crenosoma spp. polypeptides, Dictyocaulus spp. polypeptides, Dioctophyme spp. polypeptides, Dipetalonema spp. polypeptides, Diphyllobothri spp. polypeptides, um polypeptides, Diplydium polypeptides, Dirofilaria polypeptides, Dracunculus polypeptides, Enterobius polypeptides, Filaroides polypeptides, Haemonchus polypeptides, Lagochilascaris polypeptides, Loa polypeptides, Mansonella polypeptides, Muellerius polypeptides, Nanophyetus polypeptides, Necator polypeptides, Nematodirus polypeptides, Oesophagostomum polypeptides, Onchocerca polypeptides, Opisthorchis polypeptides,Polypeptides of the genus Ostertagia, Parafilaria, Paragonimus, Parascaris, Physaloptera, Protostrongylus, Setaria, Spirocerca, Spirometra, Stephanofilaria, Strongyloides, Strongylus, Thelazia, Toxascaris, Toxocara, Trichinella, Tricho strongylus, Trichuris, Uncinaria, and Wuchereria. (e.g., P. falciparum circumsporozoite (PfCSP)), sporozoite surface protein 2 (PfSSP2), carboxyl terminus of liver status antigen 1 (PfLSAl c-term), and exported protein 1 (PfExp-1), Pneumocystis spp. polypeptides, Sarcocystis spp. polypeptides, Schistosoma spp. polypeptides, Theileria spp. polypeptides, Toxoplasma spp. polypeptides, and Trypanosoma spp. polypeptides.

[0122] Examples of ectoparasite antigens include, but are not limited to, fleas; ticks, including hard mites and ulcerative mites; flies, such as midges, mosquitoes, sand flies, blow flies, bot flies, horn flies, deer flies, tsetse flies, stable flies, myiasis-causing flies and biting black flies; ants; spiders, lice; mites; and polypeptides (including antigens and allergens) from Hemiptera insects (true bugs) such as bedbugs and kissing bugs. III. Optional Proteins

[0123] In some embodiments, one or more other proteins are utilized with a particular CAR of the present disclosure. The one or more other proteins may be utilized for any reason, including to promote the efficacy of the CAR itself and / or to promote the efficacy of any type of cell expressing the CAR. In some cases, the other protein promotes the treatment of an individual receiving a cell expressing the CAR as a therapy, regardless of whether the other protein(s) directly or indirectly affect the activity of the CAR or the cell. In some cases, the other protein is a suicide gene, one or more cytokines, or both. In specific embodiments, the one or more other proteins are produced from a vector and are ultimately produced as two separate polypeptides. For example, the CAR and the other protein may be separated, for example, by a 2A sequence or an IRES.

[0124] In a specific embodiment, one or more cytokines, such as IL-15, are utilized in conjunction with a CAR.

[0125] The amino acid sequence of IL-15: ISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 8) Any polypeptide encompassed by this disclosure may comprise SEQ ID NO:8, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99%, or more percent identical to SEQ ID NO:8.

[0126] In a specific embodiment, a suicide gene product, such as caspase 9 (e.g., inducible caspase 9), is utilized in conjunction with a CAR.

[0127] Example of the amino acid sequence of caspase 9: MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS (SEQ ID NO: 9) Any polypeptide encompassed by this disclosure may comprise SEQ ID NO:9, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more percent identical to SEQ ID NO:9.

[0128] If it is intended that the CAR and another protein in the same vector be produced into two distinct polypeptides, a specific 2A sequence can be utilized.

[0129] The E2A amino acid sequence may be utilized as follows: QCTNYALLKLAGDVESNPGP (SEQ ID NO: 10)

[0130] Other 2A examples are available, as follows:

[0131] T2A: EGRGSLLTCGDVEENPGP (SEQ ID NO: 11)

[0132] P2A: ATNFSLLKQAGDVEENPGP (SEQ ID NO: 12)

[0133] F2A: VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 13)

[0134] The present disclosure also encompasses specific CAR molecules, including expression in any type of immune effector cell.

[0135] In the vector, the CAR may be expressed together with IL-15 or may be separated from the CAR by, for example, a 2A sequence. A. Cytokines

[0136] One or more cytokines can be utilized with one or more of the engineered receptors of the present disclosure, such as a CAR that includes (a) an optional hinge, and (b1) a CD28 transmembrane domain or (b2) a DAP10 transmembrane domain, and (c) a DAP10 costimulatory domain. In some cases, the one or more cytokines are present on the same vector molecule as the engineered receptor, while in other cases, they are present on separate vector molecules. In certain embodiments, the one or more cytokines are simultaneously expressed from the same vector as the engineered receptor. The one or more cytokines may be produced as a separate polypeptide from the antigen-specific receptor. As an example, interleukin-15 (IL-15) is utilized. IL-15 is utilized because, for example, IL-15 is tissue-restricted and is only observed in serum or systemically at any level under pathological conditions. IL-15 has several properties that are desirable for adoptive therapy. IL-15 is a homeostatic cytokine that promotes the eradication of established tumors and inhibits activation-induced cell death by inducing the development and cell proliferation of natural killer cells and relieving the functional suppression of tumor-resident cells. In addition to IL-15, other cytokines are also envisioned. This includes, but is not limited to, cytokines, chemokines, and other molecules that contribute to the activation and proliferation of cells used in human applications. By way of example, the one or more cytokines are IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, IL-7, or a combination thereof. NK cells expressing IL-15 can be utilized and continue supportive cytokine signaling, which is useful for survival after infusion.

[0137] In a specific embodiment, the NK cells express one or more exogenously supplied cytokines. The cytokines may be exogenously supplied to the NK cells because they are expressed from an expression vector in the cells and / or because they are supplied to the culture medium of the cells. In an alternative case, the endogenous cytokine in the cells is upregulated by manipulation of the expression regulation of the endogenous cytokine, such as genetic modification at the promoter site of the cytokine. When the cytokine is supplied to the cells on an expression construct, the cytokine may be encoded from the same vector as the suicide gene. The cytokine may be expressed as a polypeptide molecule separate from the suicide gene and as a polypeptide separate from the engineered receptor of the cell. In some embodiments, the present disclosure relates to the co-use of CAR and / or TCR vectors and IL-15, particularly in NK cells. B. Suicide gene

[0138] In certain embodiments, suicide genes are utilized in conjunction with any type of cell therapy to control its use and allow for the termination of cell therapy at a desired event and / or time. Suicide genes are used in transduced cells to induce the death of the transduced cells when necessary. The antigen-targeted cells of the present disclosure modified to carry the vectors encompassed by the present disclosure may contain one or more suicide genes. In some embodiments, the term "suicide gene" as used herein is defined as a gene that upon administration of a prodrug or other drug, results in the transition of the gene product to a compound that kills the host cell. In other embodiments, the suicide gene encodes a gene product that is targeted by a drug (e.g., an antibody) that targets the suicide gene product when desired. "Suicide gene product" describes a protein or polypeptide encoded by a suicide gene.

[0139] Examples of suicide gene / prodrug combinations that can be used are herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. The so-called suicide gene E. coli purine nucleoside phosphorylase can be used, which converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine. Other examples of suicide genes used with prodrug therapy are the E. coli cytosine deaminase gene and the HSV thymidine kinase gene.

[0140] Exemplary suicide genes include CD20, CD52, EGFRv3, or inducible caspase 9. In one embodiment, a truncated version of EGFR variant III (EGFRv3) can be used as a suicide antigen that can be excised by cetuximab. Additional suicide genes known in the art that can be used in the present disclosure include purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP). In some embodiments, inducible caspase 9 (iC9) is used. Exemplary iC9 is described, for example, in Yagyu S et al. Mol Ther. 2015 Sep;23(9):1475-85, which is incorporated by reference in its entirety.

[0141] In certain embodiments, the vector encoding the CAR or any vector in the NK cells encompassed herein comprises one or more suicide genes. The suicide gene may or may not be on the same vector as the CAR. If the suicide gene is on the same vector as the CAR, the suicide gene and the CAR may be separated, for example, by an IRES or 2A element. C. Other receptors

[0142] In some embodiments, cells comprising a CAR may express one or more other receptors, including other CAR molecules that may or may not include any one or more components encompassed herein, one or more cytokine receptors, one or more chemokine receptors (e.g., as modifications that enhance trafficking and homing to tumor sites, such as CXCR1 and CXCR2 to enhance trafficking to CXCL8-producing tumors), and / or one or more synthetic TCRs. In cases where the other receptor targets an antigen, such as a cancer antigen, the other receptor may or may not target the same antigen as the CAR of the present disclosure. IV. Vectors

[0143] CAR comprising (a) CD28 hinge, and (b1) CD28 transmembrane domain or (b2) DAP10 transmembrane domain, and (c) DAP10 costimulatory domain can be delivered to recipient immune cells by any suitable vector, including viral vector or non-viral vector.Examples of viral vectors include at least retrovirus, lentivirus, adenovirus, or adeno-associated virus vector.Examples of non-viral vectors include at least plasmid, transposon, lipid, nanoparticle, etc.

