Modular polycistronic vectors for CAR and TCR transduction

Abstract

Methods and compositions relating to polycistronic vectors are provided. [Solution] Provided herein is a modular polycistronic vector system, such as for genetic reprogramming of cells to express one or more antigen receptors. The modular nature of the system allows for the exchange of one or more components within a particular cistron as well as one or more cistrons. In specific embodiments, the modularity of the system allows for the exchange of components of a chimeric antigen receptor, such as the exchange of costimulatory domains, scFvs, hinges, signaling domains, etc.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 769,414, filed November 19, 2018; U.S. Provisional Patent Application No. 62 / 773,394, filed November 30, 2018; and U.S. Provisional Patent Application No. 62 / 791,491, filed January 11, 2019. All of these provisional applications are incorporated herein by reference in their entirety. Sequence Listing

[0002] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 13, 2019, is named UTFC_P1152WO_SL.txt and is 2,502 bytes in size. [Background technology]

[0003] 1. Field The present disclosure relates generally to at least the fields of immunology, cell biology, molecular biology, recombinant technology and medicine. More particularly, the present invention relates to polycistronic vectors, such as for the expression of antigen receptors. 2. Description of Related Technology

[0004] Despite significant technological advances in diagnostic and treatment options available to patients diagnosed with cancer, prognosis remains poor and many patients are incurable. Immunotherapy has the potential to provide more targeted and powerful treatments to patients diagnosed with a variety of tumors, with the ability to annihilate malignant tumor cells without harming normal tissues. In theory, T cells of the immune system can recognize protein patterns specific to tumor cells and mediate their destruction by various effector mechanisms.

[0005] Genetic reprogramming of immune cells, such as natural killer (NK) cells, for cancer adoptive immunotherapy has clinically relevant applications and advantages, such as innate antitumor surveillance without the need for prior sensitization, allogeneic efficacy without graft-versus-host reactivity, and direct cell-mediated cytotoxicity and lysis of targeted tumors. The administration of immune cells expressing chimeric antigen receptors (CARs) (e.g., adoptive T cell therapy or NK cell therapy) is an attempt to harness and amplify the tumor-destroying ability of the patient's own immune cells and return these effectors to the patient in a state where they effectively eliminate residual tumors without harming healthy tissues. In general, CARs comprise a single-chain variable fragment (scFv) of an antibody specific for a tumor-associated antigen (TAA) linked to the cytoplasmic domain of a T cell signaling molecule via a hinge region and a transmembrane region.

[0006] The simultaneous expression of multiple genes in desired ratios is very attractive for a wide range of basic research and biomedical applications, including gene reprogramming.Strategies aimed at the simultaneous expression of multiple genes include the introduction of multiple vectors, the use of multiple promoters in a single vector, fusion proteins, intergenic proteolytic cleavage sites, internal ribosome entry sites, and "self-cleaving" 2A peptides.However, there is still an unmet need for a method for the combinatorial expression of multiple genes, including, as some examples, CAR, T cell receptor (TCR), cytokine, cytokine receptor, chemokine receptor and homing receptor.The present disclosure meets this need. Summary of the Invention

[0007] Abstract The embodiments of the present disclosure include methods and compositions related to polycistronic vectors that contain at least two, at least three, or at least four cistrons, each flanked by one or more restriction enzyme sites, with at least one cistron encoding at least one antigen receptor. In some cases, two, three, four, or more cistrons are translated into a single polypeptide and cleaved into separate polypeptides. Adjacent cistrons on a vector may be separated by a site that provides the ability of the encoded polypeptides to become separate molecules. For example, adjacent cistrons on a vector may be separated by a self-cleaving site, such as a 2A self-cleaving site. In some cases, each cistron expresses a separate polypeptide from the vector. In certain cases, adjacent cistrons on a vector are separated by an IRES element.

[0008] In certain embodiments of the present disclosure, at least one of the cistrons on the vector contains two or more modular components, and each of the modular components in the cistron is flanked by one or more restriction enzyme sites. The cistron may contain, for example, three, four, or five modular components. In at least some cases, the cistron encodes an antigen receptor with different portions of the receptor encoded by the corresponding modular components. The first modular component of the cistron may encode the antigen binding domain of the receptor. In addition, the second modular component of the cistron may encode the hinge region of the receptor. In addition, the third modular component of the cistron may encode the transmembrane domain of the receptor. In addition, the fourth modular component of the cistron may encode the first costimulatory domain. In addition, the fifth modular component of the cistron may encode the second costimulatory domain. In addition, the sixth modular component of the cistron may encode the signaling domain.

[0009] In certain embodiments of the present disclosure, each of the two different cistrons on the vector encodes a non-identical antigen receptor. Both antigen receptors can be encoded by a cistron that contains two or more modular components, including separate cistrons that contain two or more modular components. The antigen receptor can be, for example, a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR).

[0010] In specific embodiments, the vector is a viral vector (e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector) or a non-viral vector. The vector may include the 5'LTR, 3'LTR, and / or psi packaging elements of Moloney Murine Leukemia Virus (MMLV). In specific cases, the psi packaging is integrated between the 5'LTR and the antigen receptor coding sequence. The vector may or may not include a pUC19 sequence. In some embodiments of the vector, at least one cistron encodes a secreted or membrane-bound cytokine (e.g., interleukin 15 (IL-15), IL-7, IL-21, IL-12, IL-18, or IL-2), a chemokine, a cytokine receptor, and / or a homing receptor.

[0011] When 2A cleavage sites are used in the vector, the 2A cleavage sites may include a P2A, T2A, E2A and / or F2A site.

[0012] Any cistron of the vector may contain a suicide gene. Any cistron of the vector may code for a reporter gene. In a specific embodiment, the first cistron codes for a suicide gene, the second cistron codes for an antigen receptor, the third cistron codes for a reporter gene, and the fourth cistron codes for a cytokine. In a specific embodiment, the first cistron codes for a suicide gene, the second cistron codes for a first antigen receptor, the third cistron codes for a second antigen receptor, and the fourth cistron codes for a cytokine. In a specific embodiment, different parts of the antigen receptor are coded for by corresponding modular components, with the first component of the second cistron coding for an antigen binding domain, the second component coding for a hinge and / or transmembrane domain, the third component coding for a costimulatory domain, and the fourth component coding for a signaling domain.

[0013] The methods and compositions of the disclosure include cistrons in any suitable order on a single vector.

[0014] The embodiments of the present disclosure include any type of cell with any vector encompassed herein. In a specific embodiment, there is an immune cell that includes a vector of the present disclosure, and the immune cell can be a T cell, a peripheral blood lymphocyte, a B cell, a NK cell, an invariant NK cell, a NKT cell, an iNKT, a macrophage, or a stem cell (e.g., a mesenchymal stem cell (MSC) or an induced pluripotent stem (iPS) cell). The T cell can be a CD8+ T cell, a CD4+ T cell, or a gamma-delta T cell. In a specific embodiment, the T cell is a cytotoxic T lymphocyte (CTL). Any cell can be allogeneic or autologous to the individual. The immune cell can be a human cell. The immune cell can be derived from umbilical cord blood, peripheral blood, bone marrow, CD34+ cells, or iPSC. A plurality of immune cells can be included as a population of cells. In a specific case, the immune cell is included in a pharmaceutically acceptable carrier.

[0015] In one embodiment of the present disclosure, there is a method for generating immune cells, which includes the steps of (a) obtaining a starting population of immune cells; (b) culturing the starting population of immune cells in the presence of artificial presentation cells (APCs); (c) introducing a vector encompassed in the present disclosure into the immune cells; and (d) expanding the immune cells in the presence of APCs, thereby obtaining expanded immune cells. The starting population of immune cells can be obtained by isolating mononuclear cells using a Ficoll-Paque density gradient. The APCs can be gamma-irradiated APCs. In some cases of the above method, the method further includes the steps of cryopreserving the expanded population of immune cells.

[0016] Embodiments of the present disclosure include pharmaceutical compositions comprising any population of immune cells as encompassed herein and a pharma- ceutically acceptable carrier.

[0017] In certain embodiments, compositions are provided that include an effective amount of immune cells as encompassed herein for use in treating a disease or disorder (e.g., cancer or immune-related disorder) in an individual. In specific embodiments, there is a use of a composition that includes an effective amount of immune cells as encompassed by the present disclosure for treating cancer or an immune-related disorder in an individual.

[0018] In one embodiment, there is a method of treating a disease or disorder in an individual, the method comprising administering to the individual an effective amount of immune cells encompassed by the present disclosure. In a specific embodiment, the disease or disorder is cancer (solid cancer or hematological malignancy), an autoimmune disorder, graft-versus-host disease, allograft rejection or an inflammatory condition. The autoimmune disorder may be an inflammatory condition and the immune cells may essentially have no expression of glucocorticoid receptors. The subject may have undergone or is undergoing steroid therapy. The immune cells may be autologous or allogeneic to the individual. The method may further comprise administering to the individual at least a second therapeutic agent (e.g., chemotherapy, immunotherapy, surgery, radiation therapy or biotherapy). In a specific embodiment, the immune cells are administered to the individual intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, by perfusion, or by direct injection. In certain embodiments, the second therapeutic agent is administered to the individual intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, by perfusion, or by direct injection.

[0019] In some embodiments, a polycistronic vector contains at least two, at least three, or at least four cistrons, each flanked by one or more restriction enzyme sites, and at least one of the cistrons on the vector contains two or more modular components, and each of the modular components within a cistron is flanked by one or more restriction enzyme sites.

[0020] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description, but it should be understood that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description.

[0021] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0022] [Figure 1A] An embodiment of a polycistronic vector comprising multiple modular cistrons, comprising at least one cistron having multiple modular components, where X represents at least one restriction enzyme site.

[0023] [Figure 1B] Another embodiment of a polycistronic vector comprising multiple modular cistrons, comprising at least one cistron having multiple modular components, wherein X represents at least one restriction enzyme site.

[0024] [Diagram 2] Schematic diagram showing the linear expression of an example of a polycistronic retroviral vector containing four cistrons expressing seven open reading frames (ORFs), including components within the antigen receptor cistron, via three 2A self-cleavage sites.

[0025] [Diagram 3] An example of a polycistronic plasmid containing multiple cistrons and three 2A cleavage sites. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Description of illustrative embodiments In certain embodiments, the present disclosure provides a flexible, modular system utilizing polycistronic vectors capable of expressing multiple cistrons at substantially the same level. This system can be used for cell engineering to allow combinatorial expression (including overexpression) of multiple genes. In a specific embodiment, one or more of the genes expressed by the vector includes one, two or more antigen receptors, which in a specific embodiment are non-natural receptors. The multiple genes can include, but are not limited to, CAR, TCR, cytokine, chemokine, homing receptor, CRISPR / Cas9-mediated gene mutation, decoy receptor, cytokine receptor, chimeric cytokine receptor, and the like. The vector can further include (1) one or more reporters, such as fluorescent or enzymatic reporters, for cell assays and animal imaging; (2) one or more cytokines or other signaling molecules; and / or (3) suicide genes.

[0027] Specifically, the vector may contain at least four cistrons separated by any kind of cleavage site, such as 2A cleavage sites. The vector may or may not be a Moloney Murine Leukemia Virus (MoMLV or MMLV) based vector, which contains 3' and 5' LTR with psi packaging sequence in a pUC19 backbone. The vector may contain four or more cistrons containing three or more 2A cleavage sites and multiple ORFs for gene exchange. This system allows combinatorial overexpression of multiple genes (seven or more) flanked by restriction enzyme recognition sites for rapid integration by subcloning, and in some embodiments, this system also contains at least three 2A self-cleavage sites. Thus, this system allows expression of multiple CARs, TCRs, signaling molecules, cytokines, cytokine receptors and / or homing receptors. This system may also be applied to other viral and non-viral vectors, including but not limited to lentivirus, adenovirus AAV and non-viral plasmids.

