Method of engineering natural killer cells to target CD70-positive tumors

Abstract

Methods and compositions are provided relating to engineered cell receptors that target CD70 (also known as CD27 ligand, CD27LG, and TNFSF7). [Solution] Embodiments of the present disclosure include methods and compositions related to targeting CD70-expressing cells with NK cells specifically engineered to bind to the CD70 antigen. In certain embodiments, NK cells engineered to express a CD70-targeting engineered receptor, such as a CAR, are utilized to target CD70-expressing cancers. In certain embodiments, the vector expressing the CD70-targeting CAR also expresses a specific suicide gene and / or one or more specific cytokines.

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 958,563, filed on January 8, 2020. This provisional application is hereby incorporated by reference in its entirety.

[0002] Embodiments of the present disclosure include at least the fields of cell biology, molecular biology, immunology, and medicine (including cancer medicine).

Background Art

[0003] The reprogramming of natural killer (NK) cell genes for cancer adoptive immunotherapy has several clinically relevant uses and advantages, including 1) innate antitumor surveillance that does not require prior sensitization; 2) allogeneic effectiveness without graft-versus-host reactivity; and 3) direct cell-mediated cytotoxicity and lysis of target tumors. The development of human NK cells, as well as the acquisition of self-tolerance, alloreactivity, and effector functions, is an adaptive process of licensing, calibration, and arming. At the molecular level, specific activating and inhibitory receptors integrate extracellular signals, balance them, and direct NK cell functions by integrating them into unique effector functions. Since the functional activity and responsiveness of NK cells to external stimuli follow a "rheostat" model of continuous education, they can be reprogrammed. Reprogramming the effector functions of NK cells by genetic modification is an effective way to kill tumor cells by harnessing their cytotoxic capabilities.

[0004] The present disclosure relates to the improvement of cell therapy and adoptive cell therapy targeting surface antigen classification (Cluster of Differentiation) (CD70)-positive cancers.

Summary of the Invention

[0005] Embodiments of the present disclosure include methods and compositions related to engineered cell receptors that target CD70 (also known as CD27 ligand, CD27LG, and TNFSF7 for example). In specific embodiments, the engineered receptor that targets CD70 is in the form of a polynucleotide, polypeptide, or is included on the surface of any type of cell including immune cells. In specific cases, those cells are immune cells, and in certain embodiments, the immune cells are NK cells, NKT cells, invariant NKT cells, gamma delta T cells, regulatory T cells, B cells, macrophages, mesenchymal stromal cells (MSCs), dendritic cells, etc. from any origin. In certain embodiments, cord blood-derived initialized NK cells (CB-NK) are included for targeting tumors expressing the CD70 molecule.

[0006] Since CD70 is expressed in many cancers including, by way of example, acute myeloid leukemia (AML), lymphoma, lung cancer, melanoma, breast cancer, glioblastoma, mesothelioma, head and neck cancer, renal cancer, multiple myeloma, and pancreatic tumors, it is used as a target antigen for the methods and compositions. The expression of CD70 in normal tissues is limited to subsets of T cells and dendritic cells (DCs).

[0007] Embodiments of the present disclosure include various novel specific CAR constructs incorporating a CD70scFv heterologously fused to one or more signaling domains (such as the cytoplasmic portion of CD247 (also known as CD3ζ), and signaling domains including one or more of CD28, DAP10, DAP12, and NKG2D). The scFv, in some cases, is the heavy (V H ) chain and light (V L)It may contain a fusion of variable fragments derived from a lock. The vector may also contain one or more cytokine genes that produce human interleukin 15 (IL-15), IL-2, IL-21, IL-12, IL-7, and / or IL-18, which help the survival and maintenance of NK cells. As an example, the CB-NK cells modified in this way contain a vector encoding CD70scFv in its CAR, in addition to IL15 produced as a molecule separate from the CAR containing CD28 and CD3z.

[0008] In some embodiments, the above methods and compositions are used to treat individuals with CD70-positive cancer, but in other cases, those methods and compositions are used to remove CD70-expressing (non-cancerous) immunomodulatory cells (e.g., regulatory T cells (Tregs)) as checkpoints. In a specific embodiment, there is a method of targeting CD70-expressing non-cancerous cells in an individual, the method including the step of delivering an effective amount of CD70CAR-expressing cells to the individual.

[0009] Certain embodiments of the present disclosure are allogeneic to the recipient individual, enable the use of off-the-shelf immune cells (including at least NK cells) that target any type of CD70-positive cells and may or may not be transduced to express one or more cytokines (e.g., IL15, IL-2, IL21, IL-12, IL-7, and / or IL-18).

[0010] In specific embodiments of the present disclosure, the expression of one or more endogenous genes in immune cells has been modified, for example, the expression is partially or completely reduced. The modification can be performed by any means, but in specific embodiments, the expression of one or more genes has been modified, such as by reducing the expression level, which can be performed by any suitable means including at least CRISPR. By way of mere example, the endogenous gene is selected from the group consisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, CTLA-4, TDAG8, CD38 and combinations thereof.

[0011] In one embodiment, there is an expression construct encoding an engineered receptor specific for CD70 and comprising a sequence encoding one or both of: (a) a suicide gene; and (b) a cytokine. In a specific case, the engineered receptor specific for CD70 is a chimeric antigen receptor (CAR) or a T cell receptor. The CD70-specific CAR can comprise an scFv having a heavy chain and a light chain, wherein the heavy chain within the sequence encoding the CAR is upstream of the light chain in the 5' to 3' direction. In other cases, the CD70-specific CAR comprises an scFv having a heavy chain and a light chain, and the heavy chain within the sequence encoding the CAR is downstream of the light chain in the 5' to 3' direction. In any case herein, the CD70-specific CAR can or does not comprise an scFv with optimized codons. In any case herein, the CD70-specific CAR can or does not comprise a humanized scFv. In any case herein, the CD70-specific CAR can or does not comprise a signaling peptide (e.g., a signaling peptide from CD8 alpha, Ig heavy chain or granulocyte macrophage colony-stimulating factor receptor, or one from one or more other surface receptors). In a particular embodiment, the CD70-specific CAR comprises one or more co-stimulatory domains, for example, one or more co-stimulatory domains selected from the group consisting of CD28, CD27, OX-40 (CD134), DAP10, DAP12, 4-1BB (CD137), CD40L, 2B4, DNAM, CS1, CD48, NKG2D, NKp30, NKp44, NKp46, NKp80 or combinations thereof.

[0012] Any CD70-specific CAR may or may not include a hinge between CD3 zeta and / or scFv and the transmembrane domain. In specific cases, the hinge is a CD8-alpha hinge, which may include an artificial spacer containing Gly3, or the hinge may include the CH1, CH2, and / or CH3 domains of IgG. In specific embodiments, the cytokine is IL-15, IL-12, IL-2, IL-18, IL-21, IL-7, or a combination thereof. When using a suicide gene, the suicide gene can be mutant TNF-alpha (e.g., engineered non-secretory mutant), inducible caspase 9, HSV-thymidine kinase, CD19, CD20, CD52, or EGFRv3.

[0013] Embodiments of the present disclosure include expression constructs comprising any one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

[0014] Embodiments of the present disclosure include any type of immune cell comprising any expression included herein. In specific embodiments, the immune cell is an NK cell, T cell, gamma-delta T cell, invariant NKT (iNKT) cell, B cell, macrophage, MSC, or dendritic cell. When the immune cell is an NK cell, the NK cell can be derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, bone marrow, or a cell line. In a specific aspect, the NK cell line is the NK-92 cell line or another NK cell line derived from a tumor or healthy NK cells or progenitor cells.

[0015] In a specific embodiment, the immune cells are NK cells, such as those derived from umbilical cord blood mononuclear cells, i.e., NK cells derived from umbilical cord blood. In a specific case, the NK cells can be CD56+ NK cells. The NK cells can express one or more exogenously provided cytokines (e.g., IL-15, IL-2, IL-12, IL-18, IL-21, IL-7, or a combination thereof). Certain embodiments include a population of any type of immune cell of the present disclosure, and those cells can be present in a suitable medium or in any type of suitable carrier.

[0016] In one embodiment, there is a method of killing CD70-positive cells in an individual, the method including administering to the individual an effective amount of cells having any expression construct encompassed by the present disclosure. In a specific embodiment, those cells are NK cells, T cells, gamma-delta T cells, invariant NKT (iNKT) cells, B cells, macrophages, gamma-delta T cells, or dendritic cells. The NK cells can be derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, bone marrow, or a cell line. The NK cells can be derived from umbilical cord blood mononuclear cells. In some cases, the CD70-positive cells are not cancer cells, while in other cases, they are cancer cells. The CD70-positive cells can be regulatory T cells. In certain embodiments, the individual has acute myeloid leukemia, lymphoma, lung cancer, renal cancer, bladder cancer, melanoma, glioblastoma, breast cancer, head and neck cancer, mesothelioma, or a combination thereof. The above cells can be allogeneic or autologous to an individual who may or may not be human. The above cells can be administered to the individual by injection, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, intracranially, percutaneously, subcutaneously, regionally, by perfusion, into the tumor microenvironment, or by a combination thereof.

[0017] In certain embodiments of the above method, the cells can be administered to the individual one or more times. The time between administrations of the cells to the individual can be 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, 1 to 12 months, or 1 year or longer. The method can further comprise the step of providing the individual with an effective amount of additional treatment (e.g., surgery, radiation, gene therapy, immunotherapy, and / or hormone therapy). In some cases, the additional treatment can comprise one or more antibodies or antibody-based agents. In some aspects of the method, they can further comprise the step of identifying CD70-positive cells in the individual.

[0018] In a specific embodiment, there are compositions comprising the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.

[0019] It is expressly contemplated that any limitation discussed with respect to one embodiment of the invention applies to any other embodiment of the invention. Further, any composition of the invention can be used in any method of the invention, and any method of the invention can be used to produce or use any composition of the invention. The aspects of the embodiments shown in the examples are also embodiments that can be practiced in the context of different examples or elsewhere in the present application, e.g., in the context of embodiments discussed in the brief summary, detailed description, claims, and brief description of the drawings.

[0020] Above, to better understand the following detailed description, the features and technical advantages of the present disclosure have been outlined rather broadly. Additional features and advantages that form the subject matter of the claims in this specification will be described later in this specification. Those skilled in the art will recognize that the disclosed concepts and specific embodiments can be readily utilized as a basis for modifying or designing other structures to achieve the same purposes of this concept. It will also be understood by those skilled in the art that such equivalent configurations do not depart from the spirit and scope as set forth in the appended claims. Novel features believed to be specific to the concepts disclosed herein, both as to their construction and method of operation, together with further objects and advantages, will be better understood from the following description when considered in connection with the accompanying drawings. However, it should be clearly understood that each of those drawings is provided for purposes of illustration and description only and is not intended as a definition of the limits of the present disclosure.

[0021] For a more complete understanding of the present disclosure, reference is made to the following description, which is to be interpreted in conjunction with the accompanying drawings.

Brief Description of the Drawings

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[0056] Although various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will recognize numerous variations, modifications, and substitutions without departing from the invention. It should be understood that various alternatives to the embodiments of the disclosure described herein may be used.

Modes for Carrying Out the Invention

[0057] 1. Examples of Definitions In accordance with long-standing patent law convention, when used in this specification, including in the claims, the words "a" and "an" represent "one or more" when used in conjunction with the word "comprising". 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. Any method or composition described herein can be practiced with respect to any other method or composition described herein, and it is contemplated that different embodiments may be combined.

[0058] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprising", and "include" are to be construed to mean including the stated step or element or group of steps or elements but not excluding other steps or elements or group of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the expression "consisting of" indicates that the listed elements are necessary or essential and that no other elements can exist. "Consisting essentially of" means including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified for the listed elements in the disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements.

[0059] Throughout this specification, references to "one embodiment", "an embodiment", "a particular embodiment", "related embodiments", "an embodiment", "additional embodiments", or "further embodiments", or combinations thereof, mean 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 the foregoing phrases in various places in this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0060] As used herein, the terms "or" and "and / or" are used to describe multiple components either in combination with each other or exclusively. 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 can be specifically excluded from an embodiment.

[0061] Throughout this application, the term "about" is used in accordance with its plain and ordinary meaning in the field of cell and molecular biology to indicate that it includes the standard deviation of error for the apparatus or method employed to determine the value.

[0062] As used herein, the term "engineered" refers to an entity produced by human hands, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, the engineered article is synthetic and does not exist in nature or is composed of elements configured in the methods utilized in this disclosure.

[0063] As used herein, the term "isolated" refers to a molecule or biological or cellular material that is substantially free from other substances. 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 has been separated from other DNA or RNA, or protein or polypeptide, or cell or organelle, or tissue or organ as it exists in a natural source. The term "isolated" also means a nucleic acid or peptide that substantially does not contain cell material, viral material, or culture medium when produced by recombinant DNA technology, or chemical precursors or other chemicals when chemically synthesized. Further, "isolated nucleic acid" means including nucleic acid fragments that do not exist naturally as fragments and are not found in the natural state. The term "isolated" is also used herein to refer to a polypeptide isolated from other cellular proteins and is intended to encompass both purified polypeptides and recombinant polypeptides. The term "isolated" is also used herein to refer to a cell or tissue isolated from other cells or tissues and is intended to encompass both cultured cells or tissues and engineered cells or tissues.

[0064] As used herein, similar terms such as "prevent", "prevented", and "preventing" refer to an approach for preventing, suppressing, or reducing the likelihood of the occurrence or recurrence of a disease or condition, such as cancer. It also means delaying the onset or recurrence of a disease or condition, or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, effect, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0065] As used herein, the term "sample" generally refers to a biological sample. The sample may be taken from tissue or cells from an individual. In some examples, the sample may consist of or be derived from a tissue biopsy, blood (e.g., whole blood), plasma, extracellular fluid, dried blood spot, cultured cells, discarded tissue. The sample may be separated from the source prior to collection. Non-limiting examples include blood, cerebrospinal fluid, pleural effusion, amniotic fluid, lymph fluid, saliva, urine, feces, tears, sweat, or mucosal excretions, and other body fluids separated from the primary source prior to collection. In some examples, the sample is isolated from its primary source (such as cells, tissues, body fluids such as blood, environmental samples, etc.) during sample preparation. The sample may or may not be purified or concentrated from its primary product. In some cases, the primary product is homogenized prior to further processing. The sample may be filtered or centrifuged to remove buffy coat, lipids, or particulate matter. The sample can also be purified or concentrated for nucleic acids and can be treated with RNase. The sample can contain intact, fragmented, or partially degraded tissue or cells.

[0066] As used herein, the term "subject" generally refers to an individual having a biological sample that is to be processed or analyzed, and in certain cases, refers to an individual having or suspected of having cancer. The subject can be any organism or animal subject to a method or material, such as a mammal, e.g., a human, an experimental animal (e.g., a primate, a rat, a mouse, a rabbit), a domestic animal (e.g., a cow, a sheep, a goat, a pig, a turkey, a chicken), a household pet (e.g., a dog, a cat, a rodent), a horse, and a transgenic non-human animal. The subject can be, for example, a patient having or suspected of having a benign or malignant neoplasm, or a disease (sometimes referred to as a medical condition) such as cancer. The subject may be undergoing treatment or may have been treated. The subject is asymptomatic. The subject can be a healthy individual or an individual desiring cancer prevention. The term "individual" can be used interchangeably in at least some cases. The "subject" or "individual" as used herein may or may not be housed in a medical facility and may be treated as an outpatient of a medical facility. The individual may receive one or more medicaments via the Internet. The individual can include any age of a human or non-human animal, and thus includes both adults and juveniles (i.e., children) and infants, including in utero individuals. This term is not intended to mean a medical treatment necessity, and thus an individual can be part of an experiment, either voluntarily or involuntarily, whether clinical or in support of basic scientific research.

[0067] As used herein, "treatment" or "treating" includes any beneficial or desirable effect on the symptoms or pathology of a disease or medical condition, and can include even a minimal decrease in one or more measurable markers of the disease or condition being treated (e.g., cancer). Treatment can optionally include either a reduction or amelioration of the symptoms of the disease or medical condition, or a delay in the progression of the disease or medical condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or of the symptoms associated therewith.