[0144] In cases where immune cells are transduced with a vector encoding an engineered receptor and further transduction of another gene(s) into the cells, such as a suicide gene and / or a cytokine and / or an optional therapeutic gene product, the antigen target receptor, suicide gene, cytokine and optional therapeutic gene may or may not be included on or with the same vector. In some cases, the CAR, suicide gene, cytokine and optional therapeutic gene are expressed from the same vector molecule, e.g., the same viral vector molecule. In such cases, the expression of the CAR, suicide gene, cytokine and optional therapeutic gene may or may not be regulated by the same regulatory element(s). If the CAR, suicide gene, cytokine and optional therapeutic gene are present on the same vector, they may or may not be expressed as separate polypeptides. If they are expressed as separate polypeptides, they may be separated on the vector by, for example, a 2A element or an IRES element (or both types may be used once or more on the same vector). A. General embodiment

[0145] One of ordinary skill in the art would be well within the ability to construct a vector by standard recombinant techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference) for expressing an antigen receptor of the present disclosure. 1. Regulatory Elements

[0146] The expression cassette contained in the vector useful in the present disclosure contains, inter alia, a eukaryotic transcriptional promoter operably linked to a protein coding sequence, a splice signal with intervening sequences, and a transcription termination / polyadenylation sequence (5' to 3' direction). The promoters and enhancers that control the transcription of protein-coding genes in eukaryotic cells may be composed of multiple genetic elements. The cellular machinery can collect and integrate the regulatory information conveyed by each element, and different genes can evolve distinct and often complex patterns of transcriptional regulation. Promoters used in the context of the present disclosure include, for example, constitutive promoters, inducible promoters, and tissue-specific promoters. If the vector is utilized for the production of cancer treatment, the promoter may be effective under hypoxic conditions. 2. Promoters / Enhancers

[0147] The expression constructs provided herein include promoters for driving the expression of antigen receptors and other cistron gene products. Promoters generally contain sequences that function to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters that lack a TATA box, such as the promoters of the mammalian terminal deoxynucleotidyl transferase gene and the promoters of the SV40 late genes, discrete elements that overlap the start site itself help fix the start location. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the upstream region of the start site, but some promoters have been shown to contain functional elements downstream of the start site as well. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcriptional reading frame is placed "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates transcription of DNA and promotes expression of the encoded RNA.

[0148] Spacing between promoter elements is often flexible so that promoter function is preserved when elements are inverted or moved relative to one another. For example, in the tk promoter, the spacing between promoter elements can be increased by up to 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0149] A promoter may be one that is naturally associated with a nucleic acid sequence, such as can be obtained by isolating the 5' non-coding sequence located upstream of a coding segment and / or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages may be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cells, and promoters or enhancers that are not "naturally occurring", i.e., promoters or enhancers that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. For example, promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose and tryptophan (trp-) promoter systems. In addition to producing promoter and enhancer nucleic acid sequences synthetically, sequences can be synthesized using PCR in conjunction with the compositions disclosed herein. (商標)It may be produced using recombinant cloning and / or nucleic acid amplification techniques, including those described herein. Additionally, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria, chloroplasts, etc., may similarly be employed.

[0150] Naturally, it will be important to employ a promoter and / or enhancer that effectively induces the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology are generally aware of the use of promoter, enhancer and cell type combinations for protein expression (see, for example, Sambrook et al. 1989, which is incorporated herein by reference). The promoter used is constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, which is advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0151] Additionally, any promoter / enhancer combination (e.g., from the Eukaryotic Promoter Data Base EPDB, World Wide Web at epd.isb-sib.ch / ) can be used to drive expression. The use of T3, T7 or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided as part of the delivery complex or as an additional gene expression construct.

[0152] Non-limiting examples of promoters include early or late viral promoters, such as SV40 early or late promoters near the minimal TATA box, cytomegalovirus (CMV) immediate early promoter, Rous Sarcoma Virus (RSV) early promoter; eukaryotic promoters, such as beta actin promoter, GADPH promoter, metallothionein promoter; and tethered response element promoters, such as cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA) and response element promoter (tre). It is also possible to use the human growth hormone promoter sequence (e.g., human growth hormone minimal promoter, described in GenBank®, Accession No. X05244, nucleotides 283-341) or mouse mammary tumor promoter (available from the ATCC, catalog number ATCC 45007). In certain embodiments, the promoter is a CMV IE, Dectin-1, Dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, beta-actin, MHC class I or MHC class II promoter, although any other promoter useful for driving expression of therapeutic genes is applicable to the practice of the present disclosure.

[0153] In certain aspects, the methods of the present disclosure also relate to enhancer sequences, i.e. nucleic acid sequences that increase the activity of a promoter and have the potential to act in cis and regardless of their orientation, even at relatively long distances (up to several kilobases away from the target promoter), however, enhancer function is not necessarily limited to such long distances and may also function in close proximity to a given promoter. 3. Initiation Signals and Associated Expression

[0154] Specific initiation signals may also be used in the expression constructs provided in this disclosure for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. Those skilled in the art will be able to easily determine this and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be "in frame" with the reading frame of the desired coding sequence. Exogenous translational control signals and initiation codons may be natural or synthetic. The efficiency of expression may be enhanced by including appropriate transcriptional enhancer elements.

[0155] In certain embodiments, the use of an internal ribosome entry site (IRES) element is used to create multigene, or polycistronic, messages. IRES elements can bypass the ribosome scanning model of 5' methylated Cap-dependent translation and initiate translation at an internal site. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) and from mammalian messages have been described. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. Thanks to the IRES element, each open reading frame has access to the ribosome for efficient translation. Multiple genes can be efficiently expressed using one promoter / enhancer to transcribe one message.

[0156] As detailed elsewhere herein, certain 2A sequence elements can be used to provide linked or co-expression of genes in the constructs provided in this disclosure. For example, a cleavage sequence can be used to link open reading frames to form a single cistron to allow co-expression of genes. Exemplary cleavage sequences are Equine rhinitis A virus (E2A) or F2A (foot and mouth disease virus 2A) or "2A-like" sequences (e.g., Thosea asigna virus 2A; T2A) or Porcine Teschovirus-1 (P2A). In specific embodiments, in a single vector, the multiple 2A sequences are non-identical, while in alternative embodiments, the same vector utilizes two or more of the same 2A sequences. Examples of 2A sequences are provided in US 2011 / 0065779, which is incorporated herein by reference in its entirety. 4. Origin of replication

[0157] To propagate the vector in the host cell, the vector may contain one or more origins of replication sites (often referred to as "ori"), for example, a nucleic acid sequence corresponding to the oriP of EBV as described above, or an engineered oriP with a similar or enhanced function in programming, which is the specific nucleic acid sequence from which replication is initiated. Alternatively, an origin of replication of an extrachromosomally replicating virus as described above, or an autonomously replicating sequence (ARS) may be used. 5. Selectable and Screenable Markers

[0158] In some embodiments, NK cells containing the receptor construct of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker confers an identifiable change to the cell that allows easy identification of cells containing the expression vector. In general, a selection marker confers a property that allows selection. A positive selection marker is one whose presence allows selection, and a negative selection marker is one whose presence prevents selection. An example of a positive selection marker is a drug resistance marker.

[0159] Typically, a drug selection marker is included to aid in cloning and identification of transformants; for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype that allows for the identification of transformants based on the implementation of conditions, other types of markers are contemplated, including screenable markers such as GFP based on colorimetry. Alternatively, screenable enzymes can be utilized as negative selection markers, such as thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) of herpes simplex virus. Those skilled in the art will also know how to use immunological markers, possibly in combination with FACS analysis. The marker used is not believed to be important, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection markers and screenable markers are well known to those skilled in the art. B. Multicistronic Vectors

[0160] In certain embodiments, the vector encoding the engineered receptor comprising (a) CD28 hinge, (b1) CD28 transmembrane domain or (b2) DAP10 transmembrane domain, and (c) DAP10 costimulatory domain also comprises sequences encoding an optional suicide gene, an optional cytokine, and / or an optional therapeutic gene, including those expressed from a multicistronic vector (the term "cistron" as used herein refers to a nucleic acid sequence from which a gene product can be produced). In specific embodiments, the multicistronic vector encodes a receptor, a suicide gene, and at least one cytokine, and / or an engineered receptor, such as a T cell receptor and / or an additional CAR. In some cases, the multicistronic vector encodes at least one CAR, at least one non-secreted TNF-alpha variant, and at least one cytokine. The cytokine may be a specific type of cytokine, such as human or mouse or any species. In certain cases, the cytokine is IL15, IL12, IL2, IL18, and / or IL21.

[0161] In certain embodiments, the present disclosure provides a flexible modular system (the term "modular" as used herein refers to a cistron or a component of a cistron, allowing for their interchangeability, e.g., by removing and replacing the entire cistron or the component of the cistron, respectively, by using standard recombinant techniques) that utilizes a polycistronic vector capable of expressing multiple cistrons at substantially the same level. This system can be used for cell engineering that allows for combinatorial expression (including overexpression) of multiple genes. In specific embodiments, one or more of the genes expressed by the vector include one, two, or more antigen receptors. The multiple genes may include, but are not limited to, CAR, TCR, cytokine, chemokine, homing receptor, CRISPR / Cas9-mediated gene mutation, decoy receptor, cytokine receptor, chimeric cytokine receptor, and the like. The vector may further include: (1) one or more reporters, e.g., fluorescent or enzymatic reporters, such as for cell assays and animal imaging, (2) one or more cytokines or other signaling molecules, and / or (3) a suicide gene.