[0028] The modular nature of the system allows for efficient subcloning of genes into each of the four cistrons within the polycistronic expression vector, as well as the exchange of genes for rapid testing, etc. Strategically placed restriction enzyme recognition sites within the polycistronic expression vector allow efficient gene exchange. Examples of restriction enzyme sites include, at least, the following: BlnI, BstEII, EcoRV, AccI, AluI, ApaI, BamHI, BclI, BglII, BsmI, CfoI, ClaI, DdeI, DpnI, DraI, EclXI, EcoRI, HaeIII, HindI. II, HpaI, KpnI, KspI, MvaI, NcoI, NdeI, NheI, NotI, NsiI, PstI, PvuI, PvuII, RsaI, SacI, SalI, Sau3AI, ScaI, SfiI, SmaI, SpeI, SphI, TaqI, XbaI and / or XhoI.

[0029] Further provided herein is a method for genetically engineering immune cells using the modular vectors encompassed herein. The immune cells can be of any type, including, for example, T cells, B cells, NK cells, NKT cells, or mesenchymal stromal cells (MSCs). Also provided herein is a method of immunotherapy comprising administering immune cells engineered by the vector, such as for treating cancer, infectious diseases, or autoimmune diseases. I. Definition

[0030] As used herein, "essentially free" of a particular component is used herein to mean that the particular component is not intentionally formulated in the composition and / or is not present even as a contaminant or in trace amounts.Thus, the total amount of the particular component resulting from any unintentional contamination of a composition is less than 0.05%, preferably less than 0.01%.Most preferred is a composition in which the amount of the particular component cannot be detected by standard analytical methods.

[0031] In line with long-standing patent law practice, the words "a" and "an" when used in conjunction with the word "comprising" in this specification (including the claims) refer to "one or more". Some embodiments of the present disclosure may consist of or consist essentially of one or more elements, method steps and / or methods of the present disclosure. It is contemplated that any method or composition described herein can be performed on any other method or composition described herein of the disclosed embodiments and still produce the same or similar results without departing from the spirit and scope of the present disclosure. The use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to only alternatives or unless the alternatives are mutually exclusive, but the present disclosure supports the definition to refer to only alternatives and "and / or". As used herein, "another" can mean at least a second or more.

[0032] As used herein, the term "cistron" refers to a nucleic acid sequence that can produce a gene product.

[0033] As used herein, the term "modular" refers to a cistron or a component of a cistron, respectively, that allows for their interchangeability, such as by removal and replacement of entire cistrons or components of cistrons, using standard recombinant techniques such as the use of one or more restriction enzyme sites, including in some cases unique restriction enzyme sites.

[0034] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to measure the value, or the variation that exists among the subjects in a study.

[0035] "Immune disorder," "immune-related disorder," or "immune-mediated disorder" refers to a disorder in which the immune response plays a significant role in the development and / or progression of the disease. Immune-mediated disorders include, for example, autoimmune disorders, allograft rejection, graft-versus-host disease, and inflammatory and allergic conditions.

[0036] An "immune response" is the response of a cell of the immune system, such as a B cell or T cell or an innate immune cell, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response").

[0037] "Autoimmune disease" refers to a disease in which the immune system mounts an immune response (e.g., a B cell response or a T cell response) against antigens that are part of the normal host (i.e., self-antigens), resulting in tissue damage. Self-antigens can be derived from host cells or from commensal organisms (e.g., microorganisms that normally colonize mucosal surfaces, known as commensals).

[0038] "Treating" a disease or condition or treatment of a disease or condition refers to carrying out a protocol that may include administering one or more drugs or therapies (including cells) to a patient with the goal of alleviating at least one sign or symptom of the disease. Desirable effects of treatment include slowing the rate of disease progression, reversing or alleviating the disease state, delaying the onset of at least one symptom, and remission or improvement of prognosis. Alleviation may occur before, as well as after, or both, the signs or symptoms of the disease or condition that appear. Thus, "treating" or "treatment" may include "preventing" or the "prevention" of a disease or undesirable condition. Furthermore, "treating" or "treatment" does not require complete alleviation of one or more signs or symptoms, does not require a cure, and in particular includes protocols that have only marginal effects on the patient.

[0039] The term "therapeutic effect" or "therapeutically effective" as used throughout this application refers to anything that enhances or improves the well-being of a subject with respect to a medical treatment for that condition. This includes, but is not limited to, reducing the frequency or severity of one or more signs or symptoms of a disease. For example, treating cancer can include, for example, reducing tumor size, reducing the invasiveness of a tumor, reducing the rate of cancer growth, and / or preventing metastasis. Treating cancer can also refer to extending the survival time of a subject with cancer.

[0040] "Subject" and "patient" and "individual" refer to humans or non-humans, e.g., primates, mammals, and vertebrates. In certain embodiments, the subject is a human, dog, cat, horse, cow, etc.

[0041] The phrase "pharmaceutical acceptable or pharmacologically acceptable" refers to molecular entities and compositions that do not cause adverse, allergic or other untoward reactions when appropriately administered to animals, such as humans. The preparation of pharmaceutical compositions containing antibodies or additional active ingredients is known to those skilled in the art in light of the present disclosure. Furthermore, it is understood that for administration to animals (e.g., humans), preparations should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards.

[0042] As used herein, "pharmaceutical acceptable carrier" includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents and inert gases), isotonicity agents, absorption retardants, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, flow and nutritional supplements, such similar materials and combinations thereof, as known to those skilled in the art. The pH and exact concentration of various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0043] The term "antigen-presenting cell (APC)" refers to a class of cells that can present one or more antigens in the form of peptide-MHC complexes that can be recognized by specific effector cells of the immune system, thereby inducing an effective cellular immune response against the presented antigens. The term "APC" encompasses whole intact cells (e.g., macrophages, B cells, endothelial cells, activated T cells and dendritic cells), or naturally occurring or synthetic molecules that can present antigens (e.g., purified MHC class I molecules complexed to beta2-microglobulin). II. Modular polycistronic vector system

[0044] Embodiments of the present disclosure encompass systems that utilize polycistronic vectors, at least some of which are modularized, for example, by allowing for the 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 whose identities and locations are specifically selected to facilitate modular use of the vector. The vectors also have embodiments in which multiple cistrons are translated into a single polypeptide and processed into separate polypeptides, thereby conferring the advantage of this vector that separate gene products are expressed in substantially equimolar concentrations.

[0045] The vectors of the disclosure are arranged to provide modularity that allows for alteration of one or more cistrons of the vector and / or alteration of one or more components of one or more particular cistrons. The vectors may be designed to take advantage of unique restriction enzyme sites adjacent to the ends of one or more cistrons and / or adjacent to the ends of one or more components of a particular cistron.

[0046] In certain embodiments, the present disclosure provides a system for cell engineering that allows for combinatorial expression (including overexpression) of multiple cistrons, which may include, for example, one, two or more antigen receptors. In certain embodiments, the use of polycistronic vectors as described herein allows the vector to produce equimolar levels of multiple gene products from the same mRNA. The multiple genes may include, but are not limited to, CAR, TCR, cytokines, chemokines, homing receptors, CRISPR / Cas9-mediated gene mutations, decoy receptors, cytokine receptors, chimeric cytokine receptors, and the like. The vector may further include one or more fluorescent or enzymatic reporters, such as for cell assays and animal imaging. The vector may also include a suicide gene product to terminate cells carrying the vector when they are no longer needed or become harmful to the provided host.

[0047] In a specific case, the vector can be a gamma-retroviral transfer vector. The retroviral transfer vector can include a backbone based on a plasmid, such as pUC19 plasmid (large fragment (2.63 kb) between HindIII and EcoRI restriction enzyme sites). The backbone can have viral components from Moloney Murine Leukemia Virus (MoMLV), including 5'LTR, psi packaging sequence, and 3'LTR. LTRs are terminal repeat sequences found on either side of the provirus of retroviruses, and in the case of transfer vectors, flank the genetic cargo of interest, such as CAR and related components. The psi packaging sequence, which is the target site for packaging by nucleocapsid, is also integrated in cis between the 5'LTR and the CAR coding sequence. Thus, the basic structure of one example of a transfer vector can be constructed as follows: pUC19 sequence-5'LTR-psi packaging sequence-genetic cargo of interest-3'LTR-pUC19 sequence. This system can also be applied to other viral and non-viral vectors, including but not limited to lentiviruses, adenoviruses AAV, and non-viral plasmids.

[0048] In certain embodiments, the multiple cistrons of the vector are separated by one or more elements that provide expression of genes from the corresponding multiple cistrons into a single transcript. The single transcript is then translated to produce a multiprotein polypeptide, which is processed (e.g., by cleavage) so that the proteins become separate protein molecules. An exemplary element is a site that encodes a self-cleaving peptide, such as a 2A peptide cleavage sequence. Other cleavage sites include a furin cleavage site or a tobacco etch virus (TEV) cleavage site. In other cases, the cistrons of the vector are separated by one or more elements (e.g., an IRES sequence) that provide different translation of separate cistrons. In some cases, the vector utilizes a combination of both types of elements.

[0049] The genetic cargo of interest may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cistrons, each containing at least one ORF that can be expressed from the vector. The embodiments of the present disclosure include vectors in which the genetic cargo of interest may not currently be housed in the vector, but which still retain one or more structural or housekeeping elements (e.g., promoters, multiple 2A sequences, etc.) necessary for expression and / or further processing of the cistrons when they are present. The vector may have multiple cistrons that can be translated into a single polypeptide and processed into separate polypeptides (e.g., by using 2A self-cleavage sites between adjacent cistrons). In alternative embodiments, the multiple cistrons are expressed as separate polypeptides (e.g., by using IRES elements between adjacent cistrons).

[0050] In a specific case, the structure of the genetic cargo of interest in the vector may be as follows: Cistron 1-2A-Cistron 2-2A-Cistron 3-2A-Cistron 4 Here, in specific embodiments, cistron 1, cistron 2, cistron 3 and cistron 4 are different genes. In at least some cases, the 2A sequences in the vector may or may not be identical.

[0051] In specific embodiments, at least one of the cistrons encodes a suicide gene. In some embodiments, at least one of the cistrons encodes a cytokine. In certain embodiments, at least one cistron encodes an antigen receptor. The cistron may or may not encode a reporter gene. In certain embodiments, at least two cistrons encode two different antigen receptors (e.g., CAR and / or TCR). The cistron may or may not encode a reporter gene. In certain cases, the genetic cargo of interest is as follows: suicide gene-2A-antigen receptor-2A-reporter gene-2A-cytokine; or suicide gene-2A-antigen receptor-2A-cytokine-2A reporter gene; or suicide gene-2A-cytokine-2A reporter gene-2A-antigen receptor; or suicide gene-2A-cytokine-2A antigen receptor-2A-reporter gene; or suicide gene-2A-reporter gene-2A-cytokine-2A antigen receptor; or suicide gene-2A-reporter gene-2A-antigen receptor-2A-cytokine; or antigen receptor-2A-cytokine-2A-reporter gene-2A-suicide gene; or antigen receptor-2A-cytokine-2A-suicide gene-2A-reporter gene; or antigen receptor-2A-reporter gene-2A-cytokine-2A-suicide gene; or antigen receptor-2A-reporter gene-2A-suicide gene-2A-cytokine; or antigen receptor-2A-suicide gene-2A-reporter gene-2A-cytokine; or antigen receptor-2A-suicide gene-2A-cytokine-2A-reporter gene; or reporter gene-2A-antigen receptor-2A-suicide gene-2A-cytokine; or reporter gene-2A-antigen receptor-2A-cytokine-2A-suicide gene; or reporter gene-2A-cytokine-2A-suicide gene-2A-antigen receptor; or reporter gene-2A-cytokine-2A-antigen receptor-2A-suicide gene; or reporter gene-2A-suicide gene-2A-antigen receptor-2A-cytokine; or reporter gene-2A-suicide gene-2A-cytokine-2A-antigen receptor; or cytokine-2A-reporter gene-2A-suicide gene-2A-antigen receptor; or cytokine-2A-reporter gene-2A-antigen receptor-2A-suicide gene; or cytokine-2A-suicide gene-2A-antigen receptor-2A-reporter gene; or cytokine-2A-suicide gene-2A-reporter gene-2A-antigen receptor; or cytokine-2A-antigen receptor-2A-suicide gene-2A-suicide gene; or Cytokine-2A-antigen receptor-2A-reporter gene-2A-reporter gene.