[0068] The present disclosure relates to methods and compositions directed to any type of genetically engineered mammalian immune cell (including at least human NK cells) that target CD70-positive tumors. The present disclosure encompasses any type of genetically engineered receptor (including CARs) that is directed against CD70, which is the ligand of the cytokine receptor CD27. CD70 is expressed in hematological malignancies such as acute myeloid leukemia (AML) and lymphoma, and is also expressed in many solid tumors, including cancers of the kidney, bladder, lung, breast, glioblastoma, pancreas, and melanoma, making it attractive as a "pan-cancer antigen." CD70 is present only transiently on activated T lymphocytes, B lymphocytes, and dendritic cells. Since CD70, unlike other AML targets, is not expressed on normal hematopoietic stem cells, it is particularly advantageous as a target for immunotherapy of AML because cytopenia persists after CAR therapy and the need for recipient hematopoietic stem cell transplantation is less likely to occur. In certain embodiments, a number of novel expression constructs are provided that include retroviral constructs that express a single-chain variable fragment (scFv) against CD70 in the CAR and also express one or more cytokines, such as IL15, to support the survival and proliferation of NK cells. In a series of in vitro studies provided herein, the activity of CAR70 / IL-15-transduced cord blood (CB)-NK cells against AML, lung cancer targets, and glioblastoma is demonstrated.

[0069] I. Genetically Engineered Receptors The immune cells of the present disclosure can be genetically engineered to express an antigen receptor that targets CD70, such as an engineered TCR or CAR. For example, the immune cells may be NK cells that have been modified to express a CAR and / or TCR having antigen specificity for CD70. Other CARs and / or TCRs may be expressed by the same cells that express the CD70 receptor cells, and they may be directed against different antigens. In some embodiments, the immune cells are engineered to express a CD70-specific CAR or CD70-specific TCR by knock-in of the CAR or TCR using CRISPR.

[0070] Suitable modification methods are known in the art. See, for example, Sambrook and Ausubel, supra. For example, those cells can be transduced to express a TCR having antigen specificity for a cancer antigen using the transduction methods described in Heemskerk et al., 2008 and Johnson et al., 2009.

[0071] In some embodiments, those cells comprise one or more nucleic acids introduced via genetic engineering that encode one or more antigen receptors (at least one of which is specific for CD70), and genetically engineered products of such nucleic acids. In some embodiments, those nucleic acids are heterologous, i.e., not normally present in a given cell or sample obtained from that cell, e.g., nucleic acids obtained from another organism or cell, e.g., nucleic acids not normally found in the engineered cell and / or the organism from which such cell is derived. In some embodiments, those nucleic acids do not occur naturally, e.g., are nucleic acids not found in nature (e.g., chimeric).

[0072] Exemplary antigen receptors, including CARs and recombinant TCRs, and methods for engineering those receptors and methods for introducing them into cells, include, for example, those described in 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. Patents Nos. 6,451,995, 7,446,190, 8,252,592, 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, and 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, genetically engineered antigen receptors include CARs such as those described in U.S. Patent No. 7,446,190 and those described in International Patent Application Publication No. WO / 2014055668Al. A. Chimeric Antigen Receptor

[0073] In some embodiments, the CD70-specific CAR comprises: a) one or more intracellular signaling domains, b) a transmembrane domain, and c) an extracellular domain comprising one or more antigen-binding domains that specifically bind to CD70. In certain embodiments, the antigen-binding domain is an antibody and is not a protein or protein fragment that is not an antibody.

[0074] In some embodiments, engineered antigen receptors include CARs such as activating or stimulatory CARs, co-stimulatory CARs (see WO2014 / 055668), and / or inhibitory CARs (iCARs, see Fedorov et al., 2013). These CARs generally include an extracellular antigen (or ligand) binding domain linked in some manner through a linker and / or transmembrane domain to one or more intracellular signaling components. Such molecules typically mimic or emulate signals through natural antigen receptors, signals through such receptors together with co-stimulatory receptors, and / or signals through co-stimulatory receptors alone.

[0075] Certain embodiments of the present disclosure relate to the use of nucleic acids encoding a CD70-specific CAR polypeptide (including a humanized CAR (hCAR) for reducing immunogenicity) that includes an intracellular signaling domain, a transmembrane domain, and an extracellular domain that includes one or more signaling motifs. In certain embodiments, the CD70-specific CAR can recognize an epitope that includes a space shared between one or more antigens. In certain embodiments, the binding region can include complementarity determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and / or antigen-binding fragments thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor.

[0076] The human CD70 CAR nucleic acid is contemplated to be a human gene used to enhance cellular immunotherapy for human patients. In a specific embodiment, the present disclosure includes the full-length cDNA or coding region of a CD70-specific CAR. The antigen-binding region or domain is the V H chain of a single-chain variable fragment (scFv) derived from a specific human monoclonal antibody and the V LIt may include a lock fragment (e.g., as described in U.S. Patent No. 7,109,304, which is incorporated herein by reference). The fragment can also be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is a CD70-specific scFv encoded by a sequence optimized for human codon usage for expression in human cells.

[0077] The arrangement can be multimeric (e.g., diabody or multimer). The multimer is most likely formed by cross-pairing of the variable parts of the light and heavy chains to form a diabody. There can be multiple options for the hinge part of the construct, ranging from complete deletion, maintaining the first cysteine, being a proline substitution instead of a serine substitution, to being cleaved up to the first cysteine. The Fc part can be deleted. Any protein that is stable and / or dimerizes can serve this purpose. Only one of the Fc domains, for example, the CH2 domain or CH3 domain of human immunoglobulin, can be used. The hinge, CH2, and CH3 regions of human immunoglobulin modified to improve dimerization can also be used. Only the hinge part of the immunoglobulin can be used. A part of CD8 alpha can also be used.

[0078] In some embodiments, the CD70 CAR nucleic acid includes sequences encoding other co-stimulatory receptors, such as a transmembrane domain and a modified CD28 intracellular signaling domain. Examples of other co-stimulatory receptors include, but are not limited to, one or more of CD28, CD27, OX-40 (CD134), DAP10, DAP12, and 4-1BB (CD137). In addition to the primary signal induced by CD3ζ, additional signals provided by human co-stimulatory receptors inserted into the human CAR are important for the complete activation of NK cells and can help improve in vivo persistence and the success of adoptive immunotherapy treatment.

[0079] In some embodiments, the CD70-specific CAR is constructed to have specificity for CD70 expressed in a particular cell type targeted by adoptive therapy (e.g., an antigen expressed on a normal cell type or a non-diseased cell type). Thus, the CAR typically includes, in its extracellular portion, one or more antigen-binding molecules (e.g., one or more antigen-binding fragments, antigen-binding domains, or antigen-binding portions), or one or more antibody variable domains, and / or antibody molecules. In some embodiments, the CD70 CAR includes an antigen-binding portion of an antibody molecule (e.g., a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb)).

[0080] In certain embodiments, the CD70 CAR can be co-expressed with one or more cytokines to improve persistence when the amount of tumor-associated antigen is low. For example, the CAR can be co-expressed with one or more cytokines such as IL-7, IL-2, IL-15, IL-12, IL-18, IL-21, or combinations thereof.

[0081] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA origin, cDNA origin, or can be synthesized (e.g., via PCR), or combinations thereof. Since introns have been found to stabilize mRNA, depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or combinations thereof. It may also be further beneficial to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0082] It is contemplated that the chimeric construct can be introduced into immune cells either as naked DNA or in a suitable vector. Methods for stably transfecting cells by electroporation with naked DNA are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA encoding a chimeric receptor included in a plasmid expression vector in an orientation appropriate for expression.

[0083] Alternatively, viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors or lentiviral vectors) can be used to introduce the chimeric construct into immune cells. Vectors suitable for use according to the methods of the present disclosure are non-replicative vectors in immune cells. A number of virus-based vectors are known (e.g., vectors based on HIV, SV40, EBV, HSV or BPV), and the copy number of the virus maintained intracellularly is low enough to maintain the viability of the cell.

[0084] In some embodiments, the component that specifically binds to an antigen or the antigen-specific recognition component is linked to one or more transmembrane domains and an intracellular signaling domain. In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that naturally associates with one of the domains in the CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins in order to minimize interaction with other members of the receptor complex.

[0085] The transmembrane domain is, in some embodiments, of natural or synthetic origin. When the origin is natural, the domain is, in some aspects, derived from any membrane-bound or transmembrane protein. Transmembrane regions include transmembrane regions derived from the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and DAP molecules (i.e., including at least the transmembrane regions of those molecules). Alternatively, the transmembrane domain is, in some embodiments, a synthetic transmembrane domain. In some aspects, the synthetic transmembrane domain mainly comprises hydrophobic residues such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain.

[0086] In certain embodiments, platform technologies disclosed herein for genetically modifying immune cells such as NK cells include: (i) non-viral gene transfer using an electroporation device (e.g., a nucleofector); (ii) a CAR that signals through an endodomain (e.g., CD28 / CD3-zeta, CD137 / CD3-zeta, or other combinations); (iii) a CAR having an extracellular domain of variable length that connects an antigen recognition domain to the cell surface; and in some cases, (iv) a CAR + Examples include artificial antigen-presenting cells (aAPCs) derived from K562 (Singh et al., 2008; Singh et al., 2011) that can robustly and numerically expand immune cells.

[0087] B. Examples of Specific CAR Embodiments In certain embodiments, vectors encoding a particular CD70 CAR molecule, or multiple molecules comprising a CD70-specific CAR, are encompassed herein. In some aspects, the CD70 binding domain of the CAR is an scFv, and any scFv that binds to CD70 can be utilized herein. The variable heavy and variable light chains of the scFv can be in any order from the N-terminus to the C-terminus. For example, the variable heavy chain can be on the N-terminal side of the variable light chain, or vice versa. The scFv may or may not be codon-optimized. The scFv may or may not be humanized. Specific examples of CD70 scFv include at least 42D12, Ab7, 27B3, 9D1, 57B6, or others. The scFv utilized may be at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to 42D12, Ab7, 27B3, 9D1, 57B6, or others.

[0088] In certain embodiments, the vector encodes a CD70-specific CAR and also encodes one or more other molecules. For example, the vector may encode a CD70-specific CAR that may or may not be codon-optimized (CO), and in certain aspects, the anti-CD70 scFv is a 42D12 scFv that may have the variable light chain upstream or downstream of the variable heavy chain. In a specific embodiment, the CAR comprises CD28 and no other co-stimulatory domains, and the CAR may also comprise CD3ζ. In some cases, the vector encodes one or more cytokines and one or more suicide genes.

[0089] The DNA sequence and polypeptide sequence of the codon-optimized (CO) CAR.CD70 42D12 VLVH scFv antibody sequence are as follows: DNA ATGGCCCTGCCTGTGACAGCTCTGCTCCTCCCTCTGGCCCTGCTGCTCCATGCCGCCAGACCCCAGGCAGTtGTGACCCAGGAGCCTTCCCTGACAGTGTCTCCAGGAGGGACGGTCACGCTCACCTGCGGCCTCAAATCTGGGTCTGTCACTTCCGATAACTTCCCCACTTGGTACCAGCAGACACCAGGCCAGGCTCCCCGATTGCTTATCTACAACACAAACACCCGTCACTCTGGCGTCCCCGACCGCTTCTCCGGATCCATCCTGGGCAACAAAGCCGCCCTCACCATCACGGGGGCCCAGGCCGACGACGAGGCCGAATATTTCTGTGCTCTGTTCATAAGTAATCCTAGTGTTGAGTTCGGCGGAGGGACCCAACTGACCGTCCTAGGTGGCAGCACCAGCGGCTCCGGCAAGCCTGGCTCTGGCGAGGGCAGCACAAAGGGAGAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGTCTACTACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCAGATATTAATAATGAAGGTGGTACTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACTCTAAGAACAGCCTGTATCTGCAAATGAACAGCCTGCGCGCCGAGGACACGGCCGTGTACTACTGCGCGAGAGATGCCGGATATAGCAACCATGTACCCATCTTTGATTCTTGGGGCCAGGGGACCCTGGTCACTGTCTCCTCA (Sequence number in the sequence listing: 1) Protein MALPVTALLLPLALLLHAARPQAVVTQEPSLTVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLGGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS (Sequence Listing SEQ ID NO:2)

[0090] The DNA sequence and protein sequence of the non-codon-optimized (CO) CAR.CD70 42D12 VHVL scFv antibody are as follows: DNA ATGGCCCTGCCTGTGACAGCTCTGCTCCTCCCTCTGGCCCTGCTGCTCCATGCCGCCAGACCCGAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGTCTACTACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCAGATATTAATAATGAAGGTGGTACTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACTCTAAGAACAGCCTGTATCTGCAAATGAACAGCCTGCGCGCCGAGGACACGGCCGTGTACTACTGCGCGAGAGATGCCGGATATAGCAACCATGTACCCATCTTTGATTCTTGGGGCCAGGGGACCCTGGTCACTGTCTCCTCAGGCAGCACCAGCGGCTCCGGCAAGCCTGGCTCTGGCGAGGGCAGCACAAAGGGACAGGCAGTGGTGACCCAGGAGCCTTCCCTGACAGTGTCTCCAGGAGGGACGGTCACGCTCACCTGCGGCCTCAAATCTGGGTCTGTCACTTCCGATAACTTCCCCACTTGGTACCAGCAGACACCAGGCCAGGCTCCCCGATTGCTTATCTACAACACAAACACCCGTCACTCTGGCGTCCCCGACCGCTTCTCCGGATCCATCCTGGGCAACAAAGCCGCCCTCACCATCACGGGGGCCCAGGCCGACGACGAGGCCGAATATTTCTGTGCTCTGTTCATAAGTAATCCTAGTGTTGAGTTCGGCGGAGGGACCCAACTGACCGTCCTAGGT(Sequence number in the sequence listing: 3) Protein MGMALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSSGSTSGSGKPGSGEGSTKGQAVVTQEPSLTVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLG (Sequence number in the sequence listing: 4)

[0091] The DNA and protein sequences of the scFv antibody sequence CAR.CD70 42D12 VLVH are as follows.: DNA ATGGCCCTGCCTGTGACAGCTCTGCTCCTCCCTCTGGCCCTGCTGCTCCATGCCGCCAGACCCGAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGTCTACTACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTTGAGTGGGTCTCAGATATTAATAATGAAGGTGGTACTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACTCTAAGAACAGCCTGTATCTGCAAATGAACAGCCTGCGCGCCGAGGACACGGCCGTGTACTACTGCGCGAGAGATGCCGGATATAGCAACCATGTACCCATCTTTGATTCTTGGGGCCAGGGGACCCTGGTCACTGTCTCCTCAGGCAGCACCAGCGGCTCCGGCAAGCCTGGCTCTGGCGAGGGCAGCACAAAGGGACAGGCAGTGGTGACCCAGGAGCCTTCCCTGACAGTGTCTCCAGGAGGGACGGTCACGCTCACCTGCGGCCTCAAATCTGGGTCTGTCACTTCCGATAACTTCCCCACTTGGTACCAGCAGACACCAGGCCAGGCTCCCCGATTGCTTATCTACAACACAAACACCCGTCACTCTGGCGTCCCCGACCGCTTCTCCGGATCCATCCTGGGCAACAAAGCCGCCCTCACCATCACGGGGGCCCAGGCCGACGACGAGGCCGAATATTTCTGTGCTCTGTTCATAAGTAATCCTAGTGTTGAGTTCGGCGGAGGGACCCAACTGACCGTCCTAGGT (Sequence number in the sequence listing: 5) Protein MGMALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSSGSTSGSGKPGSGEGSTKGQAVVTQEPSLTVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLG (Sequence Listing SEQ ID NO: 6)

[0092] Examples of specific vector molecules containing CAR and IL15 include at least the following: CO CAR.CD70 42D12. VLVH.IgG1.CD28.CD3z-2A-IL15 CO CAR.CD70 42D12 VHVL.IgG1.CD28.CD3z-2A-IL15 CAR.CD70 42D12 VLVH.IgG1.CD28.CD3z-2A-IL15 CAR.CD70 42D12 VHVL.IgG1.CD28.CD3z-2A-IL15