[0162] In certain cases, the vector may contain at least four cistrons separated by any kind of cleavage site, for example, 2A cleavage sites. The vector may or may not be Moloney Murine Leukemia Virus (MoMLV or MMLV) based, containing 3' and 5' LTR with psi packaging sequences in a pUC19 backbone. The vector may contain four or more cistrons with three or more 2A cleavage sites and multiple ORFs for gene swapping. This system allows for combinatorial overexpression of multiple genes (seven or more) flanked by restriction sites for rapid integration by subcloning, and this system also contains, in some embodiments, at least three 2A self-cleavage sites. Thus, this system allows for the expression of multiple CARs, TCRs, signaling molecules, cytokines, cytokine receptors, and / or homing receptors. This system can also be applied to other viral and non-viral vectors, including but not limited to lentivirus, adenovirus AAV, and non-viral plasmids.

[0163] The modular nature of this system also allows efficient subcloning of genes into each of the four cistrons in the polycistronic expression vector, allowing for rapid gene swapping, etc. Strategically placed restriction sites in the polycistronic expression vector allow for efficient gene swapping.

[0164] Embodiments of the present disclosure encompass systems that utilize polycistronic vectors, where at least some of the vectors are modular, e.g., by allowing the removal and replacement of one or more cistrons (or components of one or more cistrons), e.g., by utilizing one or more restriction enzyme sites whose identities and positions are specifically selected to facilitate modular use of the vector. The vectors also have embodiments in which multiple cistrons are translated into a single polypeptide and processed into separate polypeptides, thereby conferring the advantage that the vector expresses separate gene products at substantially equimolar concentrations.

[0165] The vectors of the present disclosure are modularly constructed to allow for alteration of one or more cistrons of the vector and / or alteration of one or more components of one or more particular cistrons. The vectors may be designed to take advantage of unique restriction enzyme sites adjacent to the ends of one or more cistrons and / or adjacent to the ends of one or more components of a particular cistron.

[0166] The embodiments of the present disclosure include polycistronic vectors, each of which comprises at least two, at least three, or at least four cistrons flanked by one or more restriction enzyme sites, with at least one cistron encoding at least one antigen receptor. In some cases, two, three, four, or more of the cistrons are translated into a single polypeptide and cleaved into separate polypeptides, while in other cases, multiple of the cistrons are translated into a single polypeptide and cleaved into separate polypeptides. Adjacent cistrons on a vector may be separated by a self-cleaving site, such as a 2A self-cleaving site. In some cases, each cistron expresses a separate polypeptide from the vector. In certain cases, adjacent cistrons on a vector are separated by an IRES element.

[0167] In certain embodiments, the present disclosure provides a system for cell engineering that allows for combinatorial expression, including overexpression, of multiple cistrons, which may include, for example, one, two, or more antigen receptors. In certain embodiments, the use of polycistronic vectors described herein allows the vector to produce equimolar levels of multiple gene products from the same mRNA. The multiple genes may include, but are not limited to, CARs, TCRs, cytokines, chemokines, homing receptors, CRISPR / Cas9-mediated gene mutations, decoy receptors, cytokine receptors, chimeric cytokine receptors, and the like. The vector may further include one or more fluorescent or enzymatic reporters for cell assays, animal imaging, and the like. The vector may also include a suicide gene product to terminate the cells carrying the vector when they are no longer needed or when they become harmful to the host to which the vector was provided.

[0168] In specific embodiments, the vector is a viral vector (e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector) or a non-viral vector. The vector may include the 5'LTR, 3'LTR, and / or psi packaging elements of Moloney Murine Leukemia Virus (MMLV). In specific cases, the psi packaging is integrated between the 5'LTR and the antigen receptor coding sequence. The vector may or may not include a pUC19 sequence. In some aspects of the vector, at least one cistron encodes a cytokine (e.g., IL-15, IL-7, IL-21, IL-23, IL-18, IL-12, or IL-2), a chemokine, a cytokine receptor, and / or a homing receptor.

[0169] If 2A cleavage sites are utilized in the vector, the 2A cleavage sites may include a P2A, T2A, E2A and / or F2A site.

[0170] The restriction enzyme site may be of any type and may contain any number of bases in its recognition site, for example, 4-8 bases; the number of bases in the recognition site may be at least 4, 5, 6, 7, 8, or more. Sites where cleavage may occur produce blunt cuts or sticky ends. Restriction enzymes may be, for example, type I, type II, type III, or type IV. Restriction enzyme sites may be obtained from available databases such as the Integrated relational Enzyme database (IntEnz) or BRENDA (The Comprehensive Enzyme Information System).

[0171] An exemplary vector is circular and, by convention, position 1 (the 12 o'clock position at the top of the circle, with the rest of the sequence in a clockwise direction) is set to the start of the 5'LTR.

[0172] In embodiments in which a self-cleaving 2A peptide is utilized, the 2A peptide can be a viral oligopeptide between 18 and 22 amino acids (aa) long that mediates the "cleavage" of the polypeptide during translation in eukaryotic cells. The designation "2A" refers to a specific region of the viral genome, and different viral 2As are generally named after the virus. The first 2A discovered was F2A (foot and mouth disease virus), followed by E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2A), and T2A (thosea asigna virus 2A). The mechanism of 2A-mediated "self-cleavage" was discovered to be ribosome skipping the formation of a glycyl-prolyl peptide bond at the C-terminus of 2A.

[0173] In a specific case, the vector may be a gamma-retroviral transfer vector. The retroviral transfer vector may include a backbone (large fragment (2.63 kb) between HindIII and EcoRI restriction enzyme sites) based on a plasmid such as pUC19 plasmid. The backbone may carry viral components from Moloney Murine Leukemia Virus (MoMLV) including 5'LTR, psi packaging sequence, and 3'LTR. LTRs are long terminal repeats found on both sides of retroviral proviruses, and in the case of transfer vectors, bracket the gene cargo of interest, such as CAR, including (a) CD28 hinge, (b1) CD28 transmembrane domain or (b2) DAP10 transmembrane domain, and (c) DAP10 co-stimulatory domain, and optionally related components. The psi packaging sequence, which is the target site for packaging by the nucleocapsid, is also integrated in cis between the 5'LTR and the CAR coding sequence. Thus, an example of the basic structure of the transfer vector can be constructed as follows: pUC19 sequence-5'LTR-psi packaging sequence-gene cargo of interest-3'LTR-pUC19 sequence. This system can also be applied to other viral and non-viral vectors, including but not limited to lentivirus, adenovirus AAV, and non-viral plasmids. V. Cell

[0174] The present disclosure encompasses any type of immune cell or stem cell carrying at least one vector encoding a genetically modified receptor comprising (a) CD28 hinge, (b1) CD28 transmembrane domain or (b2) DAP10 transmembrane domain, and (c) DAP10 costimulatory domain, and may also encode at least one cytokine and / or at least one suicide gene. In some cases, a different vector encodes the CAR, whereas the suicide gene and / or cytokine. Immune cells, including NK cells, may be derived from umbilical cord blood (including umbilical cord blood pooled from multiple sources), peripheral blood, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (hematopoietic stem cells), bone marrow, or mixtures thereof. NK cells may be derived from cell lines, such as, but not limited to, NK-92 cells. NK cells may be derived from CD56 + They may also be cord blood mononuclear cells, such as NK cells.

[0175] The present disclosure encompasses any type of immune cell or other cell, including conventional T cells, gamma-delta T cells, NKT and invariant NK T cells, regulatory T cells, macrophages, B cells, dendritic cells, mesenchymal stromal cells (MSCs), or mixtures thereof.

[0176] In some cases, the cells are expanded in the presence of an effective amount of universal antigen presenting cells (UAPC), including any suitable ratio. The cells can be cultured with UAPC at a ratio of 10:1 to 1:10, 9:1 to 1:9, 8:1 to 1:8, 7:1 to 1:7, 6:1 to 1:6, 5:1 to 1:5, 4:1 to 1:4, 3:1 to 1:3, 2:1 to 1:2, or 1:1, including, for example, a ratio of 1:2. In some cases, the NK cells were expanded in the presence of IL-2 at a concentration of, for example, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 100-500, 100-400, 100-300, 100-200, 200-500, 200-400, 200-300, 300-500, 300-400, or 400-500 U / mL.

[0177] After genetic modification with the vector, the NK cells may be fused immediately or stored. In certain embodiments, after genetic modification, the cells may be propagated ex vivo for days, weeks, or months as a bulk population, about 1, 2, 3, 4, 5, or more days after gene transfer into the cells. In further embodiments, the transfectants are cloned, clones exhibiting the presence of a single integrated or episomally maintained expression cassette or plasmid, and expression of the CAR is expanded ex vivo. Clones selected for expansion exhibit the ability to specifically recognize and lyse antigen-expressing target cells. The recombinant immune cells may be expanded by stimulation with IL-2 or other cytokines that bind to the common gamma chain (e.g., IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, etc.). The recombinant immune cells may be expanded by stimulation with artificial antigen-presenting cells. In further embodiments, the genetically modified cells may be cryopreserved.

[0178] As encompassed herein, embodiments of the present disclosure include cells expressing one or more CARs and one or more suicide genes. In a specific embodiment, the NK cells comprise a recombinant nucleic acid encoding one or more CARs and one or more engineered non-secreted membrane-bound TNF-alpha mutant polypeptides. In a specific embodiment, in addition to expressing one or more CARs and TNF-alpha mutant polypeptides, the cells also comprise a nucleic acid encoding one or more therapeutic gene products.