[0052] In a particular arrangement of the genetic cargo of interest, a single vector may contain a cistron encoding a first antigen receptor and a cistron encoding a second antigen receptor that is not identical to the first antigen receptor. In a specific embodiment, the first antigen receptor encodes a CAR and the second antigen receptor encodes a TCR, or vice versa. In a particular embodiment, a vector containing separate cistrons encoding the first and second antigen receptors, respectively, 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. In a particular case, the genetic cargo of interest is as follows: suicide gene-2A-first antigen receptor-2A-cytokine-2A-second antigen receptor; or suicide gene-2A-first antigen receptor-2A-second antigen receptor-2A-cytokine; or suicide gene-2A-cytokine-2A-first antigen receptor-2A-second antigen receptor; or suicide gene-2A-cytokine-2A-second antigen receptor-2A-first antigen receptor; or suicide gene-2A-secondary antigen receptor-2A-first antigen receptor-2A-cytokine; or suicide gene-2A-second antigen receptor-2A-cytokine-2A-first antigen receptor; or First antigen receptor-2A-cytokine-2A-second antigen receptor-2A-suicide gene; or first antigen receptor-2A-cytokine-2A-suicide gene-2A-second antigen receptor; or first antigen receptor-2A-suicide gene-2A-second antigen receptor-2A-cytokine; or first antigen receptor-2A-suicide gene-2A-cytokine-2A-second antigen receptor; or First antigen receptor-2A-second antigen receptor-2A-cytokine-2A-suicide gene; or first antigen receptor-2A-second antigen receptor-2A-suicide gene-2A-cytokine; or cytokine-2A-first antigen receptor-2A-suicide gene-2A-second antigen receptor; or cytokine-2A-primary antigen receptor-2A-secondary antigen receptor-2A-suicide gene; or cytokine-2A-suicide gene-2A-first antigen receptor-2A-second antigen receptor; or cytokine-2A-suicide gene-2A-second antigen receptor-2A-first antigen receptor; or cytokine-2A-secondary antigen receptor-2A-primary antigen receptor-2A-suicide gene; or Cytokine-2A-secondary antigen receptor-2A-suicide gene-2A-primary antigen receptor.

[0053] In some cases, one of the cistrons in the vector is not a reporter gene or is not a cytokine.In an alternative embodiment of the cistron arrangement provided above, instead of the 2A peptide sequence, there may be an alternative element (e.g., an IRES sequence) that allows simultaneous expression of various cistrons.A combination of 2A peptide cleavage sequence and IRES element may also be utilized in a single vector.

[0054] In certain embodiments, at least one cistron contains multiple components that are themselves modular. For example, a cistron may code for multiple component gene products, such as an antigen receptor with multiple portions. In a specific case, the antigen receptor is encoded from a single cistron, so that a single polypeptide is ultimately generated. 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 that are unique to the vector that contains the cistron (FIGS. 1A and 1B). In a specific embodiment, a cistron with multiple components encodes an antigen receptor with multiple corresponding portions, each of which corresponding portions ascribes a unique function to the receptor. In a specific embodiment, each or most of the components of a multi-component cistron are separated by one or more restriction enzyme digestion sites that are unique to the vector, thereby allowing for interchangeability of separate components, if desired.

[0055] By way of illustration, the modularity of one example of a multi-component cistron is organized as follows, each with one or more unique restriction enzyme sites represented by X:

[0056] Component 1--X 1 -Component 2---X 2 -Component 3---X 3 -Component 4---X 4 -Component 5---X 5 --etc.

[0057] In specific embodiments, each component of a multi-component cistron corresponds to a different portion of an encoded antigen receptor, such as a chimeric antigen receptor (CAR). In an illustrative embodiment, component 1 may encode the 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 costimulatory domain of the receptor, and component 5 may encode the signaling domain of the receptor. In specific embodiments, an antigen receptor may contain one or more costimulatory domains, each of which is separated by a unique restriction enzyme digestion site, such that the costimulatory domains within a receptor are interchangeable.

[0058] Referring to Figures 1A and 1B, these illustrations provide an example of an embodiment of at least a portion of one vector of the present disclosure, illustrating the modular nature of the vector. Figure 1A illustrates a polycistronic vector with four separate cistrons, with adjacent cistrons separated by 2A cleavage sites, however, in a specific embodiment, in place of the 2A cleavage sites, an element (e.g., an IRES sequence) is present that allows separate polypeptides to be generated directly or indirectly from the cistron. In Figure 1A, the four separate cistrons are separated by three 2A peptide cleavage sites, and each cistron has a restriction enzyme recognition site (X ) flanking each end of the cistron that allows for interchangeability of a particular cistron (e.g., interchangeability with another cistron or other types of sequences) when using standard recombinant techniques. 1 , X 2 etc. In specific embodiments, the restriction enzyme sites flanking each cistron are unique to the vector to facilitate recombination, although in alternative embodiments, the restriction enzyme sites are not unique to the vector.

[0059] In certain embodiments, vectors provide a unique second level of modularity by allowing interchangeability within a particular cistron (including interchangeability within multiple components of a particular cistron). Multiple components of a particular cistron may be separated by one or more restriction enzyme sites (including those unique to the vector) to allow interchangeability of one or more components within that cistron. In FIG. 1A, as an example, cistron 2 contains five separate components, although there may be two, three, four, five, six or more components per cistron. The example in FIG. 1A shows a unique restriction enzyme site X, each of which allows standard recombination to exchange different components 1, 2, 3, 4, and / or 5. 9 , X 10 , X 11 , X 12 , X 13 and X 14 Figure 1B includes cistron 2 having five components separated by . Figure 1B differs from Figure 1A only in illustrating that multiple restriction enzyme sites may be present between the different components (either unique, or one or more may not be unique), and that sequences may be present (or may not be present) between the multiple restriction enzyme sites. In certain embodiments, all components encoded by the cistrons are designed with the intent to be interchangeable. In certain cases, one or more components of a cistron may be designed to be interchangeable, while one or more other components of that cistron may not be designed to be interchangeable.

[0060] In a specific embodiment, in the example of Figure 1A and Figure 1B, the cistron encodes a CAR molecule or other antigen receptor with multiple components. For example, cistron 2 can contain a sequence encoding a CAR molecule with separate components represented by component 1, component 2, component 3, etc. The CAR molecule can contain two, three, four, five, six, seven, eight or more interchangeable components. In a specific example, component 1 in Figure 1A or Figure 1B encodes an scFv against an antigen of interest; component 2 encodes a hinge; component 3 encodes a transmembrane domain; component 4 encodes a costimulatory domain (although there can also be a component 4' encoding a second or more costimulatory domains flanked by restriction enzyme recognition sites for exchange); and component 5 encodes a signaling domain. In a particular example, component 1 encodes CD19scFv; component 2 encodes an IgG1 hinge and / or transmembrane domain; component 3 encodes CD28; and component 4 encodes CD3 zeta.

[0061] Those skilled in the art will recognize that in designing a vector, the various cistrons and components must be positioned so that they are maintained in frame, where necessary.

[0062] In the particular example of Figure 1A or Figure 1B, cistron 1 encodes a suicide gene; cistron 2 encodes a CAR; cistron 3 encodes a reporter gene; cistron 4 encodes a cytokine; component 1 of cistron 2 encodes an scFv; component 2 of cistron 2 encodes an IgG1 hinge; component 3 of cistron 2 encodes CD28; and component 4 encodes CD3 zeta. In alternative embodiments, none of the cistrons encodes an antigen receptor.

[0063] The restriction enzyme site may be of any type and may contain any number of bases in its recognition site (e.g., 4-8 bases). The number of bases in the recognition site may be at least 4, 5, 6, 7, 8 or more. The site may generate blunt or sticky ends upon cleavage. The restriction enzyme may be, for example, type I, type II, type III or type IV. Restriction enzyme sites may be obtained from available databases such as the Integrated relational Enzyme database (IntEnz) or BRENDA (The Comprehensive Enzyme Information System).

[0064] Exemplary vectors are shown in Figures 2 and 3. In Figure 2, the vector DNA is circular and, by convention, position 1 (the 12 o'clock position at the top of the circle, with the remaining sequences oriented clockwise) is set at the start of the 5'LTR.

[0065] In embodiments utilizing self-cleaving 2A peptides, the 2A peptides can be viral oligopeptides 18-22 amino acids (aa) long that mediate the "cleavage" of polypeptides during translation in eukaryotic cells. The "2A" designation 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), followed by E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2A), and T2A (thosea asigna virus 2A). The mechanism of 2A-mediated "self-cleavage" was discovered to be that the ribosome skips the formation of a glycyl-prolyl peptide bond at the C-terminus of 2A. The highly conserved sequence GDVEXNPGP (SEQ ID NO: 5) is shared by the C-terminus of various 2As and is useful for steric hindrance generation and ribosome skipping. Successful skipping and resumption of translation results in two "cleaved" proteins. An example of a 2A sequence is as follows: T2A: (GSG)EGRGSLLTCGDVEENPGP (SEQ ID NO: 1) P2A: (GSG)ATNFSLLKQAGDVEENPGP (SEQ ID NO: 2) E2A: (GSG)QCTNYALLKLAGDVESNPGP (SEQ ID NO: 3) F2A: (GSG)VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 4) A. Preparation method

[0066] Those of skill in the art would be well equipped 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) to express the antigen receptors of the present disclosure. 1. Regulatory elements

[0067] The expression cassette contained in the vector useful in the present disclosure includes, in particular, a eukaryotic transcription promoter operably linked to a protein coding sequence, a splice signal including an intervening sequence, and a transcription termination / polyadenylation sequence (5' to 3' direction). The promoter and enhancer that controls the transcription of protein-coding genes in eukaryotic cells can include multiple genetic elements. The cellular machinery can collect and integrate the regulatory information carried by each element, allowing different genes to develop different patterns of transcriptional control, often complex patterns. Promoters used in the context of the present disclosure include, for example, constitutive promoters, inducible promoters, and tissue-specific promoters. When the vector is used to create cancer treatment, the promoter can be effective under hypoxic conditions. A. Promoter / Enhancer

[0068] The expression constructs provided herein include promoters that drive the expression of antigen receptors and other cistron gene products. Promoters generally contain sequences that function to locate the start site for RNA synthesis. The best known example of this is the TATA box, but some promoters, such as the mammalian terminal deoxynucleotidyl transferase gene promoter and the SV40 late gene promoter, lack the TATA box and have discrete elements that overlap the start site itself to help fix the location of start. Additional promoter elements control the frequency of transcription initiation. Usually, these are located in the region upstream of the start site, but some promoters have been shown to contain functional elements downstream of the start site as well. To place a coding sequence "under the control of" a promoter, the 5' end of the transcription start site of the transcriptional reading frame is placed "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates transcription of DNA and promotes expression of the encoded RNA.

[0069] Spacing between promoter elements is often variable, and promoter function is preserved even when elements are inverted or moved relative to each other. For example, in the tk promoter, spacing between promoter elements can be increased by up to 50 bp before activity begins to decrease. Depending on the promoter, individual elements appear to be able to function cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of nucleic acid sequences.

[0070] A promoter may be a promoter naturally associated with a nucleic acid sequence, such as may be obtained by isolating 5' non-coding sequences located upstream of a coding segment and / or exon. Such a promoter may be referred to as an "endogenous" promoter. Similarly, an enhancer may be an enhancer naturally associated with a nucleic acid sequence, located downstream or upstream of the sequence. Alternatively, certain advantages may be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to 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 may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, as well as promoters or enhancers that are not "naturally occurring", i.e., that contain various elements of the various transcriptional control regions and / or mutations that alter expression. For example, promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose and tryptophan (trp-) promoter systems. In addition to synthetically producing promoter and enhancer nucleic acid sequences, sequences may be produced in conjunction with the compositions disclosed herein using recombinant cloning and / or nucleic acid amplification techniques, including PCR™. Furthermore, it is contemplated that regulatory sequences that direct transcription and / or expression of sequences in organelles other than the nucleus (e.g., mitochondria, chloroplasts, etc.) may be used as well.