[0093] An example of a plasmid map of the exemplary CO CAR.CD70 42D12 VLVH.IgG1.CD28.CD3z-2A-IL15 vector is Figure 1. The complete DNA sequence of the vector constituting CO CAR.CD70 42D12 VLVH.IgG1.CD28.CD3z-2A-IL15 is as follows:

[0094] In some embodiments, a codon-optimized CO CAR.CD70 42D12 VHVL.IgG1.CD28.CD3z-2A-IL15 vector is employed. An example of the plasmid map of the codon-optimized CO CAR.CD70 42D12 VHVL.IgG1.CD28.CD3z-2A-IL15 vector is shown in Figure 2. The complete DNA sequence of the following construct CO CAR.CD70 42D12 VHVL.IgG1.CD28.CD3z-2A-IL15 is as follows.:

[0095] Non-codon-optimized CARs can also be employed. For example, the CAR.CD70 42D12 VLVH.IgG1.CD28.CD3z-2A-IL15 vector is exemplified, and the sequence is provided as follows.:

[0096] In certain cases, specific antibodies having a CD8α signal peptide (CD8SP) are utilized. An example of the CD8SP CD70 42D12 VLVH sequence is as follows: DNA ATGGCCCTGCCTGTGACAGCTCTGCTCCTCCCTCTGGCCCTGCTGCTCCATGCCGCCAGACCCCAGGCAGTGGTGACCCAGGAGCCTTCCCTGACAGTGTCTCCAGGAGGGACGGTCACGCTCACCTGCGGCCTCAAATCTGGGTCTGTCACTTCCGATAACTTCCCCACTTGGTACCAGCAGACACCAGGCCAGGCTCCCCGATTGCTTATCTACAACACAAACACCCGTCACTCTGGCGTCCCCGACCGCTTCTCCGGATCCATCCTGGGCAACAAAGCCGCCCTCACCATCACGGGGGCCCAGGCCGACGACGAGGCCGAATATTTCTGTGCTCTGTTCATAAGTAATCCTAGTGTTGAGTTCGGCGGAGGGACCCAACTGACCGTCCTAGGTGGCAGCACCAGCGGCTCCGGCAAGCCTGGCTCTGGCGAGGGCAGCACAAAGGGAGAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGTCTACTACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCAGATATTAATAATGAAGGTGGTACTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACTCTAAGAACAGCCTGTATCTGCAAATGAACAGCCTGCGCGCCGAGGACACGGCCGTGTACTACTGCGCGAGAGATGCCGGATATAGCAACCATGTACCCATCTTTGATTCTTGGGGCCAGGGGACCCTGGTCACTGTCTCCTCA (SEQ ID NO: 10 in the Sequence Listing) Protein ARVATMGMALPVTALLLPLALLLHAARPQAVVTQEPSLTVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLGGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS (Sequence Listing SEQ ID NO: 11)

[0097] A plasmid vector map of an example of the CAR.CD70 42D12 VLVH.IgG1.CD28.CD3z-2A-IL15 CAR is provided in Figure 3.

[0098] The complete DNA sequence of CAR.CD70 42D12 VLVH.IgG1.CD28.CD3z-2A-IL15 is as follows.

[0099] The CAR.CD70 42D12 VHVL.IgG1.CD28.CD3z-2A-IL15 vector can be utilized in the methods and compositions of the present disclosure. The plasmid vector map of CAR.CD70 42D12 VHVL.IgG1.CD28.CD3z-2A-IL15 is shown in Figure 4. The complete DNA sequence of CAR.CD70 42D12 VHVL.IgG1.CD28.CD3z-2A-IL15 is as follows.: C.T cell receptor (TCR)

[0100] In some embodiments, genetically engineered antigen receptors include recombinant TCRs and / or TCRs cloned from naturally occurring T cells. A “T cell receptor” or “TCR” is a molecule that includes variable alpha and variable beta chains (also known as TCRα and TCRβ, respectively) or variable gamma and variable delta chains (also known as TCRγ and TCRδ, respectively), and that can specifically bind to an antigen peptide bound to an MHC receptor. In some embodiments, the TCR is of the αβ type.

[0101] Generally, TCRs that exist as αβ and γδ types are generally similar in structure, but the T cells expressing them can differ in anatomical location or function. TCRs can be found on the cell surface or in a soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes) and are usually involved in the recognition of antigens bound to major histocompatibility complex (MHC) molecules on their surface. In some embodiments, the TCR can also include a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, for example, Janeway et al, 1997). For example, in some aspects, each chain of the TCR can have one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Unless otherwise stated, the term “TCR” should be understood to encompass its functional TCR fragments. This term also encompasses intact or full-length TCRs, including αβ or γδ type TCRs.

[0102] Accordingly, for the purposes herein, reference to a TCR includes any TCR or functional fragment (e.g., the specific antigen peptide bound in an MHC molecule, i.e., the antigen-binding portion of the TCR that binds to an MHC-peptide complex). The "antigen-binding portion" or antigen-binding fragment of a TCR, which can be used interchangeably, refers to a molecule that contains only a part of the structural domain of the TCR but binds to the antigen (e.g., MHC-peptide complex) to which the complete TCR binds. In some cases, the antigen-binding portion includes the variable domains of the TCR (e.g., the variable alpha and variable beta chains of the TCR) sufficient to form a binding site for binding to a specific MHC-peptide complex, and generally, each chain contains three complementarity-determining regions, for example.

[0103] In some embodiments, the variable domains of the TCR chains associate to form loops or complementarity-determining regions (CDRs) similar to immunoglobulins, thereby resulting in antigen recognition, determining peptide specificity by forming the binding site of the TCR molecule, and determining peptide specificity. Usually, similar to immunoglobulins, the CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the major CDR involved in the recognition of processed antigens, whereas the CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of the antigen peptide, while the CDR1 of the beta chain interacts with the C-terminal portion of that peptide. CDR2 is thought to recognize MHC molecules. In some embodiments, the variable region of the beta chain may include an additional hypervariable (HV4) region.

[0104] In some embodiments, the TCR chains include constant domains. For example, similar to immunoglobulins, the extracellular portion of the TCR chains (e.g., alpha chain, beta chain) is two immunoglobulin domains, the variable domain at the N-terminus (e.g., V aor Vp; typically, amino acids 1-116 based on Kabat numbering of Kabat et al., “Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed. (amino acids 1-116), and one constant domain adjacent to the cell membrane (e.g., the a-chain constant domain or C a , typically amino acids 117-259 based on Kabat, the β-chain constant domain or Cp, typically amino acids 117-295 based on Kabat). For example, in some cases, the extracellular portion of the TCR formed by those two chains includes two membrane-proximal constant domains and two membrane-distal variable domains that include CDRs. The constant domains of the TCR domains include short connecting sequences where cysteine residues form disulfide bonds, thereby forming a linkage between those two chains. In some embodiments, the TCR may have additional cysteine residues in each of the α-chain and β-chain such that the TCR includes two disulfide bonds in the constant domain.

[0105] In some embodiments, the TCR chain may include a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain includes a cytoplasmic tail. In some cases, due to its structure, the TCR can associate with other molecules such as CD3. For example, a TCR that includes a constant domain along with the transmembrane region can anchor the protein to the cell membrane and associate with the invariant subunits of the CD3 signaling apparatus or complex.

[0106] In general, CD3 is a multi - protein complex that can have three different chains (γ, δ, and ε) and the ζ chain in mammals. For example, in mammals, this complex can include the CD3γ chain, the CD3δ chain, two CD3ε chains, and the homodimeric CD3ζ chain. The CD3γ chain, CD3δ chain, and CD3ε chain are highly related cell - surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ chain, CD3δ chain, and CD3ε chain are negatively charged, a property that allows these chains to associate with the positively charged T - cell receptor chains. Each intracellular tail of the CD3γ chain, CD3δ chain, and CD3ε chain contains a single conserved motif known as an immunoreceptor activation tyrosine motif or ITAM, whereas each CD3ζ chain contains three. In general, ITAM is involved in the signaling ability of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in the propagation of signals from the TCR to the cell. The CD3 chains and the ζ chain together with the TCR form a complex known as the T - cell receptor complex.

[0107] In some embodiments, the TCR can be a heterodimer of two chains α and β (or γ and δ if necessary) or a single-chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two separate chains (α chain and β chain or γ chain and δ chain) linked by, for example, disulfide bonds. In some embodiments, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into cells. In some embodiments, the nucleic acid encoding the TCR can be obtained from various sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological origin, such as a cell, e.g., a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other publicly available sources. In some embodiments, the T cell can be obtained from cells isolated in vivo. In some embodiments, high-affinity T cell clones can be isolated from a patient and the TCR can be isolated. In some embodiments, the T cell can be a cultured T cell hybridoma or clone. In some embodiments, the TCR clone against a target antigen is a clone produced in a transgenic mouse engineered with human immune system genes (e.g., the human leukocyte antigen system, i.e., HLA). See, for example, tumor antigens (e.g., Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate a TCR against a target antigen (see, e.g., Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, the TCR or its antigen-binding portion can be synthetically made based on knowledge of the TCR sequence.

[0108] II. Cytokine One or more cytokines can be utilized together with one or more CD70-targeted genetically engineered receptors such as CD70-specific CARs. In some cases, the one or more cytokines are present on the same vector molecule as the genetically engineered receptor, while in other cases they are present on separate molecules. In certain embodiments, the one or more cytokines are co-expressed from the same vector as the engineered receptor. The one or more cytokines may be produced as a polypeptide distinct from the CD70-specific receptor. As an example, interleukin-15 (IL-15) is utilized. IL-15 can be employed because, for example, it is tissue-restricted and is observed in serum or systemically at any level only under pathological conditions. IL-15 has several properties desirable for adoptive immunotherapy. IL-15 is a homeostatic cytokine that induces the development and cell proliferation of natural killer cells, alleviates the functional suppression of tumor resident cells to promote the eradication of established tumors, and inhibits activation-induced cell death. In addition to IL-15, other cytokines are envisioned. These include, but are not limited to, cytokines, chemokines, and other molecules that contribute to the activation and proliferation of cells used in human applications. As an example, the cytokine is IL-15, IL-12, IL-2, IL-18, IL-21, IL-7, or a combination thereof. NK cells expressing IL-15 may be utilized and can continue supportive cytokine signaling, which is useful for survival after injection.

[0109] In certain embodiments, the NK cells express one or more exogenously provided cytokines. Since the cytokines are expressed from an intracellular expression vector, they can be provided exogenously to the NK cells. In another aspect, the endogenous cytokines within the cell are upregulated by manipulation of the expression regulation of the endogenous cytokines, such as genetic recombination at the promoter site(s) of the cytokine. When the cytokine is supplied to the cell on an expression construct, the cytokine may be encoded from the same vector as the suicide. The cytokine may be expressed as a polypeptide molecule separate from the suicide gene and also as a polypeptide separate from the engineered receptor of the cell. In some embodiments, the present disclosure relates to the co-utilization of CAR and / or TCR vectors and IL-15, particularly in NK cells.

[0110] III. Suicide Gene In certain embodiments, a suicide gene is used with any type of cell therapy to control the use of the cell therapy and to terminate the cell therapy at a desired event and / or time point. The suicide gene is used in the transduced cells for the purpose of inducing death in the transduced cells when needed. The CD70-targeted cells of the present disclosure modified to have 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 whose gene product is changed into a compound that kills the host cell when a prodrug or other agent is administered. In other embodiments, the suicide gene encodes a gene product that is targeted by an agent (e.g., an antibody) that targets the suicide gene product when desired.

[0111] 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 cytarabine. The so-called suicide gene of E. coli, purine nucleoside phosphorylase, which converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine, can be used. Other examples of suicide genes used in combination with prodrug therapy are the cytosine deaminase gene of E. coli and the HSV thymidine kinase gene.

[0112] Exemplary suicide genes also include CD20, CD52, EGFRv3 or inducible caspase 9. In one embodiment, a truncated version of epidermal growth factor receptor variant III (EGFRv3) can be used as a suicide antigen that can be cleaved by cetuximab. Further suicide genes known in the art that can be used in the present disclosure include purine nucleoside phosphorylase (PNP), cytochrome p450 enzyme (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzyme, methionine-α,γ-lyase (MET) and thymidine phosphorylase (TP).

[0113] In certain embodiments, a vector encoding a CD70-targeted CAR, or any vector in the NK cells encompassed herein, contains one or more suicide genes. The suicide gene may or may not be present on the same vector as the CD70-targeted CAR. When the suicide gene is present on the same vector as the CD70-targeted CAR, the suicide gene and the CAR may be separated, for example, by an IRES or a 2A element.

[0114] In a specific embodiment, the suicide gene is a tumor necrosis factor (TNF)-alpha variant that cannot be cleaved by a standard enzyme that cleaves TNF in nature, such as TNF-alpha converting enzyme (also referred to as TACE). Thus, the TNF-alpha variant is, in certain embodiments, membrane-bound and non-secreted. The TNF-alpha variant used in the present disclosure can be targeted by one or more agents (including at least an antibody) that bind to the variant, and after the agent binds to the TNF-alpha variant on the surface of the cell, the cell dies. Embodiments of the present disclosure enable the TNF-alpha variant to be used as a marker for cells that express it.

[0115] Cells expressing a non-cleavable TNF-alpha variant can be targeted for selective ablation using currently clinically used FDA-approved TNF-α antibodies such as etanercept, infliximab, or adalimumab. The mutated TNF-alpha polypeptide can be co-expressed in the cell with one or more therapeutic transgenes (e.g., genes encoding a TCR or CAR, including a TCR and / or CAR that targets CD70). Furthermore, cells expressing the TNF-alpha variant have excellent activity against tumor targets mediated by the biological activity of the membrane-bound TNF-alpha protein.

[0116] With respect to the wild type, TNF-alpha has a 26 kD transmembrane form and a 17 kD secreted component. Several mutants described by Perez et al. (1990) can be used in the present disclosure. In a specific embodiment, examples of the TNF-alpha mutants of the present disclosure include at least the following with respect to 17 kD TNF: (1) deletion of Val1 and deletion of Pro112; (2) deletion of Val13; (3) deletion of Val1 and deletion of Val13; (4) deletion from Val1 to Pro112 and deletion of Val13 (a 13 aa deletion); (5) deletion from Ala-3 to Val13 (a 14 aa deletion). In a specific embodiment, the TNF-alpha mutant comprises a deletion of each amino acid at positions -3, -2, -1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or combinations thereof. Specific combinations include deletions from position -3 to position 13; from position -3 to position 12; from position -3 to position 11; from position -3 to position 10; from position -3 to position 9; from position -3 to position 8; from position -3 to position 7; from position -3 to position 6; from position -3 to position 5; from position -3 to position 4; from position -3 to position 3; from position -3 to position 2; from position -3 to position 1; from position -3 to position -1; from position -3 to position -2; from position -2 to position 13; from position -2 to position 12; from position -2 to position 11; from position -2 to position 10; from position -2 to position 9; from position -2 to position 8; from position -2 to position 7; from position -2 to position 6; from position -2 to position 5; from position -2 to position 4; from position -2 to position 3; from position -2 to position 2; from position -2 to position 1; from position -2 to position -1; from position -1 to position 13; from position -1 to position 12; from position -1 to position 11; from position -1 to position 10; from position -1 to position 9; from position -1 to position 8; from position -1 to position 7; from position -1 to position 6; from position -1 to position 5; from position -1 to position 4; from position -1 to position 3; from position -1 to position 2; from position -1 to position 1; from position 1 to position 13; from position 1 to position 12; from position 1 to position 11; from position 1 to position 10; from position 1 to position 9; from position 1 to position 8; from position 1 to position 7; from position 1 to position 6; from position 1 to position 5; from position 1 to position 4; from position 1 to position 3; from position 1 to position 2; etc.