[0179] The cells may be obtained directly from the individual, or may be obtained from a depository or other repository. Cells as a therapy may be autologous or autologous with respect to the individual to whom the cells are provided as a therapy.

[0180] The cells may be derived from an individual in need of treatment for a medical condition, and may be engineered to express a CAR, an optional suicide gene, an optional cytokine, and an optional therapeutic gene product (e.g., using standard techniques for transduction and expansion for adoptive cell therapy), and then returned to the individual from whom the cells originally came. In some cases, the cells are stored for later use in that individual or another individual.

[0181] The immune cells may be included in a population of cells, the population being a majority transduced with one or more receptors and / or one or more suicide genes and / or one or more cytokines. The cell population may comprise 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of immune cells transduced with one or more CARs and / or one or more suicide genes and / or one or more cytokines. The one or more CARs and / or the one or more suicide genes and / or the one or more cytokines may be separate polypeptides.

[0182] Immune cells may be produced with one or more CARs and / or one or more suicide genes and / or one or more cytokines, intended to be modular for a particular purpose. For example, cells expressing a CAR and / or one or more suicide genes and / or one or more cytokines (or distributed with nucleic acids encoding variants for subsequent transduction) may be produced, including for commercial distribution, and users may modify them to express one or more other genes of interest (including therapeutic genes) depending on the intended purpose. For example, individuals interested in treating antigen-positive cells, including antigen-positive cancers or cells infected with infectious agents, may obtain or produce suicide gene-expressing cells (or heterologous cytokine-expressing cells) and modify them to express a receptor containing an antigen-specific scFv, or vice versa.

[0183] In certain embodiments, NK cells are utilized, and the genome of the transduced NK cells expressing one or more CARs and / or one or more suicide genes and / or one or more cytokines can be modified. The genome can be modified in any manner, but in specific embodiments, the genome is modified by, for example, CRISPR gene editing. The genome of the cell can be modified to enhance the effectiveness of the cell for any purpose. VI. Treatment Method

[0184] In various embodiments, diseased cells or other cells expressing desired on the surface are targeted to improve the disease in individuals with disease, or to reduce the risk or delay the severity and / or onset of disease in individuals.In a specific case, cancer cells expressing endogenous antigens are targeted to kill cancer cells.In other cases, cells infected with infectious agents are targeted to kill infected cells.

[0185] In certain embodiments, the CAR constructs, nucleic acid sequences, vectors, immune cells, etc., as contemplated herein, and / or pharmaceutical compositions comprising them are used to prevent, treat, or ameliorate diseases, such as cancerous diseases. In certain embodiments, the pharmaceutical compositions of the present disclosure may be particularly useful for preventing, ameliorating, and / or treating cancers, including, for example, cancers that express specific antigens and may or may not be solid tumors.

[0186] The immune cells for which the receptor is utilized may in certain embodiments be NK cells, T cells, gamma delta T cells, alpha beta T cells, or NKT or invariant NKT (iNKT), or invariant NKT cells engineered for mammalian cell therapy. In such cases where the cells are NK cells, the NK cell therapy may be of any type and the NK cells may be of any type. In certain embodiments, the cells are NK cells engineered to express one or more CARs and / or one or more suicide genes and / or one or more cytokines. In a specific embodiment, the cells are NK cells transduced with a CAR.

[0187] In certain embodiments, the present disclosure contemplates, in part, CAR-expressing cells, CAR constructs, CAR nucleic acid molecules, and CAR vectors that can be administered, alone or in any combination, using standard vectors and / or gene delivery systems, and in at least some cases, can be administered with a pharma- ceutically acceptable carrier or excipient. In certain embodiments, after administration, the nucleic acid molecule or vector can be stably integrated into the subject's genome.

[0188] In a specific embodiment, a viral vector can be used that is specific to a certain cell or tissue and persists in NK cells.Suitable pharmaceutical carriers and excipients are well known in the art.The composition prepared according to the present disclosure can be used for the prevention or treatment or delay of the above-identified diseases.

[0189] Furthermore, the present disclosure relates to a method for the prevention, treatment or amelioration of a neoplastic disease comprising administering to a subject in need thereof an effective amount of a CAR contemplated herein, a nucleic acid sequence, a vector, and / or a cell expressing a CAR produced by a process contemplated herein.

[0190] The possible indications for administration of the exemplary CAR cell composition are cancerous diseases, including tumor diseases, including, for example, B-cell malignancies, multiple myeloma, breast cancer, glioblastoma, renal cancer, pancreatic cancer, or lung cancer. The exemplary indications for administration of the antigen-targeted CAR cell composition are cancerous diseases, including any malignant tumor that expresses an antigen. Administration of the disclosed composition is useful for all stages (I, II, III, or IV) and types of cancer, including, for example, minimal residual disease, early cancer, advanced cancer, and / or metastatic cancer, and / or refractory cancer.

[0191] The present disclosure further includes co-administration protocols with other compounds that act through immune cells, such as bispecific antibody constructs, targeted toxins, or other compounds.Clinical regimens for co-administration of the compounds of the present invention may include simultaneous co-administration before or after administration of other components.Specific combination therapies include chemotherapy, radiation, surgery, hormone therapy, and other types of immunotherapy.

[0192] The embodiment relates to a kit comprising the CAR construct defined herein, or the components contained herein, the nucleic acid sequence defined herein, the vector defined herein, and / or the host cell (such as immune cell) defined herein. In a specific embodiment, the kit comprises the nucleic acid encoding (a) CD28 hinge, (b1) CD28 transmembrane domain or (b2) DAP10 transmembrane domain, and / or (c) DAP10 costimulatory domain, or suitable primers for amplifying them. It is also contemplated that the kit of the present disclosure comprises the pharmaceutical composition described hereinabove, which is administered to an individual in need of medical treatment or intervention, alone or in combination with additional pharmaceuticals. VII. Pharmaceutical Compositions

[0193] The pharmaceutical composition of the present disclosure comprises an effective amount of a composition comprising NK cells dispersed in a pharma- ceutically acceptable carrier. The phrase "pharmaceutical or pharmacologically acceptable" refers, as appropriate, to molecular entities and compositions that do not produce adverse, allergic or other undesirable reactions when administered to an animal, such as a human. Preparation of pharmaceutical compositions comprising the present composition will be known to those skilled in the art in light of the present disclosure, as exemplified in Remington: The Science and Practice of Pharmacy, 21st ed. Lippincott Williams and Wilkins, 2005, which is incorporated herein by reference. In addition, it will be understood that for administration to animals (e.g., humans), preparations should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards.

[0194] As used herein, "pharmaceutical acceptable carrier" includes any solvent, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antibacterial, antifungal), isotonicity agent, absorption retardant, salt, preservative, drug, drug stabilizer, gel, binder, excipient, disintegrant, lubricant, sweetener, flavoring agent, dye, such materials, and combinations thereof, known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, pages 1289-1329, which is incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical composition is contemplated.

[0195] Pharmaceutical compositions can contain different types of carriers depending on whether they are administered in solid, liquid or aerosol form and whether they need to be sterile for routes of administration such as injection. The presently disclosed compositions can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, directly bathing target cells by localized perfusion, via catheter, via lavage, in creams, in lipid compositions (e.g., liposomes), or by any other method or combination thereof that would be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, which is incorporated herein by reference).

[0196] The composition comprising NK cells can be formulated into a composition in free base, neutral or salt form. Where appropriate, pharma- ceutically acceptable salts include acid addition salts, for example, those formed with free amino groups of the proteinaceous composition, for example, those formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxide, or organic bases such as isopropylamine, trimethylamine, histidine or procaine. Once formulated, the solution is administered in a manner compatible with the dosage form and in a therapeutically effective amount. The formulation is easily administered in a variety of dosage forms, including those formulated for parenteral administration, such as injection solutions, aerosols for delivery to the lungs, or those formulated for dietary administration, such as drug-releasing capsules.

[0197] Further in accordance with the present disclosure, the compositions of the present disclosure suitable for administration are provided in a pharma- ceutically acceptable carrier, with or without an inert diluent. The carrier should be assimilable, and includes liquid, semi-solid, i.e., paste, or solid carriers. Except where any conventional vehicle, agent, diluent, or carrier is deleterious to the recipient or to the therapeutic effect of the composition contained therein, its use in an administrable composition for use in practicing the method of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline, lipids, liposomes, resins, binders, fillers, and the like, or combinations thereof. The compositions may also include various antioxidants to retard oxidation of one or more components. In addition, prevention of the action of microorganisms can be brought about by preservatives, such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0198] In accordance with the present disclosure, compositions are combined with the carrier by any convenient and practical method, i.e., solution, suspension, emulsion, mixing, encapsulation, absorption, etc. Such procedures are routine to those of ordinary skill in the art.

[0199] In a specific embodiment of the present disclosure, the composition is combined or thoroughly mixed with a semi-solid or solid carrier.Mixing can be performed in any convenient manner, such as by grinding.Stabilizers can also be added in the mixing step to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach.Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, and the like.

[0200] In further embodiments, the disclosure may relate to the use of pharmaceutical lipid vehicle compositions comprising compositions comprising NK cells and optionally an aqueous solvent. As used herein, the term "lipid" is defined to include any of a wide variety of substances that are characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds is well known to those of skill in the art, and the term "lipid" as used herein is not limited to a particular structure. Examples include compounds containing long chain aliphatic hydrocarbons and their derivatives. Lipids may be naturally occurring or synthetic (i.e., artificially designed or produced). However, lipids are typically biological materials. Biological lipids are well known in the art and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with ether and ester-linked fatty acids, polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that would be understood by those of skill in the art to be lipids are also encompassed by the compositions and methods of the present invention.