[0071] Naturally, it becomes important to use a promoter and / or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ or organism selected for expression. Those skilled in the art of molecular biology are generally aware of the use of a combination of promoter, enhancer and cell type for protein expression (see, for example, Sambrook et al. 1989, which is incorporated herein by reference). The promoter used can be a constitutive promoter, a tissue-specific promoter, an inducible promoter, and / or a promoter that directs high-level expression of the introduced DNA segment and is useful under appropriate conditions (e.g., a promoter that is useful in large-scale production of recombinant proteins and / or recombinant peptides). The promoter can be heterologous or endogenous.

[0072] Additionally, any promoter / enhancer combination (e.g., according to the Eukaryotic Promoter Data Base EPDB via the World Wide Web at epd.isb-sib.ch / ) can be used to drive expression. The use of T3, T7 or SP6 cytoplasmic expression systems is another viable embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if a suitable bacterial polymerase is provided as part of the delivery complex or as an additional genetic expression construct.

[0073] Non-limiting examples of promoters include early or late viral promoters (e.g., SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus (RSV) early promoter); eukaryotic promoters (e.g., beta actin promoter, GADPH promoter, metallothionein promoter); and chain-like response element promoters (e.g., cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA) and minimal TATA box proximal response element promoter (tre)). It is also possible to use a human growth hormone promoter sequence (e.g., human growth hormone minimal promoter described in Genbank®, accession number X05244, nucleotides 283-341) or a 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 expression of therapeutic genes is applicable to the practice of the present disclosure.

[0074] In certain embodiments, the method of the present disclosure also relates to enhancer sequences, i.e., nucleic acid sequences that increase the activity of promoters, and have the ability to act in cis and in any orientation over relatively long distances (up to several kilobases away from the target promoter).However, enhancer function is not necessarily limited to such long distances, since enhancers can also function proximally to a given promoter. b. Initiation signal and ligated expression

[0075] Specific initiation signals may also be used in the expression constructs provided in this disclosure for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals (including the ATG initiation codon) may need to be provided. Those skilled in the art will be able to easily determine this and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be "in frame" with the reading frame of the desired coding sequence. The exogenous translational control signals and initiation codons may be natural or synthetic. Expression efficiency may be increased by including appropriate transcriptional enhancer elements.

[0076] In certain embodiments, the use of an internal ribosome entry site (IRES) element is used to generate multigene messages, i.e., polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated cap-dependent translation and initiate translation at internal sites. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) and IRES from mammalian messages have been reported. IRES elements can link heterologous open reading frames. Multiple open reading frames, each separated by an IRES, can be transcribed together to generate polycistronic messages. The IRES element allows each open reading frame to be accessible to ribosomes for efficient translation. Multiple genes can also be efficiently expressed using a single promoter / enhancer to transcribe a single message.

[0077] As detailed elsewhere herein, certain 2A sequence elements can be used to provide for linked or co-expression of genes in constructs provided in the present disclosure. For example, a cleavage sequence 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 specific embodiments, in a single vector, the multiple 2A sequences are not identical, but in alternative embodiments, the same vector utilizes two or more of the same 2A sequences. Examples of 2A sequences are provided in US2011 / 0065779, which is incorporated herein by reference in its entirety. c.Replication starting point To propagate a vector in a host cell, the vector may contain one or more origins of replication (often referred to as "ori"), such as a specific nucleic acid sequence at which replication is initiated, such as the oriP of EBV as described above, or a genetically engineered oriP with similar or enhanced function in programming. Alternatively, origins of replication of other viruses that replicate extrachromosomally, as described above, or autonomously replicating sequences (ARS) can be used. d. Selectable and Screenable Markers

[0078] In some embodiments, cells containing the constructs of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker brings about an identifiable change in cells that allows cells containing the expression vector to be easily identified. In general, a selection marker is a marker that confers a property that allows selection. A positive selection marker is a marker whose presence allows its selection, and a negative selection marker is a marker whose presence prevents selection. An example of a positive selection marker is a drug resistance marker.

[0079] Typically, the inclusion of a drug selection marker aids in cloning and identification of transformants; for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype that allows the differentiation of transformants based on the implementation of conditions, other types of markers are contemplated, including colorimetrically based screenable markers such as GFP. Alternatively, screenable enzymes can be utilized as negative selection markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT). Those skilled in the art will also know how to use immunological markers, possibly in conjunction with FACS analysis. The marker used is not believed to be critical, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection markers and screenable markers are well known to those skilled in the art. B. Genetically Engineered Antigen Receptors

[0080] The vector may encode one or more antigen receptors (e.g., engineered TCRs, CARs, decoy receptors, cytokine receptors, chimeric cytokine receptors, etc.). Multiple CARs and / or TCRs (e.g., against different antigens) may be used within the vector system. A single vector may encode two separate CAR molecules, or a single vector may encode one or more CAR molecules, at least one of which has specificity for two non-identical antigens (e.g., bispecific CARs, bispecific TCRs, or bispecific CAR / TCRs). The antigen receptors encoded by the vectors of the present disclosure, the vectors themselves, and the cells carrying the vectors are created by the hand of man and do not exist in nature.

[0081] In some embodiments, the CAR comprises an extracellular antigen recognition domain that specifically binds to an antigen. The CAR can be specifically designed to target an antigen of a particular tissue or cell type. In some embodiments, the antigen is a protein expressed on the cell surface. In some embodiments, the CAR is a TCR-like CAR, and the antigen is a processed peptide antigen (e.g., a peptide antigen of an intracellular protein) that is recognized on the cell surface in the context of major histocompatibility complex (MHC) molecules, similar to TCR.

[0082] Exemplary antigen receptors, including CARs and recombinant TCRs, and methods for engineering and introducing those receptors into cells are described in, for example, International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748 ... Nos. 130149337, 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. EP 2537416, and / or Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In some embodiments, the 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 / 2014055668A1. 1. Chimeric antigen receptor

[0083] In some embodiments, the CAR is encoded by the vector and comprises at least a) an intracellular signaling domain, b) a transmembrane domain, and c) an extracellular domain comprising at least one antigen-binding region.

[0084] In some embodiments, engineered antigen receptors include CARs, including activating or stimulatory CARs, costimulatory CARs (see WO2014 / 055668) and / or inhibitory CARs (iCARs, see Fedorov et al., 2013). These CARs generally comprise an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some embodiments via a linker and / or transmembrane domain. Such molecules usually mimic or mimic the signaling through a natural antigen receptor, through such receptors together with costimulatory receptors, and / or through costimulatory receptors alone. The CARs can be first, second or third generation or later.

[0085] Certain embodiments of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding antigen-specific CAR polypeptides (including humanized CARs (hCARs) to reduce immunogenicity) that include an intracellular signaling domain, a transmembrane domain, and an extracellular domain with one or more signaling motifs. In certain embodiments, the CAR may recognize an epitope that includes a space shared between one or more antigens. In certain embodiments, the binding region may include a complementarity determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor.

[0086] It is contemplated that the human CAR nucleic acid may be derived from a human gene to be used to enhance cellular immunotherapy for human patients. In a specific embodiment, the present disclosure includes the full-length cDNA or coding region of the CAR encoded by the vector. The antigen-binding region or domain is the V of a single-chain variable fragment (scFv) derived from a specific human monoclonal antibody. H Chain and V L The fragments may include fragments of the chains (e.g., those described in U.S. Patent No. 7,109,304, incorporated herein by reference). The fragments may also be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragments are antigen-specific scFvs encoded by sequences optimized for human codon usage for expression in human cells.

[0087] The arrangement may be a multimer (e.g., a diabody or multimer). The multimer is most likely formed by cross-pairing the variable portions of the light and heavy chains into a diabody. The hinge portion of the construct may have multiple options ranging from complete deletion, to maintaining the first cysteine, to proline substitution instead of serine substitution, to truncation up to the first cysteine. The Fc portion may or may not be deleted. Any protein that is stable and / or dimerizes may serve this purpose. Only one of the Fc domains may be used, for example the CH2 or CH3 domain of a human immunoglobulin. The hinge, CH2 and CH3 regions of a human immunoglobulin modified to improve dimerization may also be used. Only the hinge portion of an immunoglobulin may also be used. A portion of CD8 alpha may also be used.

[0088] In some embodiments, the CAR nucleic acid comprises a sequence encoding other costimulatory receptors, such as a transmembrane domain and one or more intracellular signaling domains (e.g., CD28 intracellular signaling domain). Other costimulatory 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 elicited by CD3ζ, the additional signal provided by the human costimulatory receptor inserted into the human CAR can be utilized to fully activate NK cells, helping to improve in vivo persistence and therapeutic success of adoptive immunotherapy.

[0089] In some embodiments, the CAR is constructed to have specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type targeted by adoptive therapy, e.g., a cancer marker and / or an antigen intended to induce an attenuated response (e.g., an antigen expressed on a normal or non-diseased cell type). Thus, the CAR typically comprises, 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 CAR comprises 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)).

[0090] In certain embodiments of the chimeric antigen receptor, the antigen-specific portion of the receptor (which may be referred to as the extracellular domain containing the antigen-binding region) comprises a tumor-associated antigen-binding domain or a pathogen-specific antigen-binding domain. Antigens include carbohydrate antigens recognized by pattern recognition receptors such as Dectin-1. The tumor-associated antigen may be of any type, so long as it is expressed on the cell surface of the tumor cell. Exemplary embodiments of tumor-associated antigens include CD19, CD20, carcinoembryonic antigen, alpha fetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, and the like. In certain embodiments, the CAR may be co-expressed with cytokines to improve persistence when the amount of tumor-associated antigen is low. For example, the CAR may be co-expressed with IL-2, IL-21, IL-12, IL-18, or IL-15.

[0091] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA sources, cDNA sources, or can be synthesized (e.g., via PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, since introns have been found to stabilize mRNA. It may also be more beneficial to use endogenous or foreign non-coding regions to stabilize mRNA.

[0092] It is contemplated that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector. Methods for stably transfecting cells by electroporation with naked DNA are known in the art. For example, see U.S. Pat. No. 6,410,319. Naked DNA generally refers to the DNA encoding the chimeric receptor contained in an expression vector in the proper orientation for expression. The modular polycistronic vector of the present disclosure may or may not be a viral vector (e.g., a plasmid). In the case of illustrative embodiments, the vector detailed herein is a retroviral vector, but in other cases, the vector is also a viral vector, but instead is, for example, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector.

[0093] Any vector can be used to introduce chimeric construct into immune cells.Vector suitable for use according to the method of the present disclosure is a vector that is non-replicative in immune cells.Many virus-based vectors are known (e.g., HIV, SV40, EBV, HSV or BPV-based vectors), and the copy number of the virus that is maintained in cells is low enough to maintain the viability of the cells.In a specific case, the vector is based on Moloney murine leukemia virus.

[0094] In some embodiments, the antigen-specific binding component or antigen-specific recognition component is linked to one or more transmembrane domains and intracellular signaling domains.In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR.In one embodiment, the 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 such domains binding to the transmembrane domain of the same or different surface membrane protein to minimize interaction with other members of the receptor complex.

[0095] The transmembrane domain, in some embodiments, is derived from natural or synthetic origin. If the origin is natural, the domain, in some embodiments, is derived from any membrane-bound or transmembrane protein. The transmembrane region includes the transmembrane region derived from (i.e., at least includes) the alpha, beta or zeta chain 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. Alternatively, the transmembrane domain, in some embodiments, is a synthetic transmembrane domain. In some embodiments, the synthetic transmembrane domain mainly comprises hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine may be found at each end of the synthetic transmembrane domain.

[0096] In certain embodiments, platform technologies disclosed herein for genetically modifying immune cells (e.g., T, NK, iNKT, B or MSC cells) include (i) non-viral gene transfer using an electroporation device (e.g., nucleofector), (ii) CARs that signal through an endodomain (e.g., CD28 / CD3-ζ, CD137 / CD3-ζ or other combinations), (iii) CARs with an extracellular domain of variable length that connects the antigen recognition domain to the cell surface, and in some cases, (iv) CARs + and artificial antigen-presenting cells (aAPCs) derived from K562, which can robustly and numerically expand immune cells ( Singh et al., 2008 ; Singh et al., 2011 ). 2. T cell receptor (TCR)

[0097] In some embodiments, genetically engineered antigen receptors include recombinant TCRs and / or TCRs cloned from naturally occurring T cells. "T cell receptor" or "TCR" refers to a molecule that contains a variable a chain and a variable β chain (also known as TCRα and TCRβ, respectively) or a variable γ chain and a variable δ chain (also known as TCRγ and TCRδ, respectively) and that can specifically bind to an antigenic peptide bound to an MHC receptor. In some embodiments, the TCR is of the αβ type.