[0117] TNF-alpha variants may be produced by any suitable method, but in specific embodiments, they are produced by site-directed mutagenesis. In some cases, TNF-alpha variants may have mutations other than those that render the protein unable to be cleaved. In specific cases, TNF-alpha variants may have one, two, three or more mutations other than deletions in Val1, Pro12 and / or Val13 or the region between them. Mutations other than those that make the variant non-secretory may be one or more of amino acid substitutions, deletions, additions, inversions, etc. If the additional mutation is an amino acid substitution, the substitution may be, for example, a substitution to a conservative amino acid or not. In some cases, one, two, three, four, five or more additional amino acids may be present at the N-terminus and / or C-terminus of the protein. In some cases, TNF-alpha variants have (1) one or more mutations that make the variant non-secretory; (2) one or more mutations that prevent outside-in signaling to the variant; and / or (3) one or more mutations that interfere with the binding of the variant to TNF receptor 1 and / or TNF receptor 2.

[0118] In certain embodiments, delivery of an effective amount of one or more agents that bind to CD70CAR-targeted cells expressing TNF-alpha variants results in the removal of most of the TNF-alpha variant-expressing cells. In specific embodiments, more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the cells expressing TNF-alpha variants are removed in an individual. After recognizing the need to remove the cells, delivery of the agent to the individual can be continued until one or more symptoms no longer exist or until a sufficient number of cells have been removed. The number of cells in an individual can be monitored using the TNF-alpha variant as a marker.

[0119] Embodiments of the methods of the present disclosure include a first step of providing an effective amount of CD70-targeted immunocytotherapy to an individual in need thereof, wherein the cells comprise one or more non-secretable TNF-alpha variants; and a second step of using the TNF-alpha variant as a suicide gene to eliminate the cells (either directly or indirectly through cell death by any mechanism). The second step can be initiated upon the occurrence of at least one adverse event to the individual, which adverse event can be recognized by any means, including routine monitoring, which may or may not be continuous since the initiation of the cell therapy. Such adverse events can be detected upon examination and / or testing. If the individual has cytokine release syndrome (which may also be referred to as cytokine storm), the individual may have, for example, high inflammatory cytokines (by way of example only: interferon-gamma, granulocyte macrophage colony-stimulating factor, IL-10, IL-6, and TNF-alpha); fever; fatigue; hypotension; hypoxia, tachycardia; nausea; capillary leak; heart / kidney / liver dysfunction; or combinations thereof. If the individual has neurotoxicity, the individual may have confusion, delirium, dysplasia, and / or seizures. In some cases, the individual is tested for markers associated with the occurrence and / or severity of cytokine release syndrome (e.g., C-reactive protein, IL-6, TNF-alpha, and / or ferritin).

[0120] In additional embodiments, administration of one or more agents that bind non-secreted TNF-α in cytokine release syndrome or neurotoxicity has the additional advantage of neutralizing, for example, high levels of soluble TNF-alpha that contribute to the toxicity of treatment. Soluble TNF-alpha is released at high levels in cytokine release syndrome and is a mediator of toxicity by CAR T cell therapy. In such cases, administration of the TNF-alpha antibodies encompassed herein has a beneficial dual effect, namely, selective deletion of TNF-alpha mutant-expressing cells, as well as neutralization of soluble TNF-alpha that causes toxicity. Accordingly, embodiments of the present disclosure include methods of precluding or reducing the severity of cytokine release syndrome in an individual who has received or has previously received adoptive cell therapy in which the cells express a non-secreted TNF-alpha mutant, the method comprising providing an effective amount of an agent that binds the non-secreted TNF-alpha mutant, the agent, in the individual, (a) removing at least a portion of the cells of the cell therapy; (b) reducing the level of soluble TNF-alpha.

[0121] Embodiments of the present disclosure include methods of reducing the impact of cytokine release syndrome in an individual who has received or is receiving cell therapy using cells that express a non-secreted TNF-alpha mutant, the method comprising providing an effective amount of one or more agents that bind the mutant, the agents, in the individual, (a) removing at least a portion of the cells of the cell therapy; (b) reducing the level of soluble TNF1-alpha.

[0122] When the need arises to use a TNF-alpha suicide gene, an individual is provided with an effective amount of one or more inhibitors that can inhibit the TNF-alpha variant on the surface of cells (e.g., by directly binding to the TNF-alpha variant). In some embodiments, those inhibitors can be provided systemically and / or locally to the individual. The inhibitor can be a polypeptide (e.g., an antibody), a nucleic acid, a small molecule (e.g., a xanthine derivative), a peptide, or a combination thereof. In specific embodiments, those antibodies are FDA-approved. When the inhibitor is an antibody, the inhibitor can be a monoclonal antibody in at least some cases. When a mixture of antibodies is used, one or more of the antibodies in the mixture can be a monoclonal antibody. Examples of small molecule TNF-alpha inhibitors include small molecules such as those described in U.S. Patent No. 5,118,500, which is hereby incorporated by reference in its entirety. Examples of polypeptide TNF-alpha inhibitors include polypeptides such as those described in U.S. Patent No. 6,143,866, which is hereby incorporated by reference in its entirety.

[0123] In certain embodiments, at least one antibody is used to target a TNF-alpha variant that triggers its activity as a suicide gene. Examples of antibodies include, for example, at least adalimumab, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, golimumab, infliximab, infliximab-dyyb, or mixtures thereof.

[0124] Embodiments of the present disclosure include a method of reducing the risk of toxicity of cell therapy to an individual by modifying the cells of the cell therapy to express a non-secreted TNF-alpha variant. The cell therapy is, in specific embodiments, a cell therapy for cancer and can include engineered receptors that target an antigen including a cancer antigen.

[0125] In certain embodiments, in addition to the NK cell therapy of the invention of the present disclosure, additional treatment for the medical condition may have been, may be, and / or may be scheduled to be provided to the individual. If the medical condition is cancer, the individual may be provided with one or more of surgery, radiation therapy, immunotherapy (other than the cell therapy of the present disclosure), hormone therapy, gene therapy, chemotherapy, and the like. IV. Vectors

[0126] The CD70-targeted CAR can be delivered to recipient immune cells by any suitable vector, including viral vectors or non-viral vectors. Examples of viral vectors include, but are not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors. Examples of non-viral vectors include, but are not limited to, plasmids, transposons, lipids, nanoparticles, and the like.

[0127] If the immune cells are transduced with a vector encoding a CD70-targeted receptor and transduction of another gene (e.g., a suicide gene and / or a cytokine and / or a freely selectable therapeutic gene product) into the cells is also required, the CD70-targeted receptor, the suicide gene, the cytokine, and the freely selectable therapeutic gene may or may not be included on the same vector or together with the same vector. In some cases, the CD70-targeted CAR, the suicide gene, the cytokine, and the freely selectable therapeutic gene are expressed from the same vector molecule such as the same viral vector molecule. In such cases, the expression of the CD70-targeted CAR, the suicide gene, the cytokine, and the freely selectable therapeutic gene may or may not be controlled by the same regulatory element. When the CD70-targeted CAR, the suicide gene, the cytokine, and the freely selectable 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 than once on the same vector). A. General embodiments

[0128] One of ordinary skill in the art will be fully equipped with the ability to construct vectors 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 the antigen receptors of the present disclosure. 1. Regulatory elements

[0129] The expression cassettes included in the vectors useful in the present disclosure particularly include a eukaryotic transcription promoter operably linked to a protein coding sequence, a splice signal including intervening sequences, and a transcription termination / polyadenylation sequence (in the 5' to 3' direction). Promoters and enhancers that control the transcription of genes encoding proteins in eukaryotic cells can include multiple genetic elements. Cellular machinery can collect and integrate the regulatory information carried by each element, thereby enabling different genes to exhibit different patterns of transcriptional control, often in complex patterns. Examples of promoters used in the context of the present disclosure include, for example, constitutive promoters, inducible promoters, and tissue-specific promoters. When the vector is utilized for the production of cancer therapy, the promoter may be effective under hypoxic conditions. 2. Promoter / Enhancer

[0130] The expression constructs provided herein include a promoter that drives the expression of antigen receptors and other cistron gene products. A promoter generally includes a sequence that functions to specify the position of the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters, such as the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 late gene, the TATA box is absent and discontinuous elements that overlap the start site itself help to fix the location of initiation. Additional promoter elements control the frequency of transcription initiation. Usually, these are located in regions upstream of the start site, although some promoters have been shown to include 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 transcription reading frame is placed "downstream" (i.e., 3') of the selected promoter. An "upstream" promoter stimulates the transcription of DNA and promotes the expression of the encoded RNA.

[0131] The spacing between promoter elements is often flexible, and the function of the promoter is conserved even when the elements are inverted or moved relative to each other. In the tk promoter, for example, the spacing between promoter elements can be increased by up to 50 bp before activity begins to decline. Depending on the promoter, individual elements may be thought to function either cooperatively or independently to activate transcription. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0132] A promoter can be a promoter that is naturally associated with a nucleic acid sequence, such as one that may be obtained by isolating the 5' non-coding sequence located upstream of a coding segment and / or exon. Such a promoter can be referred to as an "endogenous" promoter. Similarly, an enhancer can be an enhancer that is naturally associated with a nucleic acid sequence and is located downstream or upstream of that sequence. Alternatively, certain advantages can be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not naturally associated with a nucleic acid sequence in its natural environment. Also, a recombinant or heterologous enhancer refers to an enhancer that is not naturally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers from other genes, as well as promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, and promoters or enhancers that contain mutations that "do not occur naturally," i.e., that alter the various elements of the transcriptional control regions and / or expression. For example, among the most commonly used promoters in recombinant DNA construction are the β-lactamase (penicillinase), lactose, and tryptophan (trp-) promoter systems. In addition to synthesizing the nucleic acid sequences of promoters and enhancers, recombinant cloning and / or PCR TMUsing nucleic acid amplification techniques including, but not limited to, those disclosed herein, a sequence can be generated in connection with the compositions disclosed herein. Additionally, regulatory sequences that direct transcription and / or expression of a sequence within an organelle other than the nucleus (e.g., mitochondria, chloroplasts, etc.) are likewise contemplated for use.

[0133] Of course, it is important to use promoters and / or enhancers that effectively direct the expression of a DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those of ordinary skill in the art of molecular biology are generally aware of the use of combinations of promoters, enhancers, and cell types for protein expression (see, e.g., Sambrook et al. 1989, incorporated herein by reference). The promoter used can be a constitutive promoter, a tissue-specific promoter, an inducible promoter, and / or a promoter useful under appropriate conditions that directs high-level expression of an introduced DNA segment (e.g., a promoter useful in the large-scale production of recombinant proteins and / or recombinant peptides). The promoter can be heterologous or endogenous.

[0134] Furthermore, any combination of promoters / enhancers (e.g., according to the Eukaryotic Promoter Data Base EPDB via the world wide web at epd.isb-sib.ch / ) can also be used to drive expression. The use of a T3, T7, or SP6 cytoplasmic expression system is another viable embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters when an appropriate bacterial polymerase is provided as part of the delivery complex or as an additional genetic expression construct.

[0135] Non-limiting examples of promoters include early viral promoters or late viral promoters (e.g., SV40 early or late promoter, cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus (RSV) early promoter); eukaryotic cell promoters (e.g., beta-actin promoter, GADPH promoter, metallothionein promoter); and tandem response element promoters (e.g., cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA) and response element promoter near the minimal TATA box (tre)). It is also possible to use the human growth hormone promoter sequence (e.g., the human growth hormone minimal promoter described in Genbank®, accession number X05244, nucleotides 283-341) or the mouse mammary tumor promoter (available from ATCC®, Cat. No. ATCC45007). 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, however, any other promoter useful for driving the expression of a therapeutic gene is applicable to the practice of the present disclosure.

[0136] 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, that act in cis and regardless of their orientation, and have the ability to act over relatively long distances (up to several kilobases away from the target promoter). However, since an enhancer can also function proximal to a given promoter, the function of an enhancer is not necessarily limited to such long distances. 3. Initiation Signals and Linked Expression

[0137] For efficient translation of the coding sequence, specific start signals may also be used in the expression constructs provided by the present disclosure. These signals include the ATG start codon or adjacent sequences. There may be a need to provide exogenous translation control signals (including the ATG start codon). One of ordinary skill in the art will be able to readily determine this and provide the necessary signals. It is well known that in order to ensure translation of the entire insert, the start codon must be "in-frame" with the reading frame of the desired coding sequence. The exogenous translation control signals and start codons can be either natural or synthetic. Expression efficiency can be enhanced by including appropriate transcriptional enhancer elements.

[0138] In certain embodiments, the use of an internal ribosome entry site (IRES) element is used to generate a multi-gene message, i.e., a polycistronic message. The IRES element bypasses the ribosome scanning model of 5'-methylated cap-dependent translation and can initiate translation at an internal site. IRES elements from two members of the Picornaviridae family (poliovirus and encephalomyocarditis), as well as IRESs from mammalian messages, have been reported. The IRES element can be linked to a heterologous open reading frame. Multiple open reading frames, each separated by an IRES, can be transcribed together to generate a polycistronic message. Thanks to the IRES element, each open reading frame can be accessible to ribosomes for efficient translation. It is also possible to efficiently express multiple genes using a single promoter / enhancer to transcribe a single message.

[0139] Furthermore, certain 2A sequence elements can be used to effect linked or co-expression of genes within the constructs provided by this disclosure. For example, cleavage sequences can be used to co-express genes by linking open reading frames to form a single cistron. 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 certain embodiments, in a single vector, the plurality of 2A sequences are not identical, although in alternative embodiments, the same vector utilizes two or more of the same 2A sequences. Examples of 2A sequences are provided in U.S. Patent Application Publication No. 2011 / 0065779, which is hereby incorporated by reference in its entirety. 4. Origin of replication

[0140] To propagate the vector in a host cell, the vector can contain one or more origins of replication (often referred to as "ori"), e.g., a specific nucleic acid sequence at which replication is initiated, such as the oriP of EBV as described above or a nucleic acid sequence corresponding to a genetically engineered oriP that may be similar or enhanced in function in programming. Alternatively, the origin of replication of other viruses that replicate episomally as described above, or an autonomous replication sequence (ARS), can be used. e. Selectable and screenable markers

[0141] In some embodiments, NK cells comprising the CD70-targeting receptor constructs of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such markers impart identifiable changes to the cells that allow cells containing the expression vector to be readily identified. Generally, a selectable marker is a marker that confers a property that enables selection. A positive selectable marker is a marker that enables selection by virtue of the presence of that marker, and a negative selectable marker is a marker whose presence inhibits selection. An example of a positive selectable marker is a drug resistance marker.

[0142] Typically, inclusion of a drug selection marker aids in the cloning and identification of transformants. For example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers that confer phenotypes that allow discrimination of transformants based on the execution of conditions, other types of markers are contemplated, including screenable markers such as GFP, which are based on colorimetric assays. Alternatively, enzymes that can be screened as negative selectable markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT), can be utilized. Those of skill in the art will likely also be aware of methods of using immunological markers, perhaps in conjunction with FACS analysis. The marker used is thought not to be important so long as it can be expressed concomitantly with the nucleic acid encoding the gene product. Further examples of selectable and screenable markers are well known to those of skill in the art. B. Multicistronic vectors

[0143] In certain embodiments, the CD70-targeted receptor, a freely selectable suicide gene, a freely selectable cytokine, and / or a freely selectable therapeutic gene are expressed from a multi-cistronic vector (as used herein, the term "cistron" refers to a nucleic acid sequence from which a gene product can be produced). In a specific embodiment, the multi-cistronic vector encodes a CD70-targeted receptor, a suicide gene, and at least one cytokine, and / or an engineered receptor (e.g., a T cell receptor and / or an additional non-CD70-targeted CAR). In some cases, the multi-cistronic vector encodes at least one CD70-targeted CAR, at least one TNF-alpha variant, and at least one cytokine. The cytokine can be a specific type of cytokine, e.g., a human or mouse or cytokine of any species. In a specific case, the cytokine is IL15, IL12, IL2, IL18, and / or IL21.