[0201] Those skilled in the art will be familiar with the various techniques that can be used to disperse the composition in lipid vehicle.For example, the composition comprising NK cells and antibodies can be dispersed in a solution containing lipid, dissolved with lipid, emulsified with lipid, mixed with lipid, combined with lipid, covalently bound with lipid, contained in lipid as a suspension, contained or complexed with micelles or liposomes, or otherwise associated with lipid or lipid structure by any means known to those skilled in the art.Dispersion can be or can not cause liposome formation.

[0202] The actual dosage of the composition of the present disclosure administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, type of disease to be treated, previous or concurrent therapeutic intervention, characteristics of the patient, and route of administration. Depending on the dosage and route of administration, the preferred dosage and / or the number of administrations of an effective amount can vary depending on the subject's response. The physician responsible for administration will in any case determine the concentration of active ingredient in the composition and the appropriate dose for each individual subject.

[0203] In certain embodiments, the pharmaceutical composition may, for example, contain at least about 0.1% of the active compound. In other embodiments, the active compound may, for example, comprise about 2% to about 75% of the weight of the unit, or about 25% to about 60%, and any range derivable therein. Of course, the amount of active compound in each therapeutically useful composition may be prepared to obtain a suitable dosage in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological concerns will be contemplated by those skilled in the art of preparing such pharmaceutical formulations, and as such, various dosages and treatment regimens may be desirable.

[0204] In other non-limiting examples, dosages can include about 1 microgram / kg / body weight, about 5 micrograms / kg / body weight, about 10 micrograms / kg / body weight, about 50 micrograms / kg / body weight, about 100 micrograms / kg / body weight, about 200 micrograms / kg / body weight, about 350 micrograms / kg / body weight, about 500 micrograms / kg / body weight, about 1 milligram / kg / body weight, about 5 milligrams / kg / body weight, about 10 milligrams / kg / body weight, about 50 milligrams / kg / body weight, about 100 milligrams / kg / body weight, about 200 milligrams / kg / body weight, about 350 milligrams / kg / body weight, about 500 milligrams / kg / body weight to 1000 mg / kg / body weight or more per administration, and any range derivable therein. Non-limiting examples of ranges derivable from the numbers recited herein include ranges from about 5 mg / kg / body weight to about 100 mg / kg / body weight, from about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, etc., that may be administered based on the above numbers.

[0205] Therapeutic compositions comprising NK cells of the present disclosure can be administered by injection, intravenous, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, implantation, inhalation, intrathecal, intracerebroventricular, or intranasal. Appropriate dosages can be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician.

[0206] Treatment may include various "unit doses". A unit dose is defined as containing a predetermined amount of therapeutic composition. The amount administered, as well as the specific route and formulation, are within the judgement of those skilled in the art of clinical medicine. A unit dose does not have to be administered as a single injection, but may include continuous infusion over a period of time. In some embodiments, a unit dose includes a single administrable dose.

[0207] In certain embodiments, the dose for delivery to an individual in need thereof, including by infusion, is at least 10 5 to 10 10 cells / kg / dose / week, and any range derivable therein.

[0208] The amount to be administered, i.e., both the number of treatments and the unit dose, depends on the desired treatment effect. Effective dose is understood to refer to the amount required to achieve a specific effect. In the practice of certain embodiments, it is contemplated that a dose ranging from 10mg / kg to 200mg / kg can affect the protective capacity of these agents. Thus, it is contemplated that doses include about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any range derivable therein. Furthermore, such doses can be administered multiple times during the day and / or on multiple days, weeks, or months.

[0209] In certain embodiments, an effective amount of the pharmaceutical composition is one that can provide a blood level of about 1 μM to 150 μM. In other embodiments, an effective dose provides a blood level of about 4 μM to 100 μM; or about 1 μM to 100 μM; or about 1 μM to 50 μM; or about 1 μM to 40 μM; or about 1 μM to 30 μM; or about 1 μM to 20 μM; or about 1 μM to 10 μM; or about 10 μM to 150 μM; or about 10 μM to 100 μM; or about 10 μM to about 50 μM; or about 25 μM to about 150 μM; or about 25 μM to about 100 μM; or about 25 μM to about 50 μM; or about 50 μM to about 150 μM; or about 50 μM to about 100 μM (or any range derivable therein). In other embodiments, the dose may provide the following blood levels of drug resulting from the therapeutic agent administered to the subject: about, at least about, or up to about 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 110, 111, 112, 113, 114, 6, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM, or any range derivable therein. In certain embodiments, the therapeutic agent administered to the subject is metabolized in the body to become a metabolized therapeutic agent, in which case blood levels may refer to the amount of the agent. Alternatively, to the extent that the therapeutic agent is not metabolized by the subject, blood levels discussed herein may refer to the therapeutic agent that is not metabolized. A. Dietary Compositions and Formulations

[0210] In certain embodiments of the present disclosure, the composition comprising NK cells and antibodies is formulated to be administered via dietary route. Dietary route includes all possible routes of administration where the composition comes into direct contact with the digestive tract. Specifically, the pharmaceutical compositions disclosed herein can be administered orally, bucally, rectally, or sublingually. As such, these compositions can be formulated with an inert diluent or with an assimilable edible carrier, or can be enclosed in hard or soft gelatin capsules, or can be compressed into tablets, or can be directly incorporated into the diet.

[0211] In certain embodiments, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792,451, each of which is specifically incorporated herein by reference in its entirety). Tablets, troches, residues, capsules, etc. may also contain: binders, such as gum tragacanth, acacia, corn starch, gelatin, or combinations thereof; excipients, such as dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof; disintegrants, such as corn starch, potato starch, alginic acid, or combinations thereof; lubricants, such as magnesium stearate; sweeteners, such as sucrose, lactose, saccharin, or combinations thereof; flavorings, such as peppermint, oil of wintergreen, cherry flavor, orange flavor, and the like. When the dosage unit form is a capsule, in addition to the above types of materials, it may contain a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, in addition to the above types of materials, it may contain a carrier, such as a liquid carrier. Gelatin capsules, tablets, or pills may be enteric coated. Enteric coating prevents the composition from denaturing in the stomach or upper intestine where pH is acidic. For example, see U.S. Patent No. 5,629,001. Upon reaching the small intestine, the basic pH there dissolves the coating, allowing the composition to be released and absorbed by specialized cells, such as epithelial intestinal cells and Peyer's patch M cells. Syrup of elixir may contain the active compound sucrose as a sweetener, methyl and propyl parabens as preservatives, dyes and flavors, such as cherry or orange flavor.Of course, any material used in preparing any dosage form should be pharma- ceutically pure and substantially non-toxic in the amounts used.Furthermore, the active compound can be incorporated into sustained-release preparations or formulations.

[0212] For oral administration, the composition of the present disclosure may alternatively be incorporated with one or more excipients in the form of mouthwash, dentifrice, buccal tablet, oral spray, or sublingual oral administration formulation.For example, mouthwash can be prepared by incorporating the required amount of active ingredient in a suitable solvent, such as sodium borate solution (Dobell's Solution).Alternatively, the active ingredient may be incorporated into oral solution, such as one containing sodium borate, glycerin, and potassium bicarbonate, or dispersed in dentifrice, or added in a therapeutically effective amount to a composition that may include water, binder, abrasive, flavoring agent, foaming agent, and humectant.Alternatively, the composition may be in the form of a tablet or solution and placed under the tongue or otherwise dissolved in the mouth.

[0213] Additional formulations suitable for other dietary administration modes include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, the suppository softens, melts or dissolves in the cavity fluid. In general, for suppositories, conventional carriers include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories can be formed from mixtures containing, for example, about 0.5% to about 10%, preferably about 1% to about 2% of the active ingredient. B. Parenteral Compositions and Formulations

[0214] In further embodiments, the composition can be administered via parenteral route.As used herein, the term "parenteral" includes a route that bypasses the digestive tract.Specifically, the pharmaceutical composition disclosed herein can be administered, for example, but not limited to, intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneously, or intraperitoneally.See U.S. Patent Nos. 6,613,308, 5,466,468, 5,543,158, 5,641,515, and 5,399,363 (each of which is specifically incorporated herein in its entirety by reference).

[0215] Solutions of the active compounds as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms. Suitable pharmaceutical forms for injection include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, a polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride. Prolonged absorption of the injectable composition can be achieved by using an absorption-delaying agent, such as aluminum monostearate and gelatin, in the composition.

[0216] For example, for parenteral administration in an aqueous solution, the solution should be suitably buffered, if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those of skill in the art in light of the present disclosure. For example, a dose can be dissolved in an isotonic NaCl solution and added to a subcutaneous injection solution or injected at the intended site of injection (see, for example, "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). Dosages will necessarily vary somewhat depending on the condition of the subject being treated. The physician responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards.