[0098] TCRs, which usually exist as αβ and γδ types, are generally similar in structure, but the T cells expressing them may differ in anatomical location or function. TCRs may be found on the cell surface or in 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 the surface. In some embodiments, TCRs may also include a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al, 1997). For example, in some embodiments, each chain of the TCR may 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 the invariant protein of the CD3 complex, which is involved in mediating signal transduction. Unless otherwise stated, the term "TCR" should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full-length TCRs, including TCRs of the αβ or γδ types.

[0099] Thus, for purposes herein, reference to a TCR includes any TCR or functional fragment (e.g., an antigen-binding portion of a TCR that binds to a specific antigenic peptide bound in an MHC molecule, i.e., an MHC-peptide complex). An "antigen-binding portion" or antigen-binding fragment of a TCR, which may be used interchangeably, refers to a molecule that contains only a portion of the structural domain of the TCR but binds to the antigen (e.g., an MHC-peptide complex) that the complete TCR binds. In some cases, the antigen-binding portion includes sufficient variable domains of the TCR (e.g., the variable a and variable β chains of the TCR) to form a binding site for binding to a specific MHC-peptide complex, e.g., each chain generally includes three complementarity determining regions.

[0100] In some embodiments, the variable domains of the TCR chains associate to form loops, or complementarity determining regions (CDRs) similar to immunoglobulins, which provide antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule. Usually, like immunoglobulins, the CDRs are separated by framework regions (FRs) (see, for example, Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the main CDR involved in the recognition of processed antigens, but CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the beta chain interacts with the C-terminal part of the peptide. CDR2 is believed to recognize MHC molecules. In some embodiments, the variable region of the beta chain may contain an additional hypervariable (HV4) region.

[0101] In some embodiments, the TCR chain comprises a constant domain. For example, similar to an immunoglobulin, the extracellular portion of the TCR chain (e.g., a chain, β chain) comprises two immunoglobulin domains, a variable domain (e.g., V aor Vp; usually Kabat numbering, 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.), and one constant domain adjacent to the cell membrane (e.g., the a chain constant domain or C a The TCR may comprise a α-chain constant domain or Cp, typically amino acids 117-259 based on Kabat, and a 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 the two chains comprises two membrane proximal constant domains, and two membrane distal variable domains that include the CDRs. The constant domain of the TCR domain comprises a short connecting sequence in which cysteine ​​residues form disulfide bonds, thereby forming a link between the two chains. In some embodiments, the TCR may have additional cysteine ​​residues in each of the α-chain and the β-chain such that the TCR comprises two disulfide bonds in the constant domain.

[0102] 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 together with a transmembrane region can anchor the protein to the cell membrane and associate with the invariant subunit of the CD3 signaling apparatus or complex.

[0103] In general, CD3 is a multiprotein complex that may have three different chains (γ, δ, and ε) and a ζ chain in mammals. For example, in mammals, the complex may include CD3γ, CD3δ, two CD3ε, and a homodimeric CD3ζ chain. CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of CD3γ, CD3δ, and CD3ε chains are negatively charged, a property that allows these chains to associate with the positively charged T cell receptor chain. Each intracellular tail of CD3γ, CD3δ, and CD3ε chains contains a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, whereas each CD3ζ chain contains three. In general, ITAMs are 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 chain and the ζ chain associate with the TCR to form a complex known as the T cell receptor complex.

[0104] In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), or may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two separate chains (α and β or γ and δ) linked by a disulfide bond or the like. In some embodiments, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into a cell. In some embodiments, a nucleic acid encoding the TCR may be obtained from a variety of 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 source, e.g., a cell, e.g., a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other publicly available source. In some embodiments, the T cell may be obtained from an in vivo isolated cell. In some embodiments, a high affinity T cell clone may be isolated from a patient and the TCR may be isolated. In some embodiments, the T cell may be a cultured T cell hybridoma or clone. In some embodiments, TCR clones against target antigens have been produced in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system or HLA). See, for example, tumor antigens (see, for example, Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate TCRs against target antigens (see, for example, Varela-Rohena et al., 2008 and Li et al., 2005). In some embodiments, TCRs or antigen-binding portions thereof can be synthetically produced with knowledge of the sequence of the TCR. C. Cytokine Co-expression

[0105] One or more cytokines can be co-expressed from the vector as separate polypeptides from the antigen receptor. For example, interleukin-15 (IL-15) is tissue-restricted and is only observed in serum or whole body at any level under pathological conditions. IL-15 has several desirable properties for adoptive therapy. IL-15 is a homeostatic cytokine that induces the development and cell proliferation of natural killer cells, promotes the annihilation of established tumors through the relief of the functional suppression of tumor-resident cells, and inhibits AICD.

[0106] In some embodiments, the present disclosure relates to the simultaneous use of CAR and / or TCR vectors and IL-15.In addition to IL-15, other cytokines are also envisaged.These include, but are not limited to, cytokines, chemokines and other molecules that contribute to the activation and proliferation of cells used for human application.NK cells or T cells that express IL-15 are capable of sustained supportive cytokine signaling, which is essential for their survival after infusion.

[0107] After genetic modification, the cells can be injected immediately or stored. In certain embodiments, after genetic modification, within about 1, 2, 3, 4, 5 days or more after gene introduction into the cells, the cells can be expanded ex vivo as a bulk population for days, weeks or months. In further embodiments, the transfectants are cloned and clones that show the presence of a single integrated or episomally maintained expression cassette or plasmid and expression of the chimeric receptor are expanded ex vivo. Clones selected for expansion show the ability to specifically recognize and lyse target cells expressing CD19. 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. D. Antigen

[0108] Antigens targeted by genetically engineered antigen receptors include antigens expressed in the context of the disease, condition or cell type targeted via adoptive cell therapy. These diseases and conditions include proliferative, neoplastic and malignant diseases and disorders, including cancers and tumors, including blood cancers, cancers of the immune system (e.g., lymphomas, leukemias and / or myelomas, e.g., B, T and myeloid leukemias, lymphomas and multiple myelomas), and autoimmune or alloimmune conditions. In some embodiments, the antigen is selectively expressed or overexpressed on the cells of the disease or condition, e.g., on tumor cells or pathogenic cells, compared to normal cells or tissues or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or expressed on engineered cells. In some cases, the antigen is associated with an immune-related disorder.

[0109] Any suitable antigen can be used in the method. Exemplary antigens include, but are not limited to, infectious agents, auto / self antigens, tumor-associated / cancer-associated antigens, and tumor neoantigen-derived antigenic molecules (Linnemann et al., 2015). In certain embodiments, the antigens include NY-ESO, EGFRvIII, Muc-1, Her2, CA-125, WT-1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, and CEA. In certain embodiments, antigens for two or more antigen receptors include, but are not limited to, CD19, EBNA, WT1, CD123, NY-ESO, EGFRvIII, MUC1, HER2, CA-125, WT1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, and / or CEA. Sequences for these antigens, e.g., CD19 (accession number NG_007275.1), EBNA (accession number NG_002392.2), WT1 (accession number NG_009272.1), CD123 (accession number NC_000023.11), NY-ESO (accession number NC_000023.11), EGFRvIII (accession number NG_007726.3), MUC1 (accession number NG_029383.1), HER2 (accession number NG_00 7503.1), CA-125 (Accession No. NG_055257.1), WT1 (Accession No. NG_009272.1), Mage-A3 (Accession No. NG_013244.1), Mage-A4 (Accession No. NG_013245.1), Mage-A10 (Accession No. NC_000023.11), TRAIL / DR4 (Accession No. NC_000003.12), and / or CEA (Accession No. NC_000019.10) are known in the art.

[0110] Tumor-associated antigens can be derived from prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, renal cancer, mesothelioma, ovarian cancer or melanoma. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE1, 3 and MAGE4 (or other MAGE antigens, such as those disclosed in International Patent Publication No. WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of tumor types, such as melanoma, lung cancer, sarcoma and bladder cancer. See, for example, U.S. Patent No. 6,544,518. Tumor-associated antigens of prostate cancer include, for example, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphate, NKX3.1 and six-transmembrane epithelial antigen of prostate (STEAP).

[0111] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto and Criptin. Additionally, tumor antigens can be self-peptide hormones, such as full-length gonadotropin releasing hormone (GnRH), short 10 amino acid long peptides, which are useful in the treatment of many cancers.

[0112] Tumor antigens include tumor antigens derived from cancers characterized by the expression of tumor-associated antigens, such as expression of HER-2 / neu. Tumor-associated antigens of interest include lineage-specific tumor antigens, such as melanocyte-melanoma lineage antigen MART-1 / Melan-A, gp100, gp75, mda-7, tyrosinase and tyrosinase-related proteins. Illustrative tumor-associated antigens include p53, Ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MART-1, MC1R, Gp100, PSA, PSM, tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CEA, CDK-4 / m, ELF2M, GnT-V, G250 , HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signaling transduction 1 (TACSTD1), TACSTD2, receptor tyrosine kinase (TKI) For example, epidermal growth factor receptor (EGFR) (particularly EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), cytoplasmic tyrosine kinase (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducer and activator of transcription STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor-kappa B (NF-B),Notch receptors (e.g., Notch1-4), c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and their regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1, and tumor antigens derived from or including any one or more of the following idiotypes.

[0113] Antigens can include epitopic regions or epitopic peptides derived from genes mutated in tumor cells or from genes that are transcribed at different levels in tumor cells compared to normal cells (e.g., aberrantly expressed intronic sequences such as telomerase enzyme, survivin, mesothelin, mutant ras, bcr / abl rearrangements, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and N-acetylglucosaminyltransferase-V); clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myelomas and B-cell lymphomas; tumor antigens that contain epitopic regions or epitopic peptides derived from oncoviral processes (e.g., human papillomavirus proteins E6 and E7); Epstein-Barr virus protein LMP2; and non-mutated oncofetal proteins with tumor-selective expression (e.g., carcinoembryonic antigen and alpha-fetoprotein).

[0114] In other embodiments, antigens are obtained or derived from pathogenic or opportunistic pathogenic microorganisms (also referred to herein as infectious disease microorganisms) (e.g., viruses, fungi, parasites, and bacteria). In certain embodiments, antigens derived from such microorganisms include full-length proteins.

[0115] Illustrative pathogenic organisms having antigens contemplated for use in the methods described herein include human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza A, B and C, vesicular stomatitis virus (VSV), polyomaviruses (e.g., BK virus and JC virus), adenovirus, Staphylococcus species, including methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus species, including Streptococcus pneumoniae. As one of skill in the art will appreciate, proteins from these and other pathogenic microorganisms for use as antigens as described herein, as well as the nucleotide sequences encoding those proteins, may be identified in publications and public databases, such as GENBANK®, SWISS-PROT®, and TREMBL®.

[0116] Antigens derived from Human Immunodeficiency Virus (HIV) include any of the HIV virion structural proteins (e.g., gp120, gp41, p17, p24), protease, reverse transcriptase, or any of the HIV proteins encoded by tat, rev, nef, vif, vpr, and vpu.

[0117] Antigens derived from herpes simplex viruses (e.g., HSV1 and HSV2) include, but are not limited to, proteins expressed from HSV late genes. The late group of genes primarily encode proteins that form virion particles. Such proteins include the five proteins that form the viral capsid (UL): UL6, UL18, UL35, UL38, and the major capsid proteins UL19, UL45, and UL27, each of which can be used as an antigen as described herein. Other illustrative HSV proteins contemplated for use as antigens herein include ICP27 (H1, H2), glycoprotein B (gB), and glycoprotein D (gD) proteins. The HSV genome contains at least 74 genes, each of which encodes a protein that can potentially be used as an antigen.