[0144] In certain embodiments, the present disclosure provides a flexible modular system (as used herein, the term "modular" refers to each cistron or component of a cistron that allows for interchangeability, e.g., by removal and replacement of entire cistrons or components of a cistron, by using standard recombinant techniques) utilizing a polycistronic vector having the ability to express multiple cistrons at substantially the same level. This system can be used for cell engineering that enables combinatorial expression (including overexpression) of multiple genes. In specific embodiments, one or more of the genes expressed by this vector include one, two, or more antigen receptors. The multiple genes can include, but are not limited to, CAR, TCR, cytokines, chemokines, homing receptors, CRISPR / Cas9-mediated gene mutations, decoy receptors, cytokine receptors, chimeric cytokine receptors, etc. The vector can further include (1) one or more reporters, e.g., fluorescent reporters or enzyme reporters for cell assays and animal imaging; (2) one or more cytokines or other signaling molecules; and / or (3) a suicide gene.

[0145] In specific cases, the vector may contain at least four cistrons separated by any type of cleavage site, such as a 2A cleavage site. This vector may or may not be a Moloney murine leukemia virus (MoMLV or MMLV)-based vector that includes 3' and 5' LTRs together with a psi packaging sequence in a pUC19 backbone. This vector may contain three or more 2A cleavage sites and four or more cistrons containing multiple ORFs for gene exchange. This system allows for combinatorial overexpression of multiple genes (seven or more) adjacent to restriction enzyme recognition sites for rapid integration by subcloning, and this system also includes at least three 2A self-cleavage sites in some embodiments. Thus, this system enables the expression of multiple CARs, TCRs, signaling molecules, cytokines, cytokine receptors, and / or homing receptors. This system can also be applied to other viral vectors and non-viral vectors, including but not limited to lentivirus, adenovirus AAV, and non-viral plasmids.

[0146] The modular nature of the above system enables efficient subcloning of genes into each of the four cistrons in a polycistronic expression vector and also allows for gene exchange, such as for rapid testing. Gene exchange can be efficiently performed by restriction enzyme recognition sites strategically placed within the polycistronic expression vector.

[0147] Embodiments of the present disclosure include systems that utilize polycistronic vectors, at least a portion of which is modular, for example, by allowing removal and replacement of one or more cistrons (or components of one or more cistrons), for example, by utilizing one or more restriction enzyme sites that are specifically selected for identity and location to facilitate use of modules of the vector. This vector also has embodiments that confer the advantage of this vector that multiple cistrons are translated into a single polypeptide and processed into separate polypeptides, thereby expressing distinct gene products at substantially equimolar concentrations.

[0148] The vectors of the present disclosure are configured to obtain modularity such that one or more cistrons of the vector can be altered and / or one or more components of one or more specific cistrons can be altered. The vector can be designed to utilize 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 specific cistron.

[0149] Embodiments of the present disclosure include polycistronic vectors that include at least two, at least three, or at least four cistrons, each adjacent to one or more restriction enzyme sites, and at least one cistron encodes at least one antigen receptor. In some cases, two, three, four, or more cistrons are translated into a single polypeptide and cleaved into separate polypeptides, while in other cases, multiple cistrons are translated into a single polypeptide and cleaved into separate polypeptides. Adjacent cistrons on the vector may be separated by a self-cleavage site such as a 2A self-cleavage site. In some cases, each cistron expresses a separate polypeptide from the vector. In certain cases, adjacent cistrons on the vector are separated by an IRES element.

[0150] In certain embodiments, the present disclosure provides a system for cell manipulation that enables combinatorial expression (including overexpression) of multiple cistrons that can include, for example, one, two, or more antigen receptors. In certain embodiments, by using a polycistronic vector as described herein, the vector can generate equimolar levels of multiple gene products from the same mRNA. The multiple genes can 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 can further include one or more fluorescent or enzyme reporters, such as for cell assays and animal imaging. The vector can also include a suicide gene product for terminating the cells when the cells having the vector are no longer needed or become harmful to the provided host.

[0151] In certain embodiments of the present disclosure, at least one of the cistrons on the vector includes two or more modular components, and each of the modular components within the cistron is adjacent to one or more restriction enzyme sites. The cistron can include, for example, three, four, or five modular components. In at least some cases, the cistron encodes an antigen receptor having different portions of the receptor encoded by the corresponding modular components. The first modular component of the cistron can encode the antigen-binding domain of the receptor. Further, the second modular component of the cistron can encode the hinge region of the receptor. Further, the third modular component of the cistron can encode the transmembrane domain of the receptor. Further, the fourth modular component of the cistron can encode a first co-stimulatory domain. Further, the fifth modular component of the cistron can encode a second co-stimulatory domain. Further, the sixth modular component of the cistron can encode a signaling domain.

[0152] In certain embodiments of the present disclosure, two different cis - trons on a vector each encode a non - identical antigen receptor. Both antigen receptors can be encoded by a cis - tron containing two or more modular components, where each of the two antigen receptors is encoded by a separate cis - tron containing two or more modular components. The antigen receptor can be, for example, a chimeric antigen receptor (CAR) and / or a T - cell receptor (TCR).

[0153] In a specific embodiment, 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 can include the 5’LTR, 3’LTR and / or the ψ packaging element of Moloney murine leukemia virus (MMLV). In a specific case, the packaging is incorporated between the 5’LTR and the antigen receptor coding sequence. The vector may or may not include the pUC19 sequence. In some embodiments of the vector, at least one cis - tron encodes a cytokine (e.g., interleukin 15 (IL - 15), IL - 7, IL - 21, IL - 18, IL - 12 or IL - 2), a chemokine, a cytokine receptor and / or a homing receptor.

[0154] When a 2A cleavage site is used in the vector, the 2A cleavage site can include the P2A, T2A, E2A and / or F2A sites.

[0155] In addition to one cis - tron encoding the CD70 - targeted CAR, any cis - tron of the vector may contain a suicide gene. Any cis - tron of the vector may encode a reporter gene. In a specific embodiment, the first cis - tron encodes a suicide gene, the second cis - tron encodes the CD70 - targeted CAR, the third cis - tron encodes a reporter gene, and the fourth cis - tron encodes a cytokine. In a certain specific embodiment, the first cis - tron encodes a suicide gene, the second cis - tron encodes the CD70 - targeted CAR, the third cis - tron encodes a second CAR or another antigen receptor, and the fourth cis - tron encodes a cytokine. In a specific embodiment, different portions of the CD70 - targeted CAR and / or another receptor are encoded by corresponding modular components, the first component of the second cis - tron encodes an antigen - binding domain, the second component encodes a hinge and / or transmembrane domain, the third component encodes a co - stimulatory domain, and the fourth component encodes a signaling domain.

[0156] In a specific embodiment, at least one of the above - mentioned cis - trons encodes a suicide gene. In some embodiments, at least one of the above - mentioned cis - trons encodes a cytokine. In a certain specific embodiment, at least one cis - tron encodes the CD70 - targeted CAR. The cis - tron may or may not encode a reporter gene. In a certain specific embodiment, at least two cis - trons encode two different antigen receptors (e.g., CAR and / or TCR). The cis - tron may or may not encode a reporter gene.

[0157] In a particular arrangement of the genetic payload of the purpose, a single vector may contain a cistron encoding a CD70-targeted CAR and a cistron encoding a second antigen receptor that is not identical to the CD70-targeted receptor. In a specific embodiment, the first antigen receptor encodes a CD70-targeted CAR and the second antigen receptor encodes a TCR, or vice versa. In certain embodiments, a vector containing separate cistrons encoding a CD70-targeted CAR and a second antigen receptor also contains a third cistron encoding a cytokine or chemokine and a fourth cistron encoding a suicide gene. However, the suicide gene and / or cytokine (or chemokine) may not be present on the vector.

[0158] In certain embodiments, at least one cistron contains multiple components that are themselves modular. For example, one cistron may encode the gene products of multiple components, such as an antigen receptor having multiple parts. In a specific case, the antigen receptor is encoded from a single cistron, whereby a single polypeptide is ultimately produced. A cistron encoding multiple components may have multiple components separated by one, two, three, four, five or more restriction enzyme digestion sites (including one, two, three, four, five or more restriction enzyme digestion sites specific to the vector containing the cistron) (Figures 1A and 1B). In a specific embodiment, a cistron having multiple components is an antigen receptor having multiple corresponding parts, and each of the corresponding parts encodes an antigen receptor that confers a unique function to the receptor. In a specific embodiment, each or most of the components of a cistron with multiple components are separated by one or more restriction enzyme digestion sites specific to the vector, thereby allowing for the interchangeability of separate components if desired.

[0159] In a specific embodiment, each component of the cistron of a plurality of components corresponds to a different part of the encoded antigen receptor, such as a CD70-targeted CAR. In an illustrative embodiment, component 1 may encode the CD70 antigen-binding domain of the receptor; component 2 may encode the hinge domain of the receptor; component 3 may encode the transmembrane domain of the receptor; component 4 may encode the co-stimulatory domain of the receptor, and component 5 may encode the signaling domain of the receptor. In a specific embodiment, the CD70-targeted CAR may include one or more co-stimulatory domains, and each of the co-stimulatory domains is separated by unique restriction enzyme digestion sites such that the co-stimulatory domains within the receptor are interchangeable.

[0160] In a specific embodiment, there is a polycistronic vector having four separate cistrons, where adjacent cistrons are separated by 2A cleavage sites, but in a specific embodiment, instead of the 2A cleavage sites, there are elements (e.g., IRES sequences) that directly or indirectly generate separate polypeptides from the cistrons. For example, there may be a case where four separate cistrons are separated by three 2A peptide cleavage sites, and each cistron has restriction enzyme recognition sites (X 1 , X 2 etc.) adjacent to each end of the cistron that allow for interchangeability of the specific cistron (e.g., interchangeability with another cistron or other types of sequences) when using standard recombinant techniques. In a specific embodiment, the restriction enzyme sites adjacent to each cistron are unique to the vector to facilitate recombination, but in an alternative embodiment, the restriction enzyme sites are not unique to the vector.

[0161] In certain embodiments, the vector provides a unique second level of modularity by enabling interchangeability within a particular cistron (including interchangeability within multiple components of a particular cistron). The multiple components of a particular cistron can be separated by one or more restriction enzyme sites (including those unique to the vector) to enable interchangeability of one or more components within that cistron. As an example, cistron 2 can include five separate components, although there may be two, three, four, five, six or more components per cistron. As an example, the vector has a cistron 2 with five components separated by unique restriction enzyme sites X 9 , X 10 , X 11 , X 12 , X 13 and X 14 such that standard recombination can swap different components 1, 2, 3, 4 and / or 5. In some cases, there may be multiple restriction enzyme sites between different components (which may or may not be unique, although one or more are unique), and there may or may not be a sequence between the multiple restriction enzyme sites. In certain embodiments, all components encoded by a cistron are designed for interchangeability. In certain cases, one or more components of a cistron are designed for interchangeability, while one or more other components of that cistron may not be designed for interchangeability.

[0162] In a specific embodiment, the cistron encodes a CD70-targeted CAR molecule having multiple components. For example, cistron 2 may include an array encoding a CD70-targeted CAR molecule having distinct components represented by component 1, component 2, component 3, etc. The CAR molecule may include two, three, four, five, six, seven, eight or more interchangeable components. In a specific example, component 1 encodes a CD70scFv; component 2 encodes a hinge; component 3 encodes a transmembrane domain; component 4 encodes a co-stimulatory domain (for exchange, there may also be a component 4' encoding a second or more co-stimulatory domains adjacent to a restriction enzyme recognition site); component 5 encodes a signaling domain. In a specific example, component 1 encodes a CD70scFv; component 2 encodes an IgG1 hinge and / or transmembrane domain; component 3 encodes CD28; component 4 encodes CD3 zeta.

[0163] One of ordinary skill in the art will recognize that in the design of vectors, various cistrons and components must be arranged so as to be maintained in-frame if necessary.

[0164] In a specific example, cistron 1 encodes a suicide gene; cistron 2 encodes a CD70-targeted CAR; cistron 3 encodes a reporter gene; cistron 4 encodes a cytokine; component 1 of cistron 2 encodes a CD70scFv; component 2 of cistron 2 encodes an IgG1 hinge; component 3 of cistron 2 encodes CD28; component 4 encodes CD3 zeta.

[0165] The restriction enzyme site can be of any type and can contain any number of bases (e.g., 4 - 8 bases) in its recognition site. The number of bases in the recognition site can be at least 4, 5, 6, 7, 8 or more. When the site is cleaved, it can generate blunt ends or sticky ends. The restriction enzyme can be, for example, type I, II, III or IV. The restriction enzyme site can be obtained from available databases such as the Integrated relational Enzyme database (IntEnz) or BRENDA (The Comprehensive Enzyme Information System).

[0166] Exemplary vectors can be circular and, by convention, position 1 (at the 12 o'clock position at the top of the circle, with the remaining sequence in the clockwise direction) is set as the starting point of the 5’ LTR.

[0167] In embodiments utilizing the self - cleaving 2A peptide, the 2A peptide can be a viral oligopeptide 18 - 22 amino acids (aa) in length that mediates the “cleavage” of a translating polypeptide in eukaryotic cells. The designation “2A” refers to a specific region of the viral genome, and various viral 2As are generally named after the viruses from which they are derived. The first 2A discovered was F2A (foot - and - mouth disease virus), and subsequently E2A (equine rhinitis A virus), P2A (porcine teschovirus - 1 2A) and T2A (thosea asigna virus 2A) were also identified. The mechanism of 2A - mediated “self - cleavage” was discovered to be that the ribosome skips the formation of the glycyl - prolyl peptide bond at the C - terminus of 2A.

[0168] In a specific case, the vector can be a γ-retrovirus transfer vector. The retrovirus transfer vector may include a backbone based on a plasmid such as the pUC19 plasmid (a large fragment (2.63 kb) between the HindIII restriction enzyme site and the EcoRI restriction enzyme site). The backbone may have viral components derived from Moloney murine leukemia virus (MoMLV) including the 5’ LTR, the ψ packaging sequence, and the 3’ LTR. The LTR is a terminal repeat sequence found on either side of the provirus of a retrovirus and, in the case of a transfer vector, is placed on both sides of the genetic payload of interest such as the CD70-targeted CAR and related components. The ψ packaging sequence, which is the target site for packaging by the nucleocapsid, is also incorporated in a cis-sandwiched form between the 5’ LTR and the CAR coding sequence. Thus, the basic structure of an example of a transfer vector can be configured as, for example, pUC19 sequence - 5’ LTR - ψ packaging sequence - genetic payload of interest - 3’ LTR - pUC19 sequence. This system can also be applied to other viral vectors and non-viral vectors including, but not limited to, lentivirus, adenovirus AAV, and non-viral plasmids. V. Cells

[0169] The present disclosure encompasses any type of immune cell or stem cell having at least one vector that encodes a CD70-targeted receptor and may also encode at least one cytokine and / or at least one suicide gene. In some cases, various vectors encode the suicide gene and / or cytokine, while encoding the CAR. Immune cells including NK cells can be derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), bone marrow, or mixtures thereof. NK cells can be derived from cell lines such as, but not limited to, NK-92 cells. NK cells can be umbilical cord blood mononuclear cells such as CD56+ NK cells.

[0170] The present disclosure encompasses any type of immune cells or other cells including conventional T cells, gamma-delta T cells, NKT and invariant NKT cells, regulatory T cells, macrophages, B cells, dendritic cells, mesenchymal stromal cells (MSCs) or mixtures thereof.

[0171] In some cases, those cells are expanded in the presence of an effective amount of universal antigen-presenting cells (UAPCs) (including any suitable ratio). Those cells can be cultured with UAPCs 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, NK cells are expanded in the presence of IL-2 at a concentration of, for example, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 100 to 500, 100 to 400, 100 to 300, 100 to 200, 200 to 500, 200 to 400, 200 to 300, 300 to 500, 300 to 400 or 400 to 500 U / mL.

[0172] Those NK cells can be injected immediately or stored after genetic modification by a vector. In certain embodiments, after genetic modification, within about 1, 2, 3, 4, 5 days or later after gene introduction into the cells, those cells can be expanded ex vivo as a bulk population for several days, weeks or months. In further embodiments, the transfectants are cloned and clones showing the presence of an expression cassette or plasmid retained in a single integrated or episomal form and the expression of the CD70-targeted CAR are expanded ex vivo. The clones selected for expansion show the ability to specifically recognize and lyse target cells expressing CD70. The recombinant immune cells can 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-21, etc.). The recombinant immune cells can be expanded by stimulation with artificial antigen-presenting cells. In further embodiments, the genetically modified cells can be cryopreserved.