[0217] Sterile injection solution can be prepared by incorporating the active compound in the required amount in a suitable solvent with various other components listed above, and then sterilizing by filtration as necessary.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other components required from those listed above.In the case of sterile powder for preparing sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying technology, which obtains powder of active ingredient plus any additional desired ingredient from the solution that has been previously sterilized and filtered.Powder composition is combined with liquid carrier, such as water or physiological saline, with or without stabilizer. C. Various Pharmaceutical Compositions and Formulations

[0218] In other specific embodiments of the present disclosure, the active compound compositions comprising NK cells and antibodies may be formulated for administration via a variety of different routes, for example, topical (i.e., transdermal), mucosal (intranasal, intravaginal, etc.) and / or inhalation.

[0219] Pharmaceutical compositions for topical administration may include active compounds formulated for medicinal application such as ointments, pastes, creams or powders. Ointments include all oleaginous, adsorbent, emulsion and water-soluble based compositions for topical application, while creams and lotions are compositions that contain only emulsion bases. Topically administered drugs may contain penetration enhancers to promote the adsorption of the active ingredient from the skin. Suitable penetration enhancers include glycerin, alcohol, alkyl methyl sulfoxides, pyrrolidone and lurocapram. Bases that can be used in compositions for topical preparations include polyethylene glycol, lanolin, cold cream and petrolatum, as well as any other suitable absorbent, emulsion or water-soluble ointment bases. Topical preparations may also include emulsifiers, gelling agents, antimicrobial preservatives as needed to preserve the active ingredient and provide a homogenous mixture. Transdermal administration of the present invention may also consist of the use of a "patch". For example, a patch can deliver one or more active agents continuously at a predetermined rate and for a period of time. Transdermal administration of the present invention may also involve the use of a "patch." For example, a patch can deliver one or more active agents continuously at a predetermined rate for a period of time.

[0220] In certain embodiments, pharmaceutical compositions can be delivered by eye drops, intranasal sprays, inhalation, and / or other aerosol delivery vehicles.Methods for delivering compositions directly to the lungs via nasal aerosol sprays are described, for example, in U.S. Patent Nos. 5,756,353 and 5,804,212 (each of which is specifically incorporated herein by reference in its entirety).Similarly, the delivery of drugs using nasal microparticle resins (Takenaga et al., 1998) and lysophosphatidylglycerol compounds (U.S. Patent No. 5,725,871, which is specifically incorporated herein by reference in its entirety) are also well known in the pharmaceutical art.Similarly, transmucosal drug delivery in the form of polytetrafluoroethylene support matrix is ​​described in U.S. Patent No. 5,780,045 (which is specifically incorporated herein by reference in its entirety).

[0221] The term aerosol refers to a colloidal system of finely divided solid liquid particles dispersed in a liquefied or pressurized gas propellant. A typical aerosol of the present invention for inhalation consists of a suspension of the active ingredient in a liquid propellant or a mixture of a liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers vary depending on the pressure requirements of the propellant. The administration of the aerosol varies depending on the age, weight, and severity and response of the subject. VIII. Combination Therapy

[0222] In certain embodiments, the compositions and methods of the present invention comprise immune cell populations (including NK cell populations) in combination with at least one additional therapy.Additional therapy can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, virus therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, hormone therapy, oncolytic virus, or combinations thereof.Additional therapy can be in the form of adjuvant therapy or neoadjuvant therapy.

[0223] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of treatment, such as an anti-nausea agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy may be one or more of the chemotherapeutic agents known in the art.

[0224] In certain embodiments, in addition to the inventive cell therapy of the present disclosure, an individual may have been provided, may be provided, and / or may be scheduled to be provided with certain additional therapies for cancer, including one or more of surgery, radiation, immunotherapy (other than the cell therapy of the present disclosure), hormone therapy, gene therapy, chemotherapy, and the like.

[0225] The immune cell therapy may be administered before, during, after, or in various combinations with the additional cancer therapy. The interval between administrations may range from the same time, to minutes, days, or weeks. In embodiments in which the immune cell therapy is provided to the patient separately from the additional therapeutic agent, generally no significant period of time will elapse between each delivery time, allowing the two compounds to still exert a beneficial combined effect on the patient. In such instances, it is contemplated that the antibody therapy and the anti-cancer therapy may be provided to the patient within about 12-24 hours or 72 hours of each other, more particularly within about 6-12 hours of each other. In some circumstances, it may be desirable to significantly extend the duration of treatment, such that days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapse between the respective administrations.

[0226] Various combinations can be employed. In the following examples, immune cell therapy is "A" and anti-cancer drug therapy is "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

[0227] Administration of any compound or cell therapy of the present embodiments to a patient follows general protocols for administration of such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring for toxicity resulting from the combination therapy. A. Chemotherapy

[0228] A wide variety of chemotherapeutic agents can be used according to this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic 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 the cell, for example, whether and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, to intercalate into DNA, and to induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0229] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethyleneethylethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dora statins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictine; spongiostatins; nitrogen mustards such as chlorambucil, chlornaphazine, collophosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma II and calicheamicin omega II); dynemicins such as dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnicin, olivomycin, peplomycin, potofilomycin, puromycin, keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folates, such as denopterin, pteropterin, and trimetrexate analogs; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenergics such as mitotane and trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide Glycosides;Aminolevulinic acid;Eniluracil;Amsacrine;Bestravcil;Bisantrene;Edatraxate;Defofamine;Demecolcine;Diazicon;Elformitin;Elliptinium acetate;Epothilone;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidynin;Maytansinoids such as maytansine and ansamitocin;Mitoguazone;Mitoxantrone;Mopidammol;Nitraerin;Pentostatin;Phenamet;Pirarubicin;Rosoxantrone;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK polysaccharide complex; Razoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triazicon; 2,2',2"-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, veracrine A, roridin A and anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitrol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxoids, e.g., Paclitaxel cel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, 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 inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above. B. Radiation therapy

[0230] Other agents that cause DNA damage and have been widely used include what are commonly known as gamma radiation, X-rays, and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents such as microwaves, proton beam radiation (U.S. Patents 5,760,395 and 4,870,287) and UV radiation are also contemplated. All of these agents most likely cause a wide range of damage to DNA, the precursors of DNA, DNA replication and repair, and chromosome assembly and maintenance. Dose ranges for X-rays range from daily doses of 50-200 roentgens over a prolonged period (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells. C. Immunotherapy

[0231] Those skilled in the art will understand that additional immunotherapy may be used in combination or in conjunction with the methods of the embodiments. In the context of cancer treatment, immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody may function alone as an effector of treatment, or may recruit other cells to actually affect cell killing. The antibody may also be conjugated with a drug or toxin (such as a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and act as a targeting agent. Alternatively, the effector may be a lymphocyte with a surface molecule that directly or indirectly interacts with a target on the tumor cell. Various effector cells include cytotoxic T cells and NK cells.

[0232] Antibody-drug conjugates have emerged as a breakthrough approach in the development of cancer therapeutics. Cancer is one of the leading causes of death worldwide. Antibody-drug conjugates (ADCs) contain a monoclonal antibody (MAb) covalently linked to a cytotoxic drug. This approach combines the high specificity of the MAb for an antigen target with a highly potent cytotoxic drug, resulting in an "armed" MAb that delivers the payload (drug) to tumor cells where antigen levels are enriched. Targeted delivery of the drug also minimizes drug exposure in normal tissues, reducing toxicity and improving the therapeutic index. The FDA approval of two ADC drugs, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, has proven this approach effective. Currently, there are more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become increasingly mature, the discovery and development of new ADCs increasingly relies on the identification and validation of new targets suitable for this approach, and the generation of targeted MAbs. Two criteria for ADC targets are up-regulated / high levels of expression and robust internalization in tumor cells.

[0233] In one aspect of immunotherapy, the tumor cells must have some marker suitable for targeting, i.e., not present on the majority of other cells. Many tumor markers exist, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, p155. An alternative aspect of immunotherapy is to combine anti-cancer and immune stimulatory effects. There are also immune stimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0234] Examples of immunotherapies currently under investigation or in use include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapies, such as interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998). ); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0235] In some embodiments, the immunotherapy is an immune checkpoint inhibitor. Immune checkpoints either raise or lower signals (e.g., costimulatory molecules). Inhibitory immune checkpoints that can be targeted by immune checkpoint inhibitors include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuating factor (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0236] Immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligands or receptors, or in particular antibodies, such as human antibodies (e.g., International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4).252-64, 2012, both of which are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof may be used, in particular chimeric, humanized or human forms of antibodies. As will be appreciated by those skilled in the art, alternative and / or equivalent names may be used for certain antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, it is known that lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0237] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a specific aspect, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in US Patents US8735553, US8354509, and US8008449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, as described in U.S. Patent Application Nos. US20140294898, US2014022021, and US20110008369, all of which are incorporated herein by reference.

[0238] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin that includes an extracellular portion or a PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab (MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO (登録商標) Also known as MK-3475, Merck 3475, Lambrolizumab, KEYTRUDA, is an anti-PD-1 antibody described in WO2006 / 121168. (登録商標) Pembrolizumab, also known as SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0239] Another immune checkpoint that can be targeted by the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when it binds to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily, is expressed on the surface of helper T cells, and transmits inhibitory signals to T cells. CTLA4 is similar to CD28, a T cell costimulatory protein, and both molecules bind to CD80 and CD86 (also called B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found on regulatory T cells and may be important for their function. Activation of T cells via the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0240] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0241] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, the following anti-CTLA-4 antibodies can be used in the methods disclosed herein: US8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, also known as tremelimumab; formerly known as titilimumab), USP 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17):10067-10071; Camacho et al. (2004) J Clin Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304. The teachings of each of the above publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001014424, WO2000037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0242] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the antibody described above. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the antibody described above (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0243] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors such as those described in U.S. Pat. Nos. US5844905, US5885796, and International Patent Applications WO1995001994 and WO1998042752, all of which are incorporated herein by reference, and immunoadhesins such as those described in U.S. Pat. No. US8329867, which is incorporated herein by reference. D.Surgery

[0244] Approximately 60% of patients with cancer undergo some type of surgery, including preventive surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection surgery, which physically removes, excises, and / or destroys all or part of cancerous tissue, and can be used in combination with other therapies, such as the treatment of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapy. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery).