[0118] Antigens derived from cytomegalovirus (CMV) include CMV structural proteins, viral antigens expressed during immediate early and early viral replication, glycoproteins I and III, capsid proteins, coat proteins, lower matrix proteins pp65 (ppUL83), p52 (ppUL44), IE1 and 1E2 (UL123 and UL122), protein products of the UL128-UL150 gene cluster (Rykman, et al., 2006), envelope glycoproteins B (gB), gH, gN, and pp150. As one of skill in the art will appreciate, CMV proteins for use as antigens described herein may be identified in public databases such as GENBANK®, SWISS-PROT®, and TREMBL® (see, e.g., Bennekov et al., 2004; Loewendorf et al., 2010; Marschall et al., 2009).

[0119] Antigens derived from Epstein-Van virus (EBV) contemplated for use in certain embodiments include EBV proteins produced during the latent infection cycle, including EBV lytic proteins gp350 and gp110, Epstein-Van nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP), and latent membrane protein (LMP)-1, LMP-2A, and LMP-2B (see, e.g., Lockey et al., 2008).

[0120] Antigens derived from respiratory syncytial virus (RSV) contemplated for use herein include any of 11 proteins or antigenic fragments thereof encoded by the RSV genome: NS1, NS2, N (nucleocapsid protein), M (matrix protein) SH, G and F (viral coat proteins), M2 (second matrix protein), M2-1 (elongation factor), M2-2 (transcriptional control), RNA polymerase, and phosphoprotein P.

[0121] Antigens derived from vesicular stomatitis virus (VSV) contemplated for use include any one of the five major proteins and antigenic fragments thereof encoded by the VSV genome: large protein (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P) and matrix protein (M) (see, e.g., Rieder et al., 1999).

[0122] Antigens derived from influenza virus that are contemplated for use in certain embodiments include hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins M1 and M2, NS1, NS2 (NEP), PA, PB1, PB1-F2, and PB2.

[0123] Exemplary viral antigens include adenovirus polypeptides, alphavirus polypeptides, calicivirus polypeptides (e.g., calicivirus capsid antigens), coronavirus polypeptides, distemper virus polypeptides, Ebola virus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis virus (AE) polypeptides (including hepatitis B core or surface antigens, hepatitis C virus E1 or E2 glycoproteins, core or nonstructural proteins), herpesvirus polypeptides (including herpes simplex virus or varicella zoster virus glycoproteins), infectious peritonitis virus polypeptides, leukemia virus polypeptides, Marburg virus polypeptides, and the like. Also included are, but are not limited to, polypeptides of viruses, orthomyxoviruses, papillomaviruses, polypeptides of parainfluenza viruses (e.g., hemagglutinin and neuraminidase polypeptides), polypeptides of paramyxoviruses, polypeptides of parvoviruses, polypeptides of pestiviruses, polypeptides of picornaviruses (e.g., capsid polypeptides of polioviruses), polypeptides of poxviruses (e.g., polypeptides of vaccinia viruses), polypeptides of rabies viruses (e.g., glycoprotein G of rabies virus), polypeptides of reoviruses, polypeptides of retroviruses, and polypeptides of rotaviruses.

[0124] In certain embodiments, the antigen may be a bacterial antigen. In certain embodiments, the bacterial antigen of interest may be a secreted polypeptide. In certain other embodiments, the bacterial antigen includes an antigen that has a portion of a polypeptide exposed on the extracellular surface of the bacteria.

[0125] Antigens from Staphylococcus species, including Methicillin-resistant Staphylococcus aureus (MRSA), that are intended for use include virulence regulators, such as Agr system, Sar and Sae, Arl system, Sar homologues (Rot, MgrA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ and TcaR), Srr system and TRAP.Other Staphylococcus proteins that can serve as antigens include Clp protein, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA and CfvB (see, for example, Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, Ed. Jodi Lindsay). The genomes of two species of Staphylococcus aureus (N315 and Mu50) have been sequenced and are publicly available, for example, at PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, Snyder et al., 2007). As one of skill in the art will appreciate, Staphylococcus proteins for use as antigens may also be identified in other public databases, such as GenBank®, Swiss-Prot®, and TrEMBL®.

[0126] Antigens from Streptococcus pneumoniae contemplated for use in certain embodiments described herein include pneumolysin, PspA, choline-binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and pilin proteins (RrgA; RrgB; RrgC). Antigenic proteins of Streptococcus pneumoniae are also known in the art and may be used as antigens in some embodiments (see, for example, Zysk et al., 2000). The complete genome sequence of a virulent strain of Streptococcus pneumoniae has been sequenced, and as one of skill in the art will appreciate, S. pneumoniae proteins for use herein may also be identified in other public databases, such as GENBANK®, SWISS-PROT®, and TREMBL®. Proteins of particular interest as antigens according to the present disclosure include virulence factors and proteins predicted to be exposed on the surface of S. pneumoniae (see, e.g., Frolet et al., 2010).

[0127] Examples of bacterial antigens that can be used as antigens include Actinomyces polypeptides, Bacillus polypeptides, Bacteroides polypeptides, Bordetella polypeptides, Bartonella polypeptides, Borrelia polypeptides (e.g., B. burgdorferi OspA), Brucella polypeptides, Campylobacter polypeptides, Capnocytophaga polypeptides, Chlamydia polypeptides, Corynebacterium polypeptides, Coxiella polypeptides, Dermatophilus polypeptides, Enterococcus polypeptides, Ehrlichia polypeptides, Escherichia polypeptides, Francisella polypeptides, Fusobacterium polypeptides, Haemobartonella polypeptides, Haemophilus polypeptides (e.g., H. influenzae b-type outer membrane proteins), Helicobacter polypeptides, Klebsiella polypeptides, L-type bacteria polypeptides, Leptospira polypeptides, Listeria polypeptides, Mycobacterium polypeptides, Mycoplasma polypeptides, Neisseria polypeptides, Neorickettsia polypeptides, Nocardia polypeptides, Pasteurella polypeptides, Peptococcus polypeptides, Peptostreptococcus polypeptides, Streptococcus pneumoniae polypeptides (i.e., S. pn eumoniae polypeptides) (see description herein), Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Roshalimea polypeptides, Salmonella polypeptides, Shigella polypeptides, Staphylococcus polypeptides, Group A Streptococcus polypeptides (e.g., the M protein of S. pyogenes), Group B Streptococcus (S. agalactiae) polypeptides, Treponema polypeptides, and Yersinia polypeptides (e.g., the F1 and V antigens of Y pestis).

[0128] Examples of fungal antigens include Absidia polypeptides, Acremonium polypeptides, Alternaria polypeptides, Aspergillus polypeptides, Basidiobolus polypeptides, Bipolaris polypeptides, Blastomyces polypeptides, Candida polypeptides, Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum polypeptides, Histoplasma polypeptides, Madurella polypeptides, Malassezia polypeptides, Microsporum polypeptides, Moniliella polypeptides, Mortierella polypeptides, Mucous membrane polypeptides, Pecorinolytic enzymes, and the like. These include, but are not limited to, polypeptides of the genus Silomyces, Polypeptides of the genus Penicillium, Polypeptides of the genus Phialemonium, Polypeptides of the genus Phialophora, Polypeptides of the genus Prototheca, Polypeptides of the genus Pseudoalescheria, Polypeptides of the genus Pseudomicrodochium, Polypeptides of the genus Phythium, Polypeptides of the genus Rhinosporidium, Polypeptides of the genus Rhizopus, Polypeptides of the genus Scolecobasidium, Polypeptides of the genus Sporothrix, Polypeptides of the genus Stemphylium, Polypeptides of the genus Trichosporon, Polypeptides of the genus Xylohypha.

[0129] Examples of protozoan parasite antigens include, but are not limited to, Babesia polypeptides, Balantidium polypeptides, Besnoitia polypeptides, Cryptosporidium polypeptides, Eimeria polypeptides, Encephalitozoon polypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammondia polypeptides, Hepatozoon polypeptides, Isospora polypeptides, Leishmania polypeptides, Microsporidia polypeptides, Neospora polypeptides, Nosema polypeptides, Pentatrichomonas polypeptides, and Plasmodium polypeptides. Examples of helminth parasite antigens include Acanthocheilonema polypeptides, Aelurostrongylus polypeptides, Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascaris polypeptides, Brugia polypeptides, Bunostomum polypeptides, Capillaria polypeptides, Cabertia polypeptides, Cooperia polypeptides, Crenosoma polypeptides, Dictyocaulus polypeptides, Dioctophyme polypeptides, Dipetalonema polypeptides, Diphyllobothrium polypeptides, Dipridium polypeptides, Dirofilaria polypeptides, Dracunculus polypeptides, Enterobius polypeptides, Filaroides polypeptides, Haemonchus polypeptides, Lagochilascaris polypeptides, Loa polypeptides, Mansonella polypeptides, Muellerius polypeptides, Nanophyetus polypeptides, Ancylostoma polypeptides, Nematogyrus polypeptides, Heterodera polypeptides, Onchocerca polypeptides, Opisthorchis polypeptides, Ostertagia polypeptides, Parafilaria polypeptides, Paragonimus polypeptides, Parascaris polypeptides, Physaloptera polypeptides,Protostrongylus polypeptides, Setaria polypeptides, Spirocerca polypeptides, Spirometra polypeptides, Stephanofilaria polypeptides, Strongyloides polypeptides, Strongylus polypeptides, Thelazia polypeptides, Toxascaris polypeptides, Toxocara polypeptides, Trichinella polypeptides, Trichuris polypeptides, Trichuria polypeptides, Uncinaria polypeptides and Uchleria polypeptides (e.g., P. falciparum circumsporozoite polypeptide (PfCSP)), sporozoite surface protein 2 (PfSSP2), carboxyl terminus of liver state antigen 1 (PfLSA1 c-terminus), and export protein 1 (PfExp-1), Pneumocystis polypeptide, Sarcocystis polypeptide, Schistosoma polypeptide, Theileria polypeptide, Toxoplasma polypeptide, and Trypanosoma polypeptide.

[0130] Examples of ectoparasite antigens include, but are not limited to, polypeptides (including antigens and allergens) from fleas; ticks, including hard mites and ulcerative mites; flies, such as midges, mosquitoes, sand flies, black flies, bot flies, horn flies, deer flies, tsetse flies, stable flies, flies that cause myiasis and small biting gnats; ants; spiders, lice; mites; and hemipteran insects, such as bedbugs and assassin bugs. E. Suicide gene

[0131] The cells of the present disclosure modified to carry the vectors encompassed by the present disclosure may contain one or more suicide genes. As used herein, the term "suicide gene" is defined as a gene whose gene product changes into a compound that kills host cells when a prodrug or other agent is administered. Examples of suicide gene / prodrug combinations that can be used are herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside.

[0132] The so-called suicide gene, purine nucleoside phosphorylase of E. coli, which converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine, can be used. Other examples of suicide genes used with prodrug therapy are the E. coli cytosine deaminase gene and the HSV thymidine kinase gene.

[0133] Exemplary suicide genes include CD20, CD52, EGFRv3 or inducible caspase 9. In one embodiment, a truncated version of EGFR variant III (EGFRv3) can be used as a suicide antigen that can be cleaved by cetuximab. Additional suicide genes known in the art that can be used in the present disclosure include purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-α,γ-lyase (MET) and thymidine phosphorylase (TP). In a specific embodiment, a mutated TNF-alpha suicide gene is used that encodes a non-secreted TNF-alpha protein that is expressed on the cell membrane and therefore can be targeted by inhibitors such as antibodies, as described in U.S. Provisional Patent Application No. 62 / 769,405, filed November 19, 2018, U.S. Provisional Patent Application No. 62 / 773,372, filed November 30, 2018, and U.S. Provisional Patent Application No. 62 / 791,464, filed January 11, 2019, all of which are incorporated by reference in their entireties. III. Immune cells

[0134] Certain embodiments of the present disclosure relate to immune cells expressing one or more antigen receptors, such as chimeric antigen receptors (CARs) and / or T cell receptors (TCRs). The immune cells can be of any type, such as T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma-delta T cells), NK cells, invariant NK cells, NKT cells, B cells, stem cells (e.g., mesenchymal stem cells (MSCs) or induced pluripotent stem (iPSC) cells). In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. Methods of making and manipulating immune cells, as well as methods of using and administering the cells, such as for adoptive cell therapy, are also provided herein, where the cells can be autologous or allogeneic to the recipient. Thus, the immune cells can be used as immunotherapy, such as to target cancer cells.