[0173] Embodiments of the present disclosure include cells expressing one or more CD70-targeted CARs and one or more suicide genes as included herein. The NK cells, in specific embodiments, contain a recombinant nucleic acid encoding one or more CD70-targeted CARs and one or more engineered non-secreted membrane-bound TNF-alpha mutant polypeptides. In specific embodiments, the cells also contain a nucleic acid encoding one or more therapeutic gene products in addition to the expression of one or more CD70-targeted CARs and TNF-alpha mutant polypeptides.

[0174] The above cells can be obtained directly from an individual or from a depository or other storage facility. The above cells as a treatment can be autologous or allogeneic to the individual to whom those cells are provided as a treatment.

[0175] The above cells can be derived from an individual in need of treatment for a certain medical condition and, after being manipulated (e.g., using standard techniques for transduction and expansion for adoptive cell therapy) to express a CD70-targeted CAR, a freely selectable suicide gene, a freely selectable cytokine, and a freely selectable therapeutic gene product, can be returned to the individual from which they originally originated. In some cases, those cells are stored for later use in that individual or another individual.

[0176] The above immune cells can be included in a cell population, and the population can be predominantly composed of cells transduced with one or more CD70-targeted receptors and / or one or more suicide genes and / or one or more cytokines. The cell population can contain 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% immune cells transduced with one or more CD70-targeted receptors and / or one or more suicide genes and / or one or more cytokines. The one or more CD70-targeted receptors and / or one or more suicide genes and / or one or more cytokines can be separate polypeptides.

[0177] The above immune cells can be produced using one or more CD70-targeting receptors and / or one or more suicide genes and / or one or more cytokines so as to be modular with respect to a specific purpose. For example, for commercial distribution etc., cells expressing a CD70-targeting CAR and / or one or more suicide genes and / or one or more cytokines (or distributed together with nucleic acids encoding mutants for subsequent transduction) can be produced, and depending on the intended purpose, the user may modify them to express one or more other target genes (including therapeutic genes). For example, an individual interested in the treatment of CD70-positive cells including CD70-positive cancer can obtain or produce suicide gene-expressing cells (or heterologous cytokine-expressing cells) and modify them to express a receptor containing a CD70-specific scFv, or vice versa.

[0178] In certain embodiments, NK cells are used and the genome of the transduced NK cells expressing one or more CD70-targeting 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 a specific embodiment, the genome is modified, for example, by CRISPR gene editing. The genome of those cells can be modified to enhance the effectiveness of those cells for any purpose. IV. Gene Editing of CD70-Specific CAR Cells

[0179] In certain embodiments, cells comprising at least an engineered receptor specific for CD70 are gene-edited to modify the expression of one or more endogenous genes in the cells. In a specific case, CD70-specific CAR cells are modified such that the expression level of one or more endogenous genes, such as inhibition of the expression of one or more endogenous genes (sometimes referred to as knockout), is reduced. Such cells may or may not be expanded.

[0180] In certain cases, one or more endogenous genes of the above CD70-specific CAR cells are modified, such as being disrupted in expression, where the expression is partially or completely reduced. In a specific case, one or more genes are knocked down or knocked out using the process of the present disclosure. In a specific case, multiple genes are knocked down or knocked out, which may or may not be performed in the same step in their production. The genes edited in the CD70-specific CAR cells can be of any kind, but in a specific embodiment, those genes are genes whose gene products inhibit the activity and / or proliferation of CD70-specific CAR cells (including, for example, CD70-specific CAR NK cells derived from umbilical cord blood). In a specific case, the genes edited in the CD70-specific CAR cells enable the CD70-specific CAR cells to function more effectively in the tumor microenvironment. In a specific case, those genes are one or more of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5 and CD7. In a specific embodiment, the TGFBR2 gene is knocked out or knocked down in the CD70-specific CAR cells.

[0181] In some embodiments, gene editing is performed using one or more DNA-binding nucleic acids, such as modifications via RNA-guided endonucleases (RGENs). For example, the modifications can be performed using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins; in some embodiments, CpF1 is used instead of Cas9. Generally, a “CRISPR system” collectively refers to CRISPR-associated (“Cas”) genes (including sequences encoding Cas genes), tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (which in the context of an endogenous CRISPR system include “direct repeats” and partial direct repeats processed by tracrRNA), guide sequences (also referred to as “spacers” in the context of an endogenous CRISPR system), and / or other sequences from the CRISPR locus and transcripts and other elements involved in or directing the expression or activity of such transcripts.

[0182] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA that binds sequence-specifically to DNA, and a Cas protein (e.g., Cas9) having nuclease activity (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a type I, type II, or type III CRISPR system, and can be derived, for example, from a particular organism (e.g., Streptococcus pyogenes) that includes an endogenous CRISPR system.

[0183] In some embodiments, a Cas nuclease and a gRNA (including a fusion of a crRNA specific for a target sequence and a predefined tracrRNA) are introduced into a cell. Generally, the target site at the 5' end of the gRNA targets the Cas nuclease to its target site, e.g., a gene, by complementary base pairing. The target site can be selected based on the position immediately 5' of a protospacer adjacent motif (PAM) sequence (e.g., typically NGG or NAG). In this regard, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Generally, the CRISPR system is characterized by elements that promote the formation of the CRISPR complex at the site of the target sequence. Usually, the "target sequence" generally refers to a sequence that is designed such that the guide sequence has complementarity, and hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. Complete complementarity is not necessarily required as long as there is sufficient complementarity to cause hybridization and promote the formation of the CRISPR complex.

[0184] The CRISPR system can induce disruptions or modifications as discussed herein following a double-strand break (DSB) at the target site. In other embodiments, a Cas9 variant considered a "nickase" is used to introduce a single-strand nick at the target site. For example, to improve specificity, a pair of nickases can be used, each of which is directed by a pair of different gRNAs that target the sequence, and when the nicks are introduced simultaneously, a 5' overhang is introduced. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor or transcriptional activator, to affect gene expression.

[0185] The target sequence can include any polynucleotide, such as a DNA polynucleotide or an RNA polynucleotide. The target sequence can be located in the nucleus or cytoplasm of a cell, such as within an organelle of the cell. Generally, a sequence or template that can be used for recombination into a targeted locus containing the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In some embodiments, an exogenous template polynucleotide may be referred to as an editing template. In some embodiments, the recombination is homologous recombination.

[0186] Typically, in the context of an endogenous CRISPR system, cleavage of one or both strands occurs at or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 base pairs or more of the target sequence) by formation of a CRISPR complex (including a guide sequence that hybridizes to the target sequence and complexes with one or more Cas proteins). A tracr sequence that can include all or part of a wild-type tracr sequence (e.g., about 20 nucleotides, about 26 nucleotides, about 32 nucleotides, about 45 nucleotides, about 48 nucleotides, about 54 nucleotides, about 63 nucleotides, about 67 nucleotides, about 85 nucleotides or more or more than about 20 nucleotides, more than about 26 nucleotides, more than about 32 nucleotides, more than about 45 nucleotides, more than about 48 nucleotides, more than about 54 nucleotides, more than about 63 nucleotides, more than about 67 nucleotides, more than about 85 nucleotides or more) or consists of them can also form part of a CRISPR complex, such as by hybridization along at least part of the tracr sequence to all or part of a tracr mate sequence operably linked to the guide sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence (e.g., at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned) to hybridize and participate in the formation of the CRISPR complex.

[0187] One or more vectors that drive the expression of those elements of the CRISPR system can be introduced into a cell such that the expression of one or more elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. Also, the components can be delivered to the cell as proteins and / or RNAs. For example, a Cas enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in a single vector, and one or more additional vectors provide any components of the CRISPR system not included in the first vector. The vector can include one or more insertion sites (also referred to as "cloning sites"), such as restriction endonuclease recognition sequences. In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences within the cell.

[0188] The vector can include a regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of the Cas9 protein of S. pyogenes can be found in the SwissProt database under accession number Q99ZW2.

[0189] The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumoniae). In certain cases, CpF1 can be used as an endonuclease in place of Cas9. The CRISPR enzyme can direct cleavage of one or both strands at a position of a target sequence, such as within the target sequence and / or within the complementary strand of the target sequence. The vector can encode a CRISPR enzyme that is mutated compared to the corresponding wild-type enzyme, and the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, a substitution of aspartic acid to alanine (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves a single strand). In some embodiments, the Cas9 nickase can be used in combination with a guide sequence, e.g., two guide sequences that each target the sense and antisense strands of its DNA target. This combination can be used to nick both strands and to induce NHEJ or HDR.

[0190] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell can be a cell of a particular organism (e.g., a mammal including, but not limited to, human, mouse, rat, rabbit, dog or non-human primate) or a cell derived from those organisms. Generally, codon optimization refers to the process of modifying a nucleic acid sequence so that expression is enhanced in a target host cell by replacing at least one codon of a native sequence with a codon that is more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Different species exhibit a particular bias for a particular codon of a particular amino acid. Codon bias (differences in codon usage frequency among organisms) often correlates with the translation efficiency of messenger RNA (mRNA), and that translation efficiency is thought to depend, inter alia, on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The fact that the selected tRNA is dominant within the cell usually reflects that it is the codon most frequently used in peptide synthesis. Thus, based on codon optimization, genes can be adapted for optimal gene expression in a given organism.

[0191] Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity to the target polynucleotide sequence to hybridize thereto and sufficient complementarity to direct sequence-specific binding of the CRISPR complex to that target sequence. In some embodiments, the degree of complementarity between the guide sequence and the corresponding target sequence is about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99% or more, or greater than about 50%, greater than about 60%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 97%, greater than about 99% or more when optimally aligned using a suitable alignment algorithm.

[0192] Optimal alignment can be determined using any suitable algorithm for aligning sequences. Non-limiting examples of such algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0193] The CRISPR enzyme can be part of a fusion protein that includes one or more heterologous protein domains. The CRISPR enzyme fusion protein can include any additional protein sequence and, optionally, a linker sequence between any two domains. Examples of protein domains that can be fused to the CRISPR enzyme include epitope tags, reporter gene sequences, and proteins having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcription termination factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity, but are not limited thereto. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP), but are not limited thereto. The CRISPR enzyme can be fused to a gene sequence encoding a protein or a fragment of a protein that binds to a DNA molecule or to other cellular molecules (including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions). Additional domains that can form part of the fusion protein containing the CRISPR enzyme are described in U.S. Patent Application Publication No. 20110059502, which is incorporated herein by reference. VII. Treatment Methods

[0194] In various embodiments, cells expressing endogenous CD70 on their surface are targeted for the purpose of improving a medical condition in an individual having the medical condition, or for the purpose of reducing and / or delaying the risk or severity of the medical condition in the individual. In specific cases, cancer cells expressing endogenous CD70 are targeted for the purpose of killing the cancer cells. In other cases, CD70 is targeted as CD70-positive cells, but the CD70-positive cells are not cancer cells. In such cases, the CD70-positive cells can be immune regulatory cells such as regulatory T cells. Targeting and depletion of CD70+ regulatory T cells can further enhance cancer immunotherapy by eliminating the immunosuppressive effect of this cell subset. Thus, in a specific embodiment, there is a method of reducing immunosuppression in cancer treatment by providing an effective amount of cells that target CD70 as described herein.

[0195] CD70-targeted CAR constructs, nucleic acid sequences, vectors, immune cells, etc., as contemplated herein, and / or pharmaceutical compositions containing the same, are used for the prevention, treatment or recovery of cancerous diseases such as neoplastic diseases. In certain embodiments, the pharmaceutical compositions of the present disclosure may be particularly useful in the prevention, recovery and / or treatment of cancers including cancers that express CD70, which may or may not be, for example, solid tumors.

[0196] The immune cells in which the CD70-targeted receptor is used can be NK, T cells, gamma-delta T cells, or NKT or invariant NKT (iNKT), or in certain embodiments, inducible NKT cells engineered for cell therapy in mammals. In such cases where those cells are NK cells, the NK cell therapy can be of any type, and those NK cells can also be of any type. In a specific embodiment, those cells are NK cells engineered to express one or more CD70-targeted CARs and / or one or more suicide genes and / or one or more cytokines. In a specific embodiment, those cells are NK cells transduced with a CD70-targeted CAR.

[0197] In certain embodiments, the present disclosure contemplates, in part, CD70CAR-expressing cells, CD70-targeted CAR constructs, CD70-targeted CAR nucleic acid molecules, and CD70-targeted CAR vectors, which can be administered alone or in any combination using standard vectors and / or gene delivery systems, and in at least some aspects, together with a pharmaceutically acceptable carrier or excipient. In certain specific embodiments, after administration, the nucleic acid molecule or vector can be stably integrated into the genome of the subject.

[0198] In a specific embodiment, a viral vector that is specific for a particular cell or tissue and that persists in NK cells can be used. Suitable pharmaceutical carriers and excipients are well known in the art. The compositions prepared according to the present disclosure can be used to prevent or treat or delay the diseases specified above.

[0199] Furthermore, the present disclosure relates to a method for preventing, treating, or restoring a neoplastic disease, the method comprising administering to a subject in need thereof an effective amount of cells expressing a CD70-targeted CAR, nucleic acid sequence, vector, produced by a process as contemplated herein and / or as contemplated herein.

[0200] Exemplary indications expected for the administration of a composition of CD70-targeted CAR cells are cancerous diseases, including neoplastic diseases (e.g., including B-cell malignancies, multiple myeloma, breast cancer, glioblastoma, renal cancer, pancreatic cancer, or lung cancer). Exemplary indications for the administration of a composition of CD70-targeted CAR cells are cancerous diseases including any malignant tumor that expresses CD70. Administration of the compositions of the present disclosure is useful for all stages (I, II, III, or IV) and types of cancer (e.g., including minimal residual disease, early-stage cancer, advanced cancer, and / or metastatic cancer and / or treatment-resistant cancer).

[0201] The present disclosure further encompasses co-administration protocols with other compounds that act via immune cells, such as bispecific antibody constructs, targeted toxins, or other compounds. Clinical regimens for co-administration of the compounds of the invention can include co-administration simultaneous with, prior to, or following the administration of other components. Specific combination therapies include chemotherapy, radiation, surgery, hormonal therapy, or other types of immunotherapy.

[0202] Embodiments relate to kits comprising a CD70-targeted CAR construct as defined herein, a nucleic acid sequence as defined herein, a vector as defined herein, and / or a host cell (e.g., an immune cell) as defined herein. Kits of the present disclosure are also contemplated to include, alone or together with additional drugs to be administered to an individual in need of a medical treatment or intervention, a pharmaceutical composition as described above in this specification. A. Pharmaceutical Composition

[0203] Also provided herein are pharmaceutical compositions and formulations comprising the transduced NK cells and a pharmaceutically acceptable carrier included herein. The transduced cells can be prepared by processes included herein and can be included in a medium suitable for transfer into an individual and / or a medium suitable for storage, such as cryopreservation, prior to transfer into the individual. Also provided herein are pharmaceutical compositions and formulations comprising NK cells and a pharmaceutically acceptable carrier that can be prepared by processes included herein.

[0204] Pharmaceutical compositions and formulations as described herein include an active ingredient (e.g., a cell) having a desired degree of purity in one or more freely selected pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences 22 ndIt can be prepared in the form of a lyophilized formulation or an aqueous solution by mixing with (edition, 2012). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations used, and such carriers include buffering agents (e.g., phosphoric acid, citric acid and other organic acids); antioxidants (including ascorbic acid and methionine); preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenolic alcohol, butyl alcohol or benzyl alcohol; alkyl parabens (e.g., methyl paraben or propyl paraben); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); polypeptides of low molecular weight (less than about 10 residues); proteins (e.g., serum albumin, gelatin or immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose or dextrin); chelating agents (e.g., EDTA); saccharides (e.g., sucrose, mannitol, trehalose or sorbitol); salt-forming counterions (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants (e.g., polyethylene glycol (PEG)), but are not limited thereto. Exemplary pharmaceutically acceptable carriers in the present specification further include interstitial drug dispersants, such as neutral and active soluble hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGP containing rHuPH20 and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is used in combination with one or more additional glycosaminoglycanases, such as chondroitinase. B. Combination Therapy

[0205] In certain embodiments, the compositions and methods of the present embodiments utilize an immune cell population (including an NK cell population) in combination with at least one additional therapy. The additional therapy can be radiotherapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nano-therapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy can be in the form of adjuvant therapy or neoadjuvant therapy.