[0245] When part or all of a cancerous cell, tissue, or tumor is removed, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of additional anti-cancer treatment to the area. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also be administered at various dosages. E. Other drugs

[0246] It is contemplated that other agents may be used in combination with certain aspects of the present embodiment to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cell activators and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing the number of GAP junctions will increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cell activators or differentiation agents may be used in combination with certain aspects of the present embodiment to improve the anti-hyperproliferative efficacy of the treatment. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, may be used in combination with certain aspects of the present embodiment to improve the efficacy of the treatment. IX. Kits of the Disclosure

[0247] Any of the compositions described herein may be included in the kit. In a non-limiting example, cells, reagents for producing cells, vectors, and reagents for producing vectors and / or their components may be included in the kit. In certain embodiments, NK cells may be included in the kit, which may or may not yet express a CAR comprising (a) CD28 hinge, (b1) CD28 transmembrane domain or (b2) DAP10 transmembrane domain, and (c) DAP10 costimulatory domain, optional cytokine, or optional suicide gene. Such kits may or may not have one or more reagents for engineering cells. Such reagents include, for example, small molecules, proteins, nucleic acids, antibodies, buffers, primers, nucleotides, salts, and / or combinations thereof. Nucleotides encoding one or more CARs, suicide gene products, and / or cytokines may be included in the kit. Proteins such as cytokines or antibodies, including monoclonal antibodies, may be included in the kit. Nucleotides encoding components or all of the engineered CAR receptors may be included in the kit, including reagents for producing the same.

[0248] In certain aspects, the kit includes another cancer therapy in addition to the NK cell therapy of the present disclosure. In some cases, the kit includes a second cancer therapy, such as chemotherapy, hormonal therapy, and / or immunotherapy, in addition to the cell therapy embodiment. The kit can be tailored to the specific cancer of an individual and includes the respective second cancer therapy for the individual.

[0249] The kit may include a suitable aliquot of the composition of the present disclosure. The components of the kit may be packaged either in aqueous media or in lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe or other container means into which the components may be placed and preferably suitably aliquoted. If more than one component is included in the kit, the kit will generally include a second, third or other additional container into which the additional components may be placed separately. However, various combinations of components may be included in the vials. The kits of the present invention will also typically include a means for containing the composition and optional other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained. X. Example EXAMPLES

[0250] The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art should recognize that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to work well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those skilled in the art should recognize in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention. Example 1

[0251] CAR construct components with improved signaling Figure 1A-B demonstrate that the DAP10 signaling domain confers an activated phenotype to CD5 CAR-NK cells, and phenotypes were shown using a mass cytometry panel. Using t-distributed stochastic neighbor embedding (TSNE), a statistical method for visualizing high-dimensional data by giving each data point a location in a two- or three-dimensional map, distinct clusters are generated compared to non-transduced NT cells (left) and NK cells expressing a CD5 CAR containing IgG hinge, CD28 transmembrane domain, DAP10, and CD3 zeta (CD5CAR-NKCD28TMDAP10CD3z) (Figure 1A). Two new clusters, clusters 8 and 11, expressed in CARCD5 NK cells, are highlighted by circles and rectangles. The heatmap in Figure 1B shows the normalized expression of various markers (indicated on the X-axis) in each cluster (indicated on the Y-axis). Activation, cytotoxicity, and maturation markers highly expressed in clusters #8 and #11 are highlighted by blue rectangles. This indicates that DAP10-transduced NK cells have two populations or clusters that are not present in non-transduced NK cells: these clusters contain NK cells that express high markers of activation, such as granzyme B, perforin, etc.

[0252] Figures 2A-C demonstrate that CD5 CAR-NK cells with DAP10 costimulatory domains are capable of producing multiple effector cytokines and chemokines, exhibiting enhanced multifunctionality. Figure 2A demonstrates isoplexy single-cell secretome data showing the multifunctionality of CD5 CAR-NK cells with DAP10 costimulatory domains, comparing different CD5 CAR-NK cells to non-transduced (NT) NK cells. CD5 CAR#5 (containing IgG hinge, CD28 transmembrane domain, DAP10, and CD3z) shows the highest multifunctionality with the highest percentage of single cells secreting 2, 3, 4, or 5+ proteins at a time. Figure 2B provides a bar graph showing the multifunctionality intensity index between different CD5 CAR-NK cells compared to non-transduced (NT) NK cells. Here, CD5 CAR#5 also shows the highest multifunctionality with the highest percentage of effector and chemoattractant cytokines. The multifunctional heatmap illustrates that CD5 CAR-NK cells carrying the DAP10 costimulatory domain are most capable of secreting a diverse combination of cytokines at the single-cell level (Figure 2C).

[0253] CD5 CAR-NK cells bearing both DAP10 TM and DAP10 costimulatory domains continue to kill CD5+ T-ALL cell lines (CCRF) after multiple reloading in an Incucyte killing assay (Figure 3A-C). A schematic of one of the reloading studies in the Incucyte killing assay is provided in Figure 3A. Measurements of the number of red cells (an indicator of viable tumor numbers) after each tumor reload (indicated by pink arrows) between the various CD5 CAR-NK cell conditions are provided in Figure 3B. Percent confluence, a measure of tumor burden, after each tumor reload (timings indicated by pink arrows) is provided in Figure 3C. These data show that CD5 CAR-NK cells with DAP10 TM and DAP10 costimulatory domains (represented by the red line with a box) continue to kill CD5+ T-ALL cell lines (CCRF) after multiple re-challenges compared to other CD5 CAR NK cell designs, compared to NT NK cells, and compared to irrelevant CD19 CAR-NK cells.

[0254] CD5 CAR-NK cells with DAP10 TM and DAP10 costimulatory domains show higher metabolic compatibility compared to other construct designs based on the seahorse metabolic assay, which measures the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of various CD5 CAR-NK cells (Figure 4A-4C). In Figure 4A, the OCR between various CD5 CAR-NK cell designs was compared to non-transduced (NT) NK cells. The ECAR between various CD5 CAR-NK cell designs was compared to non-transduced (NT) NK cells (Figure 4B). These data show significantly higher OCR for CD5 CAR-NK cells with DAP10 TM and DAP10 costimulatory domains compared to other designs and also compared to NT NK cells, yet the same constructs have the highest ECAR conferring higher metabolic compatibility.

[0255] Figure 5A-C show that CD5 CAR-NK cells with DAP10 costimulatory domain improve tumor control in a PDX mouse model of CD5+ mantle cell lymphoma. The absolute number of CD45+CD5+ cells in subcutaneous tumors of mice that received tumor alone (left) and tumor + CD5CAR-NK (right) in the subcutaneous tumor is shown (Figure 5A). Figure 5B provides a bar graph showing the absolute number of CD45+CD5+ cells in subcutaneous tumors of mice that received tumor alone (left) and tumor + CD5CAR-NK (right) in the spleen. The absolute number of CD45+CD5+ cells in subcutaneous tumors of mice that received tumor alone (left) and tumor + CD5CAR-NK (right) in the bone marrow is shown (Figure 5C).

[0256] Figures 6A-B demonstrate that CD27 CAR-NK cells with DAP10 costimulatory domains improve tumor control and survival in the NSG mouse model of acute myeloid leukemia (THP-1 transduced with firefly luciferase (FFLuc)). In Figure 6A, tumor burden among various groups of mice is shown as THP-1 FFLuc luminescence by serial bioluminescence imaging (BLI). Survival curves showing survival over time of various groups of mice are provided in Figure 6B, indicating that the DAP10 costimulatory domain improves the in vivo potency of CD27 CAR-NK cells. Example 2

[0257] Advantages of CD28 hinge-CD28 transmembrane domain-DAP10 costimulatory domain CAR This example relates to specific CAR constructs for characterization, including various constructs comprising a CD28 hinge, a construct comprising a CD28 transmembrane domain, and a construct comprising a DAP10 costimulatory domain. In a specific embodiment, activity is provided for a CAR construct comprising a CD28 hinge, a CD28 transmembrane domain, and a DAP10 costimulatory domain.

[0258] Figure 7A illustrates the identification of various constructs and the corresponding transduction efficiency (Figure 7B). CB-NK cells were transduced with various CD5 CAR constructs as shown in Figure 7A, and transduction efficiency was measured by flow cytometry. Transduction efficiency is based on the percentage of positive cells (Figure 7B).

[0259] Figure 8 provides an example of an experimental design in which different CD5 constructs are injected into mice, along with the corresponding timeline. The schematic shows the testing of the in vivo antitumor activity of different CD5 CAR NK cells against the T lymphoblastoid cell line CCRF-CEM as a target.