[0135] The immune cells can be isolated from subjects, particularly human subjects, including individuals who need treatment. The immune cells can be obtained from subjects of interest (e.g., subjects suspected of having a particular disease or condition, subjects suspected of having a predisposition to a particular disease or condition, or subjects undergoing treatment for a particular disease or condition). The immune cells can be collected from any location where they are present in a subject, including, but not limited to, blood, umbilical cord blood, spleen, thymus, lymph nodes, and bone marrow. The isolated immune cells can be used directly or can be stored for a period of time, such as by freezing.

[0136] The immune cells may be enriched / purified from any tissue in which they reside, including, but not limited to, blood (including blood collected by blood banks or umbilical cord blood banks), spleen, bone marrow, tissues removed and / or exposed during surgical procedures, and tissues obtained via biopsy procedures. The tissues / organs from which immune cells are enriched, isolated and / or purified may be isolated from both living and non-living subjects, where the non-living subject is an organ donor. In certain embodiments, immune cells are isolated from blood, such as peripheral blood or umbilical cord blood. In some embodiments, immune cells isolated from umbilical cord blood have high immunoregulatory capacity, as measured by suppression of CD4+ or CD8+ T cells. In a specific embodiment, immune cells are isolated from pooled blood, particularly pooled umbilical cord blood, for high immunoregulatory capacity. Pooled blood may be blood from two or more sources, such as 3, 4, 5, 6, 7, 8, 9, 10 or more sources (e.g., donor subjects).

[0137] The population of immune cells can be obtained from a subject in need of treatment or from a subject suffering from a disease associated with low immune cell activity. Thus, the cells can be autologous to the subject in need of treatment. Alternatively, the population of immune cells can be obtained from a donor, preferably a histocompatibility-matched donor. The immune cell population can be collected from peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells are present in the subject or donor. The immune cells can be isolated from a pool of subjects and / or donors, for example, pooled umbilical cord blood.

[0138] When the immune cell population is obtained from a donor different from the subject, the donor is preferably allogeneic, but the obtained cells are matched with the subject in that they can be introduced into the subject. Allogeneic donor cells may or may not be human leukocyte antigen (HLA) matched. To make them compatible with the subject, allogeneic cells can be treated to reduce immunogenicity (Fast et al., 2004). IV. Treatment Method

[0139] In some embodiments, the present disclosure provides a method for treatment, including immunotherapy, comprising administering an effective amount of immune cells encompassed in the present disclosure engineered to express the modular vector system provided herein. In some embodiments, a medical disease or disorder is treated by the transfer of an immune cell population that induces an immune response in a recipient individual. In certain embodiments of the present disclosure, a cancer or infectious disease is treated by the transfer of an immune cell population that induces an immune response. Provided herein is a method for treating cancer or delaying the progression of cancer in an individual, comprising administering to the individual an effective amount of an antigen-specific cell therapy (cell therapy specific for one or more antigens). The method may be applied to the treatment of immune disorders, solid cancers, hematological cancers, and viral infections.

[0140] When the individual in need of treatment has cancer, the cancer may be a blood cancer or may include a solid tumor. The tumors for which the present treatment method is useful include any malignant cell type, such as those found in solid tumors or blood malignancies. Exemplary solid tumors may include, but are not limited to, tumors of organs or tissues selected from the group consisting of pancreas, colon, appendix, stomach, brain, head, neck, ovaries, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, skin, thyroid, gallbladder, spleen, liver, bone, endometrium, testis, cervix, esophagus, prostate and breast. Exemplary blood tumors include tumors of bone marrow, T or B cell malignancies, leukemia, lymphoma, blastoma, myeloma, etc. Further examples of cancers that can be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric or stomach cancer (including digestive and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0141] The cancer may specifically be cancer of the following histological types, but is not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma-cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; adenocarcinoma, familial polyposis coli; solid tumor; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophilic carcinoma; acidophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cellular carcinoma. follicular carcinoma;follicular adenocarcinoma;papillary-follicular adenocarcinoma;nonencapsulated sclerosing carcinoma;adrenal cortical carcinoma;endometrioid carcinoma;adnexal carcinoma;apocrine gland carcinoma;sebaceous gland carcinoma;earwax gland carcinoma;mucoepidermoid carcinoma;cystadenocarcinoma;papillary cystadenocarcinoma;papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma;mucinous adenocarcinoma;signet ring cell carcinoma;invasive ductal carcinoma;medullary carcinoma;lobular carcinoma;inflammatory carcinoma;Paget's disease, breast;acinic cell carcinoma;adenosquamous carcinoma;adenocarcinoma with squamous metaplasia;thymoma, malignant;ovarian stromal tumor, malignant;theca cell tumor, malignant;granulosa cell tumor, malignant;androblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;lipid cell tumor, malignant;paraganglioma tumor, malignant;extramammary paraganglioma, malignant;pheochromocytoma;glomus angiosarcoma;malignant melanoma;amelanotic melanoma;superficial spreading melanoma;lentigo maligna melanoma;acral lentigo melanoma;nodular melanoma;malignant melanoma in giant pigmented nevus;epithelioid cell melanoma;blue nevus, malignant;sarcoma;fibrosarcoma;fibrous histiocytoma, malignant;myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;mixed tumor, malignant;mixed Müllerian tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;mesenchymoma, malignant;Brenner tumor, malignant;phyllodes tumor, malignant;synovial sarcoma;mesothelioma, malignant;dysgerminoma;fetal carcinoma;teratoma, malignant;ovarial goiter, malignant;choriocarcinoma;mesonephroma, malignant;angiosarcoma;hemangioendothelioma, malignant;Kaposi's sarcoma;hemangiopericytoma, malignant;lymphangiosarcoma;osteosarcoma;paracortical osteosarcoma;chondrosarcoma;chondroblastoma, malignant;mesenchymal chondrosarcoma;giant cell tumor of bone;Ewing's sarcoma;odontogenic tumor, malignant;ameloblastoma, malignant;ameloblastoma, malignant;ameloblastoma fibrosarcoma;pinealoma, malignant;chordoma;glioma, malignant;ependymoma;astrocytoma;protoplasmic astrocytoma;fibrillary astrocytoma;astroblastoma;glioblastoma;oligodendroglioma;primitive neuroectodermal;Cerebellar sarcoma;Ganglioneuroblastoma;Neuroblastoma;Retinoblastoma;Olfactory neurogenic tumor;Meningioma, malignant;Neurofibrosarcoma;Neurilemoma, malignant;Granular cell tumor, malignant;Malignant lymphoma;Hodgkin's disease;Hodgkin;Side granuloma;Malignant lymphoma, small lymphocytic;Malignant lymphoma, large cell, diffuse;Malignant lymphoma, follicular;Mycosis fungoides;Other specified non-Hodgkin's lymphoma;B-cell lymphoma;Low-grade / follicular non-Hodgkin's lymphoma (NHL);Small lymphocytic (SL) NHL;Intermediate-grade / follicular NHL;Intermediate-grade diffuse NHL;High-grade immunoblastic NHL;High-grade lymphoblastic NHL;High-grade small noncleaved cell NHL;Bulky disease disease)NHL;mantle cell lymphoma;AIDS-related lymphoma;Waldenstrom's macroglobulinemia;malignant histiocytosis;multiple myeloma;mast cell sarcoma;immunoproliferative small intestinal disease;leukemia;lymphocytic leukemia;plasma cell leukemia;erythroleukemia;lymphoblastic cell leukemia;myeloid leukemia;basophilic leukemia;eosinophilic leukemia;monocytic leukemia;mast cell leukemia;megakaryoblastic leukemia;myeloid sarcoma;hairy cell leukemia;chronic lymphocytic leukemia (CLL);acute lymphoblastic leukemia (ALL);acute myeloid leukemia (AML);and chronic myeloblastic leukemia. ;

[0142] Certain embodiments relate to methods of treating leukemia. Leukemia is a cancer of the blood or bone marrow, characterized by abnormal proliferation (production by division) of blood cells, usually white blood cells (leukocytes). Leukemia is part of a broad group of diseases called hematological neoplasms. Leukemia is a broad term that covers a wide variety of diseases. Leukemia is clinically and pathologically divided into acute and chronic forms.

[0143] In certain embodiments of the present disclosure, immune cells are delivered to an individual in need thereof (e.g., an individual with cancer or infectious disease). These cells then boost the individual's immune system to attack the respective cancer cells or pathogenic cells. In some cases, the individual is provided with immune cells one or more times. When the individual is provided with immune cells two or more times, the time between administrations should be sufficient for the immune cells to spread in the individual, and in specific embodiments, the time between administrations is 1, 2, 3, 4, 5, 6, 7 days or more.

[0144] Certain embodiments of the present disclosure provide a method for treating or preventing immune-mediated disorders. In one embodiment, the subject has an autoimmune disease. Non-limiting examples of autoimmune diseases include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune disease of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac polydermatitis, and the like. spate-dermatitis), chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, nephrotic syndrome (e.g., microvascular fibrosis, pulmonary ... These include: inflammatory bowel disease (e.g., pulmonary edema, focal glomerulosclerosis or membranous nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff man syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveitis, vasculitis (e.g., polyarteritis nodosa, Takayasu's arteritis, temporal arteritis / giant cell arteritis or dermatitis herpetiformis vasculitis), vitiligo and Wegener's granulomatosis. Thus, some examples of autoimmune diseases that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, Crohn's disease; ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis or psoriasis.Subjects can also have allergic disorders, such as asthma.

[0145] In yet another embodiment, the subject is a recipient of a transplanted organ or stem cells, and the immune cells are used to prevent and / or treat rejection. In certain embodiments, the subject has or is at risk of developing graft-versus-host disease. GVHD is a possible complication of any transplant that uses or includes stem cells from related or unrelated donors. There are two types of GVHD: acute and chronic. Acute GVHD appears within the first three months after transplant. Signs of acute GVHD include a reddish rash on the hands and feet, which may spread with peeling or blistering of the skin and may become more severe. Acute GVHD may also affect the stomach and intestines, where muscle cramps, nausea, and diarrhea are observed. Yellowing of the skin and eyes (jaundice) indicates that acute GVHD is affecting the liver. Chronic GVHD is graded based on its severity: stage / grade 1 is mild; stage / grade 4 is severe. Chronic GVHD develops 3 months or later after transplantation. Symptoms of chronic GVHD are similar to those of acute GVHD, but in addition, chronic GVHD can affect the mucous glands of the eye, the salivary glands of the mouth, and the glands that lubricate the stomach wall and intestine. Any immune cell population disclosed herein can be used. Examples of transplanted organs include organ grafts, such as kidney, liver, skin, pancreas, lung, and / or heart, or cell grafts, such as islets, hepatocytes, myoblasts, bone marrow, or hematopoietic or other stem cells. The graft can be a composite graft, such as facial tissue. Immune cells can be administered before, at the same time, or after transplantation. In some embodiments, the immune cells are administered prior to transplantation, e.g., at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 1 month prior to transplantation. In one specific, non-limiting example, administration of a therapeutically effective amount of immune cells occurs 3-5 days prior to transplantation.

[0146] In some embodiments, the subject may be administered a non-myeloablative lymphodepleting chemotherapy prior to the immune cell therapy. The non-myeloablative lymphodepleting chemotherapy may be any suitable such treatment that may be administered by any suitable route. The non-myeloablative lymphodepleting chemotherapy may include, for example, administration of cyclophosphamide and fludarabine, particularly when the cancer is melanoma that may be metastatic. An exemplary administration route for cyclophosphamide and fludarabine is intravenous. Similarly, any suitable dose of cyclophosphamide and fludarabine may be administered. In certain embodiments, approximately 60 mg / kg of cyclophosphamide is administered for two days, followed by approximately 25 mg / m 2 of fludarabine is administered for five days.