[0206] In some embodiments, the additional therapy is the administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is the administration of a side effect limiting agent (e.g., an agent aimed at reducing the incidence and / or severity of the side effects of the treatment, such as an anti-nausea agent, etc.). In some embodiments, the additional therapy is radiotherapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiotherapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a treatment targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy can be one or more of the chemotherapeutic agents known in the art.

[0207] The immunotherapy of the present disclosure can be administered before, during, after, or in various combinations with additional cancer treatments such as immune checkpoint therapy. Those administrations can be performed at intervals ranging from simultaneous to several minutes, days, or weeks. In embodiments where immunocyte therapy is provided to a patient separately from an additional therapeutic agent, it is common to ensure that a significant period does not elapse between each delivery time so that the two compounds can still exert a beneficial combined effect on the patient. In such cases, it is contemplated that antibody therapy and anti-cancer therapy can be provided to the patient within about 12 to 24 or 72 hours of each other, and more particularly, within about 6 to 12 hours of each other. In some situations, when several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) have elapsed between each administration, it may be desirable to significantly extend the treatment period.

[0208] Various combinations can be used. In the case of the following examples, the immunocyte therapy is "A" and the anti-cancer therapy is "B".

[0209] A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / B

[0210] B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A

[0211] B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A

[0212] The administration of any compound or cell therapy of the present embodiment to a patient follows the general protocol for administering such a compound, taking into account toxicity to their active substances if any. Thus, in some embodiments, there is a step of monitoring the toxicity that may result from the combination therapy. 1. Chemotherapy

[0213] A variety of chemotherapeutic agents can be used in accordance with this embodiment. The term "chemotherapy" refers to treating cancer using drugs. "Chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. These agents or drugs are classified by their mode of activity within the cell, for example, by whether they affect the cell cycle and at which stage they affect the cell cycle. Alternatively, the agents can be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0214] Examples of chemotherapeutic agents include alkylating agents (e.g., thiotepa and cyclophosphamide); alkyl sulfonates (e.g., busulfan, improsulfan and piposulfan); aziridines (e.g., benzodopa, carboquone, meturedopa and uredopa); ethyleneimines and methylamelamines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine); acetogenins (particularly, bullatacin and bullatacinone); camptothecin (including topotecan, a synthetic analog); bryostatin; calicheamicin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including KW-2189 and CB1-TM1, synthetic analogs); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards (e.g., chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide and uracil mustard); nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine); antibiotics (e.g., enediyne antibiotics (e.g., calicheamicin, particularly, calicheamicin gamma1I and calicheamicin omegaI1)); dynemicin (including dynemicin A); bisphosphonates (e.g., clodronate); esperamicin;and neocarzinostatin chromophore and related chromoproteins, engomycin antibiotics chromophore, actinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, daunorubicin, daunomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (e.g., mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin and zorubicin; antimetabolites (e.g., methotrexate and 5-fluorouracil (5-FU)); folic acid analogs (e.g., denopterin, pteropterin and trimethoprim); purine analogs (e.g., fludarabine, 6-mercaptopurine, thiamiprine and thioguanine); pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine and floxuridine); androgens (e.g., calusterone, drostanolone propionate, epitestosterone, mepitiostane and testolactone); anti-adrenals (e.g., mitotane and trilostane); folic acid supplements (e.g., frolinic acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;Bisantrene; Edatraxate; Defofamine; Demecortin; Diazicon; Elformithine; Elliptinium acetate; Epothilone; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids (e.g., Maytansine and Ansamitocins); Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex; Razoxane; Rizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2’,2”-Trichlorotriethylamine; Trichothecins (especially, T-2 toxin, Verracurin A, Roridin A and Anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside (“Ara-C”); Cyclophosphamide; Taxoids, e.g., Paclitaxel and Docetaxel Gemcitabine; 6-Thioguanine; Mercaptopurine; Platinum coordination complexes (e.g., 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 inhibitor RFS2000; Difluoromethylornithine (DMFO); Retinoids (e.g., Retinoic acid); Capecitabine;Examples include carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids or derivatives of any of the above.; 2. Radiation therapy

[0215] Other widely used factors that cause DNA damage include γ-rays, X-rays and / or those commonly known as the directed delivery of radioisotopes to tumor cells. Other forms of DNA damage factors such as microwaves, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287) and UV irradiation are also contemplated. It is most likely that all of these factors will cause extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosomal assembly and maintenance. The range of X-ray doses is from a daily dose of 50 to 200 roentgens over a long period (3 to 4 weeks) to a single dose of 2,000 to 6,000 roentgens. The range of radioisotope doses varies widely and depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by tumor cells. 3. Immunotherapy

[0216] One of ordinary skill in the art will understand that additional immunotherapies can be used in combination with or in conjunction with the methods of the above embodiments. In the context of treating cancer, immunotherapeutic agents typically rely on using immune effector cells and immune effector molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is an example of such. The immune effector can be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody can function as a therapeutic effector alone or recruit other cells to actually affect cell killing. The antibody can also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector can be a lymphocyte having surface molecules that interact directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0217] Antibody-drug conjugates have emerged as an epoch-making approach in the development of cancer therapeutics. Cancer is one of the leading causes of death worldwide. Antibody-drug conjugates (ADCs) contain monoclonal antibodies (MAbs) covalently linked to a cytotoxic drug. This approach combines the high specificity of the MAb for an antigen target with a very potent cytotoxic drug to yield an "armed" MAb that delivers a payload (drug) to tumor cells having abundant levels of the antigen. Also, the targeted delivery of the drug reduces toxicity and improves the therapeutic index by minimizing exposure to normal tissues. Exemplary ADC drugs include ADCETRIS® (brentuximab vedotin) and KADCYLA® (trastuzumab emtansine or T-DM1), approved by the FDA in 2013. 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 are becoming increasingly dependent on the identification and validation of new targets suitable for this approach and the generation of targeting MAbs. Two criteria for ADC targets are increased / high levels of expression in tumor cells and robust internalization.

[0218] In one aspect of immunotherapy, tumor cells must have some marker that is susceptible to targeting, i.e., some marker that is not present on most other cells. There are many tumor markers, and any of these 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, and p155. An alternative aspect of immunotherapy is to combine an anti-cancer effect with an immune-stimulatory effect. There are also immune-stimulatory molecules such as cytokines like IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines like MIP-1, MCP-1, IL-8, and growth factors like FLT3 ligand.

[0219] Examples of immunotherapies currently under investigation include immunoadjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy such as interferon, and IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945); and monoclonal antibodies such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in combination with the antibody therapies described herein.

[0220] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. An immune checkpoint either enhances a signal (e.g., a costimulatory molecule) or attenuates a signal. Inhibitory immune checkpoints that can be targeted by blocking an immune checkpoint include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (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, the immune checkpoint inhibitor targets the PD-1 axis and / or CTLA-4.

[0221] The immune checkpoint inhibitor can be a drug such as a small molecule, a recombinant ligand or receptor, or in particular, an antibody such as a human antibody (e.g., International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof can be used, in particular, chimeric, humanized or human antibodies can be used. As those skilled in the art will appreciate, alternative and / or equivalent names can be used for certain antibodies described in the present disclosure. Such alternative and / or equivalent names are interchangeable in the context of the present disclosure. For example, it is known that pembrolizumab is also known by the alternative and equivalent names MK-3475 and lambrolizumab.

[0222] 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 embodiment, 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 embodiment, 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 embodiment, the PDL2 binding partner is PD-1. The antagonist can be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. US8735553, US8354509, and US8008449, which are all 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.

[0223] 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 comprising the extracellular portion or the PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody that can be used (disclosed in WO2006 / 121168). Pembrolizumab, also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an exemplary anti-PD-1 antibody (WO2009 / 114335). CT-011, also known as hBAT or hBAT-1, is also an anti-PD-1 antibody (WO2009 / 101611). AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor (WO2010 / 027827 and WO2011 / 066342).

[0224] Another immune checkpoint that can be targeted in 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 bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily expressed on the surface of helper T cells and transmits an inhibitory signal to T cells. CTLA4 resembles the T cell co-stimulatory protein CD28, and both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found in regulatory T cells and may be important for the function of those cells. Activation of T cells via the T cell receptor and CD28 results in high expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0225] 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.

[0226] An anti-human CTLA-4 antibody (or VH and / or VL domains derived therefrom) suitable for use in the present method can be produced using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (also known as tremelimumab; previously also known as ticilimumab, CP675,206), U.S. Patent No. 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 can be used in the methods disclosed herein. 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.

[0227] Exemplary anti-CTLA-4 antibodies are 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 chain CDRs and light chain CDRs or heavy chain VRs and light chain 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 the same epitope on CTLA-4 as the antibody described above 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 to the antibody described above (e.g., at least about 90%, 95%, or 99% variable region identity to ipilimumab).

[0228] Other molecules for modulating CTLA-4 include CTLA-4 ligands and CTLA-4 receptors (e.g., those described in U.S. Patent Nos. 5,844,905, 5,885,796, and International Patent Application Nos. WO1995001994 and WO1998042752, all of which are incorporated herein by reference), as well as immunoadhesins (e.g., those described in U.S. Patent No. 8,329,867, which is incorporated herein by reference). 4. Surgery

[0229] Approximately 60% of people with cancer undergo some type of surgery, including prophylactic surgery, diagnostic surgery or staging surgery, radical surgery, and palliative surgery. Radical surgery includes excision procedures that physically remove, excise, and / or destroy all or part of the cancerous tissue, and can be used in combination with other treatments (e.g., the procedures of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies). Tumor excision refers to physically removing at least part of the tumor. Surgical procedures include, in addition to tumor excision, laser surgery, cryosurgery, electrocautery, and microsurgery (Mohs surgery).

[0230] When excising part or all of the cancerous cells, tissue, or tumor, a cavity can form in the body. The treatment can be achieved by perfusion, direct injection, or topical application of that area by further anti-cancer therapies. Such treatments can 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 can also be treatments at various dosages. 5. Other agents

[0231] It is contemplated that other agents may be used in combination with certain aspects of the present embodiment to improve the therapeutic effect of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cell division inhibitors and differentiating agents, inhibitors of cell adhesion, agents that enhance the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. An increase in intercellular signaling by increasing the number of gap junctions can enhance the antiproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, a cell division inhibitor or differentiating agent may be used in combination with certain aspects of the present embodiment to improve the antiproliferative effectiveness of the treatment. An inhibitor of cell adhesion is contemplated to improve the effectiveness of the present embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents (e.g., the antibody c225) that enhance the sensitivity of hyperproliferative cells to apoptosis may be used in combination with certain aspects of the present embodiment to improve the effectiveness of the treatment. VIII. Kits of the Present Disclosure

[0232] Any composition described herein can be included in a kit. By way of non-limiting example, cells, reagents for making cells, vectors, and reagents for making vectors and / or their components can be included in the kit. In certain embodiments, NK cells may be included in the kit, which may or may not express a CD70-targeting receptor, a cytokine of choice, or a suicide gene of choice. Such a kit may or may not have one or more reagents for manipulating the 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 CD70-targeting CARs, suicide gene products, and / or cytokines can be included in the kit. Proteins such as cytokines or antibodies (including monoclonal antibodies) can be included in the kit. Nucleotides encoding components of the engineered CAR receptor can be included in the kit, along with the reagents for making it.

[0233] In certain aspects, the kit includes the NK cell therapy of the disclosure, and also another cancer treatment. In some cases, the kit includes, in addition to the cell therapy embodiment, a second cancer treatment (e.g., chemotherapy, hormonal therapy, and / or immunotherapy). The kit can be tailored to a particular cancer in an individual and include each respective second cancer treatment for that individual.

[0234] The kit may include a composition appropriately aliquoted of the present disclosure. The components of the kit may be packaged in an aqueous medium or in a lyophilized form. The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe or other container means into which the components can be placed, preferably can be appropriately aliquoted. When two or more components are present in the kit, the kit may also include a second, third or other additional container into which the additional components can be separately placed. However, various combinations of the components may be included in one vial. The kit of the present invention also typically includes means for containing the composition and other optional reagent containers in a tightly enclosed state for commercial sale. Such containers may include injection-molded or blow-molded plastic containers that hold the desired vials.

Examples

[0235] IX. Examples The following examples are included to demonstrate certain non-limiting aspects of the present disclosure. It should be recognized by those skilled in the art that the techniques disclosed in the following examples are those that the inventors have found to function well in the practice of the subject matter of the present disclosure. However, those skilled in the art will recognize that, in view of the present disclosure, many modifications can be made to the specific embodiments disclosed, and such modifications will still result in similar or analogous results without departing from the spirit and scope of the subject matter of the present disclosure. Example 1 CAR.CD70 NK cells for targeting AML

[0236] In certain embodiments, CD70-specific CAR NK cells are used to target acute myeloid leukemia (AML). Figure 5A shows the transduction efficiency of CAR-CD70 NK cells compared to non-transduced cells. Figure 5B shows the expression of CD70 in various AML cell lines. Figure 6 shows a functional assay of the activity of CD70 CAR in CD70 CAR / IL-15-expressing NK cells versus non-transduced cells against Molm13 and Molm14 AML cell lines. The annexin V assay showed more killing of various AML cell lines compared to non-transduced cells (Figure 7). The chromium release assay also showed more killing of AML cell lines when using CD70 CAR-expressing NK cells that also express IL-15. Example 2 CAR.CD70 NK cells targeting lung cancer

[0237] In some embodiments, as an example of a solid tumor, a reagent that targets and kills lung cancer expressing CD70 is used. Figure 9 shows the expression of CD70 in various lung cancer cell lines. The use of CD70CAR-expressing NK cells resulted in higher toxicity against various lung cancer cell lines compared to non-transduced cells and NK cells transduced with IL-15 (Figures 10A and 10B). Annexin staining showed higher toxicity in CD70CAR-expressing NK cells compared to non-transduced cells and NK cells transduced with IL-15 when compared in various lung cancer cell lines (Figure 11). In Figure 12, when evaluated by caspase expression in lung cancer cell line spheroids, CD70CAR-expressing NK cells showed higher toxicity than non-transduced NK cells or NK cells transduced with IL-15 only (without CAR). When using the Incucyte® assay, CD70CAR / IL-15-expressing NK cells exhibit higher cytotoxicity against the ER1 lung cancer cell line compared to non-transduced NK cells (NT) and NK cells transduced with IL-15 (Figure 13). When using the Incucyte® assay, CD70CAR / IL-15-expressing NK cells exhibit higher cytotoxicity against the ER3 lung cancer cell line compared to non-transduced NK cells (NT) and NK cells transduced with IL-15 (Figure 14).

[0238] CD70-positive cancers other than lung cancer can also be treated with the methods and compositions of the present disclosure (see, for example, Figure 15). Example 3 Cord blood-derived natural killer (CBNK) cells transduced with CD70CAR against various cancers Acute myeloid leukemia (AML)

[0239] Figures 16A - 16B show the CD70 CAR transduction efficiency in CBNK cells and the expression of CD70 on various acute myeloid leukemia (AML) targets. Figure 16A shows that CD70 CAR was successfully transduced into CBNK cells with a transduction efficiency of 98% compared to non - transduced cells. Figure 16B shows that CD70 was expressed on the surface of various AML targets.

[0240] Figure 17 shows the expression of intracellular cytokines and degranulation markers in CBNK CD70CAR cells when co - cultured with Molm13 and Molm14 cells. Compared to non - transduced (NT) cells, CBNK cells transduced with CD70CAR showed increased secretion of cytokines (interferon - gamma and tumor necrosis factor - alpha) and expression of the degranulation marker CD107a when co - cultured with Molm13 (left) and Molm14 (right), suggesting high cytotoxic activity against AML cells expressing CD70.