[0260] Figure 9A and Figure 9B show that mice treated with anti-CD5 CAR NK with IgG1 hinge cells survive significantly longer than NT NK cells and tumor alone. CD5 CAR NK cells reduced tumor burden in a mouse model of T-acute lymphoblastic leukemia. CCRF-CEM cells transduced with firefly luciferase (FFLuc) were inoculated with 1x10 5 Mice were injected with 1000 mg / mouse and monitored by bioluminescence imaging between each group. Mice in the treatment groups were injected with the respective NK CAR cells 3M two days after tumor injection. Bioluminescence images (Figure 9A) and quantification of luciferase signals (Figure 9B) of mice in each group show that mice receiving various CD5 NK CARs with IgG1 hinges showed enhanced CCRF-CEM tumor control compared to tumor alone, NT NK cells, but succumbed to tumors over time due to lack of NK cell persistence.

[0261] Figure 10A and Figure 10B demonstrate that mice treated with anti-CD5 CAR NK with a CD28 hinge have significantly reduced tumor burden compared to tumor alone, NT NK cells, and CD5 CAR NK cells with an IgG1 hinge. CD5 CAR NK cells reduced tumor burden in a mouse model of T-acute lymphoblastic leukemia. CCRF-CEM cells transduced with firefly luciferase (FFLuc) were inoculated with 1x10 5 Mice were injected with 1000 mg / mouse and monitored by bioluminescence imaging between each group. Mice in the treatment group were injected with the respective NK CAR cells 3M two days after tumor injection. Bioluminescence images of mice in each group (Figure 10A), and quantification of luciferase signals (Figure 10B) show that mice receiving various CD5 NK CARs with CD28 hinges showed enhanced CCRF-CEM tumor control compared to tumor alone, NT NK cells, and CD5 NK CARs with IgG1 hinges. Furthermore, CD5 NK CARs with DAP10 costimulatory domains significantly improved tumor control compared to other costimulatory domains. Example 3

[0262] CD5 CAR-NK cells with DAP10 signaling exhibit a signature of high proliferative potential, metabolic activity and memory features at the transcriptomic, epigenetic and proteomic levels. A CD5 CAR construct containing the CD28 transmembrane (TM) domain and DAP10 costimulatory domain with a CD3z signaling domain (CD28TMDAP10CD3ζ) outperformed other examples of CD5 CAR-NK cell constructs incorporating other costimulatory molecules, exhibiting superior in vitro and in vivo antitumor activity, enhanced multifunctionality and metabolic fitness, and less functional attrition after multiple tumor rechallenges in vitro. To further characterize why DAP10 signaling confers such a significant advantage to CAR-NK cells, we performed single-cell RNA sequencing (scRNAseq), single-cell ATAC sequencing (scATACseq) and reverse-phase protein array (RPPA) on CD5 CAR-NK cells with DAP10-CD3ζ signaling compared with CD5 CAR-NK cells engineered without a costimulatory domain (CD3ζ signal only) and non-transduced (NT) NK cells generated from the same donor. At the single-cell transcriptome level, pathway enrichment analysis of scRNAseq data showed that DAP10 signaling confers CAR-NK cells superior proliferative potential evidenced by E2F target and G2M checkpoint pathways, as well as IL-2 / STAT5 signaling, and enhanced metabolic activity evidenced by enrichment of metabolic pathways such as Myc, mTORC1, and oxidative phosphorylation (Figure 11). At the epigenetic level, scATACseq data show that CD5 CAR-NK cells harboring the DAP10-CD3z signaling domain exhibit enrichment of AP-1 complex and BATF transcription factors, which are associated with memory formation and attrition resistance, respectively (Figure 12).At the proteomic level, pathway enrichment analysis of the RPPA data confirmed the transcriptomic results by showing that DAP10 signaling enhances the proliferation and cytokine production capacity of CAR-NK cells (ITGA and INSR protein pathways) after stimulation with the CD5 target antigen, providing stem cell potential (COPS5 pathway), enhancing metabolic activity at both the glycolytic and mitochondrial levels (GAPDH and PARK7 pathways), enhancing membrane polarization and the ability to form immune synapses with target cells (Cav1 pathway), and conferring memory potential (FOXM1 pathway) (Figure 13A and Figure 13B). Example 4

[0263] CD5 CAR-NK cells with DAP10 signaling persist after tumor rechallenge and show evidence of a memory response in vivo. CAR-NK cells were assessed for their ability to mount a memory response in vivo after tumor rechallenge. A well-established NSG mouse model of the CD5+ T-ALL cell line, CCRF-CEM, was used. The CCRF-CEM tumor cell line was transduced with firefly luciferase and GFP (CCRF-Ffluc-GFP) to allow monitoring of tumor growth by bioluminescence imaging (BLI) and flow cytometry, respectively. Mice were irradiated (225 cGy) on day -1 and injected intravenously with 100,000 CCRF-Ffluc tumor cells per mouse on day 0. On day 2, treatment groups received 5 × 10 CD5 CAR-NK cells with DAP10 signaling. 6Mice were intravenously injected with 50,000 CCRF-CEM tumor cells. Blood was collected on day 93 after CCRF-CEM tumor injection to assess the percentage of human NK cells (hCD45+CD56+GFP-). On day 100, mice that were cured and showed no evidence of tumor on BLI or flow were rechallenged with 50,000 CCRF-CEM tumor cells. One week later (day 107), blood was collected again to check the percentage of human NK cells. As seen in Figures 14A and 14B, after tumor rechallenge, the percentage of human NK cells (hCD45+CD56+GFP-) in the blood increased from 13% to 91%, indicating that CAR-NK cells are able to mount a memory response to tumor rechallenge in vivo. References

[0264] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0265] 1. Maude SL, Laetsch TW, Buechner J et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med. 2018;378(5):439-448.

[0266] 2.Neelapu SS, Locke FL, Bartlett NL, et al. Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma. N Engl J Med. 2017;377(26):2531-2544.

[0267] 3. Schuster SJ, Bishop MR, Tam CS, et al. Tisagenlecleucel in Adult Relapsed or Refractory Diffuse Large B-Cell Lymphoma. N Engl J Med. 2019;380(1):45 - 56.

[0268] 4. Hartmann J, Schussler-Lenz M, Bondanza A, Buchholz CJ. Clinical development of CAR T cells - challenges and opportunities in translating innovative treatment concepts. EMBO Mol Med. 2017;9(9):1183 - 1197.

[0269] 5. Daher M, Rezvani K. Next generation natural killer cells for cancer immunotherapy: the promise of genetic engineering. Curr Opin Immunol. 2018;51:146 - 153.

[0270] 6. Mehta RS, Rezvani K. Chimeric Antigen Receptor Expressing Natural Killer Cells for the Immunotherapy of Cancer. Front Immunol. 2018;9:283.

[0271] 7.Liu E AS, Kerbauy L, et al. GMP-compliant universal antigen presenting cells (uAPC) promote the metabolic fitness and antitumor activity of armored cord blood CAR-NK cell. Front Immunol doi:103389 / fimmu2021626098. 2021.

[0272] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of this disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications may be applied to the methods and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein with the same or similar results. All such similar substitutes and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

1. A polynucleotide encoding a fusion protein, the fusion protein comprising: (a) optionally a hinge; and (b1) the CD28 transmembrane domain, or (b2) the transmembrane domain of DAP10; (c) a DAP10 costimulatory domain; and (d) CD3 zeta A polynucleotide comprising:

2. 2. The polynucleotide of claim 1, wherein the fusion protein is further defined as a chimeric antigen receptor (CAR).

3. 3. The polynucleotide of claim 2, wherein the CAR further comprises one or more antigen-binding domains.

4. The polynucleotide of claim 3 , wherein the antigen-binding domain targets a tumor antigen or an infectious agent.

5. 3. The polynucleotide of claim 2, wherein the CAR further comprises one or more additional costimulatory domains.

6. 6. The polynucleotide of claim 5, wherein the one or more additional costimulatory domains are selected from the group consisting of CD28, DAP12, 4-1BB, NKG2D, 2B4, and combinations thereof.

7. The polynucleotide of claim 2 further encoding an additional polypeptide of interest.

8. The polynucleotide of claim 7, wherein the additional polypeptide of interest is a therapeutic protein or a protein that enhances the activity, expansion, and / or persistence of a cell.

9. 8. The polynucleotide of claim 7, wherein the additional polypeptide of interest is a suicide gene product, one or more cytokines, or one or more human or viral proteins that enhance proliferation, expression and / or metabolic fitness.

10. 10. The polynucleotide of claim 9, wherein the cytokine is IL-15, IL-2, IL-12, IL-18, IL-21, IL-23, or IL-7.

11. A vector comprising the polynucleotide described in claim 1.

12. A natural killer cell containing the polynucleotide described in claim 1.

13. A natural killer cell as described in claim 12, which is derived from a stem cell, a hematopoietic stem cell, bone marrow, or a cell line.

14. The natural killer cell of claim 12 , wherein the NK cell expresses a recombinant cytokine.

15. The natural killer cell of claim 14, wherein the cytokine is IL-15, IL-2, IL-12, IL-18, IL-21, IL-7, or IL-23.

16. A population of natural killer cells comprising the natural killer cells described in claim 12.

17. A pharmaceutical composition comprising an effective amount of natural killer cells carrying the vector described in claim 11 for killing cancer cells.

18. The pharmaceutical composition described in claim 17, wherein the natural killer cells are derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, hematopoietic stem cells, bone marrow, cell lines, or mixtures thereof.

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