[0241] Figure 18 shows annexin V staining to evaluate apoptosis of AML target cells after co - culture with CBNK CD70CAR cells. Compared to non - transduced (NT) cells, CBNK cells transduced with CD70CAR showed increased apoptosis of THP - 1, Molm13, and Molm14 cells, as shown by the annexin V - LIVE / DEAD TM Fixable Aqua staining assay, suggesting high cytotoxic activity of CBNK cells transduced with CD70CAR against AML cells.

[0242] Figure 19 shows a chromium release assay for evaluating the cytotoxic activity of CBNK CD70CAR against AML target cells. Compared with non-transduced (NT) cells, CBNK cells transduced with CD70CAR showed high cytotoxicity against THP-1 (left) and Molm13 (right) cells, as shown by the chromium release assay, suggesting that CBNK CD70CAR cells have high killing activity against AML cells.

[0243] Figures 20A - 20B show an IncuCyte® cytotoxicity assay against THP-1 and OCI-AML3 cells when co-cultured with CBNK CD70CAR cells. As shown by the IncuCyte® assay, CBNK cells transduced with CD70CAR showed high cytotoxicity against THP-1 (Figure 20A) and OCI-AML3 (Figure 20B) cells compared with non-transduced (NT) cells, suggesting that CBNK CD70CAR cells have high killing activity against AML cells. In this assay, CBNK cells transduced with the IL15 construct were also used as a control, which showed higher cytotoxic activity than NT but was less effective than CD70CAR. Lung cancer

[0244] Figure 21 shows the expression of CD70 in various lung cancer cell lines. Flow cytometry was used to detect the surface expression of CD70 in various lung cancer cell lines.

[0245] Figure 22 shows the expression of intracellular cytokines and degranulation markers in CBNK CD70CAR cells when co-cultured with various lung cancer cell lines. Compared with non-transduced (NT) cells, CBNK cells transduced with CD70CAR showed an increase in the secretion of cytokines (interferon gamma and tumor necrosis factor alpha) and the expression of the degranulation marker CD107a when co-cultured with various lung cancer cell lines, suggesting high cytotoxic activity of CBNK CD70CAR against lung cancer.

[0246] Figure 23 shows annexin V staining for evaluating apoptosis of lung cancer cells after co - culture with CBNK CD70CAR cells. Compared with non - transduced (NT) cells, CBNK cells transduced with CD70CAR showed high apoptosis of various lung cancer cells as shown by the annexin V - LIVE / DEAD TM Fixable Aqua staining assay, suggesting high cytotoxic activity of CBNK cells transduced with CD70CAR against lung cancer cells.

[0247] Figure 24 shows the IncuCyt (registered trademark) e cytotoxicity assay in ER1 cells when co - cultured with CBNK CD70CAR cells. Compared with non - transduced (NT) cells, CBNK cells transduced with CD70CAR showed high cytotoxicity against ER1 cells as shown by the IncuCyte (registered trademark) assay evaluated by the measured value of the green (caspase 3 / 7) signal, suggesting that CBNK CD70CAR cells have high killing activity against lung cancer cells. In this assay, CBNK cells transduced with the CD19CAR construct were also used as a control, which showed higher cytotoxic activity than NT but was less effective than CD70CAR. Quantification of the IncuCyte (registered trademark) cytotoxicity assay over 54 hours is shown in the left panel, and representative images are shown in the right panel.

[0248] Figure 25 shows the IncuCyte® cytotoxicity assay in ER3 cells when co-cultured with CBNK CD70CAR cells. Compared to non-transduced (NT) cells, CBNK cells transduced with CD70CAR showed high cytotoxicity against ER3 cells as shown by the IncuCyte® assay evaluated by the measurement of the green (caspase 3 / 7) signal, suggesting that CBNK CD70CAR cells have high killing activity against lung cancer cells. CBNK cells transduced with the CD19CAR construct were also used as a control in this assay, which showed higher cytotoxic activity than NT but was less effective than CD70CAR. Breast cancer

[0249] Figure 26 shows a chromium release assay evaluating the cytotoxic activity of CBNK CD70CAR against breast cancer cell lines with various CD70 expressions. (Left) Surface expression of CD70 was detected in various breast cancer cell lines using flow cytometry. MBA-MB-231 has low / no CD70 expression, whereas BT549 and BCX010 have high CD70 expression. (Right) Compared to non-transduced (NT) cells, CBNK cells transduced with CD70CAR showed high cytotoxicity against BT549 and BCX010 cells as shown by the chromium release assay, suggesting that CBNK CD70CAR cells have high killing activity against breast cancer cells with high CD70 expression. K562 cells sensitive to NK cells were used as a positive control. n.s. not significant; *** , P<0.001

[0250] Figures 27A-27E show the expression of intracellular cytokines and degranulation markers in CBNK CD70CAR cells when co-cultured with various breast cancer cells. Compared with non-transduced (NT) cells, CBNK cells transduced with CD70CAR showed high secretion of cytokines (interferon gamma and tumor necrosis factor alpha) and expression of the degranulation marker CD107a when co-cultured with breast cancer cell lines with high CD70 surface expression, suggesting high cytotoxic activity of CBNK CD70CAR against breast cancer. n.s. not significant; * , p<0.05; ** , p<0.01; *** , p<0.001. Multiple myeloma

[0251] Figures 28A and 28B provide a chromium release assay to evaluate the cytotoxic activity of CBNK CD70CAR against multiple myeloma. (Figure 28A) Surface expression of CD70 was high in the multiple myeloma cell line MM1s when detected by using flow cytometry. (Figure 28B) Compared with non-transduced (NT) cells, CBNK cells transduced with CD70CAR showed high cytotoxicity against MM1s cells as shown by the chromium release assay, suggesting that CBNK CD70CAR cells have high killing activity against multiple myeloma cells. Renal cell carcinoma (RCC)

[0252] Figures 29A-29B show a chromium release assay evaluating the cytotoxic activity of CBNK CD70CAR against RCC. (Figure 29A) Surface expression of CD70 was detected in various RCC and other cancer cell lines using flow cytometry. A498, SN12C, and 786-O are a few RCC cell lines with high CD70 expression. (Figure 29B) Compared to non-transduced (NT) cells, CBNK cells transduced with CD70CAR showed high cytotoxicity against A498 and SN12C cells as shown by the chromium release assay, suggesting that CBNK CD70CAR cells have high killing activity against RCC cells with high CD70 expression.

[0253] Figure 30 shows the production of intracellular cytokines and the expression of degranulation markers in CBNK CD70CAR cells when co-cultured with RCC cells. Compared to non-transduced (NT) cells, CBNK cells transduced with CD70CAR showed increased secretion of cytokines (interferon gamma and tumor necrosis factor alpha) and expression of the degranulation marker CD107a when co-cultured with the RCC cell line 786-O, which has high CD70 surface expression, suggesting high cytotoxic activity of CBNK CD70CAR against breast cancer. ** , p<0.01

[0254] Figure 31 shows an IncuCyte® cytotoxicity assay against 786-O RCC cells when co-cultured with CBNK CD70CAR cells. Compared to non-transduced (NT) cells, CBNK cells transduced with CD70CAR showed high cytotoxicity against 786-O cells as shown by the IncuCyte® assay evaluated by the measurement of the green (caspase 3 / 7) signal, suggesting that CBNK CD70CAR cells have high killing activity against RCC. ** , p<0.01; *** , p<0.001 Pancreatic cancer

[0255] Figures 32A - 32B show the intracellular cytokine expression in CBNK CD70CAR cells when co - cultured with pancreatic cancer cells. (Figure 32A) The surface expression of CD70 was detected in various pancreatic cancer cell lines using flow cytometry. MIA - Paca2 has low or no CD70 expression, while PANC - 1 has high CD70 expression. (Figure 32B) Compared with non - transduced (NT) cells, CBNK cells transduced with CD70CAR showed an increase in the secretion of cytokines (interferon - gamma and tumor necrosis factor - alpha) when co - cultured with the PANC - 1 cell line (high CD70 expression), but not when co - cultured with the MIA - Paca2 cell line (low CD70 expression), suggesting high cytotoxic activity of CBNK CD70CAR against pancreatic cells with high CD70 expression. Glioblastoma (GBM)

[0256] Figure 33 shows the IncuCyte® cytotoxicity assay against GSC20 GBM cells when co - cultured with CBNK CD70CAR cells. When the surface expression of CD70 was detected in various GBM cell lines using flow cytometry, the GSC20 cell line showed the highest CD70 surface expression (panel i). Compared with non - transduced (NT) cells, CBNK cells transduced with CD70CAR showed high cytotoxicity against GSC20 cells, as shown by the IncuCyte® assay evaluated by the measurement of green (caspase 3 / 7) signal intensity, suggesting that CBNK CD70CAR cells have high killing activity against GBM cells. The quantification of the IncuCyte® cytotoxicity assay over 57 hours is shown in panel ii, and representative images up to 23 hours are shown in panel iii.

[0257] The survival curves of NSG mice (immunodeficient) transplanted with Raji WT or CD70 KO cells and treated with CBNK CD70CAR cells are provided in Figure 34. The Kaplan Meier plot demonstrates that CBNK cells transduced with the CD70CAR construct show improved survival of mice transplanted with Raji wild-type (WT) tumors compared to non-transduced CBNK cells. Since no improvement in survival was seen in mice transplanted with CD70 knockout (KO) Raji cells, it is suggested that the improvement in mouse survival is specific to the CD70 antigen present on the tumor cells.

[0258] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the spirit and scope of the concepts defined by the appended claims. Furthermore, the scope of the present application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, compositions, means, methods, and steps described herein. Those skilled in the art will immediately recognize from this disclosure that processes, machines, manufactures, compositions, means, methods, or steps that presently exist or will later be developed and that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized in accordance with this disclosure. Accordingly, the appended claims are intended to include such processes, machines, manufactures, compositions, means, methods, or steps within their scope.

Claims

**Claim 1** An expression construct comprising an array encoding an engineered receptor specific for CD70 and an array encoding a cytokine, wherein the cytokine is IL-15, IL-12, IL-2, IL-18, IL-21, IL-7 or a combination thereof. **Claim 2** The construct according to claim 1, wherein the engineered receptor specific for CD70 is a chimeric antigen receptor (CAR) or a T cell receptor. **Claim 3** The CD70-specific CAR comprises an scFv having a heavy chain and a light chain, and the heavy chain in the array encoding the CAR is present upstream of the light chain in the 5' to 3' direction. The expression construct according to claim 2. **Claim 4** The CD70-specific CAR comprises an scFv having a heavy chain and a light chain, and the heavy chain in the array encoding the CAR is present downstream of the light chain in the 5' to 3' direction. The expression construct according to claim 2. **Claim 5** The CD70-specific CAR comprises an scFv with optimized codons. The expression construct according to any one of claims 1 to 4. **Claim 6** The CD70-specific CAR comprises a humanized scFv. The expression construct according to any one of claims 1 to 4. **Claim 7** The CD70-specific CAR comprises a signaling peptide. The expression construct according to any one of claims 1 to 6. **Claim 8** The signaling peptide is derived from a signal peptide derived from CD8 alpha, Ig heavy chain or granulocyte macrophage colony-stimulating factor receptor, or one or more other surface receptors. The expression construct according to claim 7. **Claim 9** The CD70-specific CAR comprises one or more co-stimulatory domains. The expression construct according to any one of claims 1 to 8. **Claim 10** The co-stimulatory domain is selected from the group consisting of CD28, CD27, OX-40 (CD134), DAP10, DAP12, 4-1BB (CD137), CD40L, 2B4, DNAM, CS1, CD48, NKG2D, NKp30, NKp44, NKp46, NKp80 and combinations thereof. The expression construct according to claim 9. **Claim 11** The CD70-specific CAR comprises CD3 zeta. The expression construct according to any one of claims 1 to 10. **Claim 12** The expression construct according to any one of claims 1 to 11, wherein the CD70-specific CAR comprises a hinge between the scFv and the transmembrane domain.

13. The expression construct according to claim 12, wherein the hinge is a CD8-alpha hinge, the hinge comprises an artificial spacer containing Gly3, or the hinge comprises CH1, CH2 and / or CH3 domains of IgG.

14. The expression construct according to any one of claims 1 to 13, wherein the cytokine is IL-15.

15. The expression construct according to any one of claims 1 to 14, further comprising a suicide gene, wherein the suicide gene is mutant TNF-alpha, inducible caspase 9, HSV-thymidine kinase, CD19, CD20, CD52 or EGFRv3.

16. The expression construct according to claim 15, wherein the mutant TNF-alpha is engineered non-secretable mutant TNF-alpha.

17. The expression construct according to any one of claims 1 to 16, wherein the expression construct comprises any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:

13.

18. A natural killer (NK) cell comprising the expression construct according to any one of claims 1 to 17.

19. The NK cell according to claim 18, wherein the NK cell is derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, bone marrow or a cell line.

20. The NK cell according to claim 19, wherein the NK cell line is the NK-92 cell line or another NK cell line derived from a tumor or healthy NK cells or progenitor cells.

21. The NK cell according to any one of claims 18 to 20, wherein the NK cell is umbilical cord blood mononuclear cells.

22. The NK cell according to any one of claims 18 to 21, wherein the NK cell is a CD56+ NK cell.

23. The NK cell according to any one of claims 18 to 22, wherein the NK cell expresses one or more exogenously provided cytokines.

24. The NK cell according to claim 23, wherein the cytokine is IL-15.

25. The NK cell according to any one of claims 18 to 24, wherein the expression of one or more endogenous genes in the NK cell is modified.

26. The NK cell according to claim 25, wherein the expression is partially or completely reduced.

27. The NK cell according to claim 25 or 26, wherein the expression of the one or more genes is modified using CRISPR.

28. The NK cell according to any one of claims 25 to 27, wherein the gene is selected from the group consisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, CTLA-4, TDAG8, CD38 and combinations thereof.

29. The population of NK cells according to any one of claims 18 to 28, wherein the cells are present in a suitable medium.

30. A composition for killing CD70-positive cells in an individual, the composition comprising cells having the expression construct according to any one of claims 1 to 17.

31. The composition according to claim 30, wherein the cells are NK cells, T cells, gamma delta T cells, induced NKT (iNKT) cells, B cells, macrophages, gamma delta T cells or dendritic cells.

32. The composition according to claim 31, wherein the NK cells are derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, bone marrow or cell lines.

33. The composition according to claim 31 or 32, wherein the NK cells are derived from umbilical cord blood mononuclear cells.

34. The composition according to any one of claims 30 to 33, wherein the CD70-positive cells are not cancer cells. Composition.

35. The composition according to claim 34, wherein the CD70-positive cells are regulatory T cells.

36. The composition according to any one of claims 30 to 33, wherein the individual has acute myeloid leukemia, lymphoma, lung cancer, kidney cancer, bladder cancer, melanoma, glioblastoma, breast cancer, head and neck cancer, mesothelioma, multiple myeloma, pancreatic cancer or combinations thereof.

37. The composition according to any one of claims 30 to 36, wherein the cells are allogeneic to the individual.

38. The composition according to any one of claims 30 to 36, wherein the cells are autologous to the individual.

39. The composition according to any one of claims 30 to 38, wherein the individual is a human.

40. The composition according to any one of claims 30 to 39, wherein the composition is administered to the individual once or more than once.

41. The composition according to claim 40, wherein the time between administrations to the individual is 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, 1 to 12 months, or 1 year or more.

42. The composition according to any one of claims 30 to 41, wherein the individual undergoes further treatment.

43. The composition according to claim 42, wherein the further treatment includes surgery, radiation therapy, gene therapy, immunotherapy, or hormone therapy.

44. The composition according to claim 42 or 43, wherein the further treatment includes one or more antibodies.

45. The composition according to any one of claims 30 to 44, wherein the composition is administered to the individual by injection, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, intracranially, percutaneously, subcutaneously, regionally, by perfusion, into the tumor microenvironment, or by a combination thereof.

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