[0147] In certain embodiments, a growth factor that promotes the proliferation and activation of immune cells is administered to the subject simultaneously with or subsequent to the immune cells. The immune cell growth factor can be any suitable growth factor that promotes the proliferation and activation of immune cells. Examples of suitable immune cell growth factors include interleukin (IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations (e.g., IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2).

[0148] A therapeutically effective amount of the immune cells can be administered by several routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal or intraarticular injection or infusion.

[0149] The therapeutically effective amount of immune cells for use in adoptive cell therapy is the amount that achieves the desired effect in the subject being treated.For example, it can be the amount of immune cells that is required to inhibit the progression or the amount of immune cells that is required to reverse autoimmune disease or alloimmune disease, or the amount of immune cells that can relieve symptoms caused by autoimmune disease, such as pain and inflammation.It can be the amount that is required to relieve symptoms associated with inflammation, such as pain, edema and elevated body temperature.It can also be the amount that is required to reduce or prevent rejection of transplanted organs.

[0150] The immune cell population may be administered in a treatment regimen consistent with the disease, for example, once or several times over one to several days, to ameliorate the disease state, or in regular administration over an extended period of time to inhibit disease progression and prevent disease recurrence. The exact dose employed in the formulation will also depend on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the physician and each patient's circumstances. The therapeutically effective amount of immune cells will depend on the subject being treated, the severity and type of affliction, and the mode of administration. In some embodiments, the dose that may be used in the treatment of a human subject is at least 3.8×10 4 , at least 3.8 × 10 5 , at least 3.8 × 10 6 , at least 3.8 × 10 7 , at least 3.8 × 10 8 , at least 3.8 × 10 9 or at least 3.8×10 10 immune cells / m 2 In certain embodiments, the dose used in treating a human subject is about 3.8×10 9 ~Approx. 3.8×10 10 immune cells / m 2 In a further embodiment, the therapeutically effective amount of immune cells varies from about 5×10 6 cells / kg body weight~approx. 7.5×10 8 Cells / kg body weight, e.g., about 2×10 7 Cells ~ approx. 5 x 108 Cells / kg body weight or approximately 5 x 10 7 cells ~ approx. 2 x 10 8 The amount of immune cells may vary by cell / kg body weight.The exact amount of immune cells can be readily determined by those skilled in the art based on the age, weight, sex and physiological condition of the subject.Effective amounts can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0151] The immune cells can be administered in combination with one or more other therapeutic agents for treating an immune-mediated disorder. Combination therapy may include, but is not limited to, one or more antibacterial agents (e.g., antibiotics, antiviral agents, and antifungal agents), antitumor agents (e.g., fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immunodepleting agents (e.g., fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressants (e.g., azathioprine or glucocorticoids, such as dexamethasone or prednisone), anti-inflammatory agents (e.g., glucocorticoids (e.g., hydrocortisone, dexamethasone, or prednisone) or nonsteroidal anti-inflammatory agents (e.g., acetylsalicylic acid, ibuprofen, or naproxen sodium)), cytokines (e.g., interleukin-10 or transforming growth factor-beta), hormones (e.g., estrogen), or vaccines. In addition, immunosuppressants or immune tolerance inducers may be administered, including but not limited to calcineurin inhibitors (e.g., cyclosporine and tacrolimus); mTOR inhibitors (e.g., rapamycin); mycophenolate mofetil, antibodies (e.g., antibodies that recognize CD3, CD4, CD40, CD154, CD45, IVIG or B cells); chemotherapeutic agents (e.g., methotrexate, treosulfan, busulfan); irradiation; or chemokines, interleukins or their inhibitors (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors). Such additional pharmaceutical agents may be administered before, during or after administration of the immune cells, depending on the desired effect. This administration of the cells and agents may be by the same route or different routes, and at the same site or different sites. A. Pharmaceutical Compositions

[0152] Also provided herein are pharmaceutical compositions and formulations comprising immune cells (eg, T cells or NK cells) and a pharma- ceutically acceptable carrier.

[0153] Pharmaceutical compositions and formulations as described herein comprise the active ingredient (e.g., the cells) having a desired degree of purity in one or more optional pharma- ceutical acceptable carriers (Remington's Pharmaceutical Sciences 22 ndPharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include buffers (e.g., phosphate, citric acid, and other organic acids); antioxidants (including ascorbic acid and methionine); preservatives (e.g., octadecyldimethylbenzylammonium chloride); hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol alcohol, butyl alcohol, or benzyl alcohol; alkyl parabens (e.g., methylparaben or propylparaben); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight (less than about 10 residues). polypeptides; 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 dextrins; chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants (e.g., polyethylene glycol (PEG)). Exemplary pharmacologic acceptable carriers herein further include interstitial drug dispersion agents, such as neutral active soluble hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is used in combination with one or more additional glycosaminoglycanases, such as chondroitinases. B. Combination Therapy

[0154] In certain embodiments, the compositions and methods of the present embodiment comprise immune cell populations that are combined with at least one additional treatment.The additional treatment can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy or the combination of the above.The additional treatment can be in the form of adjuvant therapy or neoadjuvant therapy.

[0155] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent aimed at reducing the incidence and / or severity of side effects of treatment, such as an anti-nausea agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy can be one or more chemotherapeutic agents known in the art.

[0156] Immune cell therapy may be administered before, during, after, or in various combinations with additional cancer therapy, such as immune checkpoint therapy. Their administration may occur at intervals ranging from simultaneously to minutes, days, or weeks. In embodiments in which immune cell therapy is provided to a patient separately from an additional therapeutic agent, it is common to ensure that no significant period of time passes between each delivery time point so that the two compounds can still exert a beneficial combined effect on the patient. In such cases, it is contemplated that the antibody therapy and the anti-cancer therapy may be provided to the patient within about 12-24 or 72 hours of each other, more particularly within about 6-12 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) pass between the respective administrations, it may be desirable to extend the treatment period considerably.

[0157] Various combinations can be used. In the following example, the immune cell therapy is "A" and the anti-cancer therapy is "B": A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0158] Administration of any compound or treatment of the present embodiments to a patient follows general protocols for administering such compounds, taking into account any toxicity of those agents. Thus, in some embodiments, there is a step of monitoring for toxicity that may result from the combination therapy. 1.Chemotherapy

[0159] A wide variety of chemotherapeutic agents can be used according to this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity in cells, for example, whether they affect the cell cycle and at what stage they affect the cell cycle. Alternatively, 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.

[0160] 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); ethylenimines and methylamelamines (altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylol melamine); trimethylolomelamine); acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatins; kallistatins; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eleuthecins. eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards (e.g., chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenestrine, rine, prednimustine, trofosfamide and uracil mustard); nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine); antibiotics (e.g., enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega 11)); dynemicins (including dynemicin A); bisphosphonates (e.g., clodronate); esperamicin;and the neocarzinostatin chromophore and related chromoproteins, the enediyne antibiotic chromophores, aclacinomycin, actinomycin, autarubicin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, 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, nogalarnicin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites (e.g., methotrexate and 5-fluorouracil (5-FU)); folate analogs (e.g., denopterin, pteropterin, and trimetrexate); purine analogs (e.g., fludarabine, 6-mercaptopurine, thiamiprin e) and thioguanine); pyrimidine analogues (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxuridine); androgens (e.g., calsterone, dromostanolone propionate, epithiostanol, 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; demecolcine; diazicon; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids (e.g., maytansine and ansamitocins); mitoguazone; mitoxantrone; mopidanmol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid acid);2-ethylhydrazide;procarbazine;PSK polysaccharide complex;razoxane;rhizoxin;schizophyllan;spirogermanium;tenuazonic acid;triaziquone;2,2',2"-trichlorotriethylamine;trichothecines (especially T-2 toxin, veracrine A, roridin A and anguidine);urethane;vindesine;dacarbazine;mannomustine;mitobronitol;mitolactol;pipobroman;gacytosine;arabinosides ("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 inhibitors RFS2000; difluoromethylornithine (DMFO); retinoids (e.g., retinoic acid); capecitabine;Carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, as well as pharmaceutically acceptable salts, acids or derivatives of any of the above; 2. Radiation therapy

[0161] Other agents that have been widely used to cause DNA damage include gamma radiation, X-rays and / or what are commonly known as directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287) and UV irradiation are also contemplated. All of these agents most likely cause widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens over prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dosage ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by the neoplastic cells. 3. Immunotherapy

[0162] Those skilled in the art will understand that additional immunotherapy may be used in conjunction or in conjunction with the methods of the above embodiments. In the context of cancer treatment, immunotherapeutics usually rely on the use of immune effector cells and immune effector molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector may be, for example, an antibody specific to some marker on the surface of tumor cells. The antibody may function alone as an effector of therapy or may recruit other cells to actually affect cell killing. The antibody may 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 may be a lymphocyte with a surface molecule that directly or indirectly interacts with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0163] Antibody-drug conjugates have emerged as a breakthrough approach to the development of cancer therapy. Cancer is one of the leading causes of death worldwide. Antibody-drug conjugates (ADCs) contain a monoclonal antibody (MAb) covalently linked to a cytotoxic drug. This approach combines the high specificity of MAbs for antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells that harbor abundant levels of the antigen. Targeted delivery of the drug also minimizes exposure to normal tissues, resulting in reduced toxicity and improved therapeutic index. The approval of two ADC drugs by the FDA, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, validated this approach. Currently, more than 30 ADC drug candidates are in various stages of clinical trials to treat cancer (Leal et al., 2014). As antibody engineering and linker-payload optimization become more sophisticated, the discovery and development of new ADCs will depend more and more on the identification and validation of new targets suitable for this approach and the generation of targeted Mabs. Two criteria for ADC targets are upregulated / high level expression in tumor cells and robust internalization.

[0164] In one aspect of immunotherapy, the tumor cells must have some marker that is amenable to targeting, i.e., some marker that is not present on most other cells. Many tumor markers exist, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine anti-cancer effects with immune stimulatory effects. There are also immune stimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0165] Examples of immunotherapies currently under investigation or in use include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, such as interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy, such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2 and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0166] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints either strengthen or weaken signals (e.g., costimulatory molecules). Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade 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, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0167] The immune checkpoint inhibitor may be a drug, such as a small molecule, a recombinant ligand or receptor, or may be an antibody, such as a human antibody (e.g., International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012; both of which are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof may be used, and in particular chimeric, humanized or human antibodies may be used. As those skilled in the art will recognize, alternative and / or equivalent names may be used for certain antibodies described in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, it is known that lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0168] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits PD-1 from binding 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 PDL1 from binding 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 PDL2 from binding to its binding partner. In a specific embodiment, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art and are described, for example, in U.S. Patent Application Nos. 20140294898, 2014022021, and 20110008369, all of which are incorporated herein by reference.

[0169] 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 an extracellular portion or a PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, Lambrolizumab, KEYTRUDA® and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0170] 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 inhibitory signals to T cells. CTLA4 resembles CD28, a T cell costimulatory protein, and both molecules bind to CD80 and CD86 (also called B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. Intracellular CTLA4 is also found on regulatory T cells and may be important for the function of those cells. Activation of T cells via the T cell receptor and CD28 leads to high expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0171] 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.

[0172] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in US8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (also known as tremelimumab; previously 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.

[0173] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or the heavy 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 above-mentioned antibodies and / or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity to the above-mentioned antibody (eg, at least about 90%, 95% or 99% variable region identity to ipilimumab).

[0174] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors (e.g., those described in U.S. Pat. Nos. 5,844,905, 5,885,796 and International Patent Application Nos. WO1995001994 and WO1998042752, all of which are incorporated by reference herein) and immunoadhesins (e.g., those described in U.S. Pat. No. 8,329,867, which is incorporated by reference herein). 4.Surgery

[0175] Approximately 60% of people with cancer undergo some type of surgery, including preventive surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection, which physically removes, excises, and / or destroys all or part of the cancerous tissue, and may be used in conjunction with other therapies (e.g., the treatment of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies). Lumpectomy refers to the physical removal of at least a portion of the tumor. Surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery) in addition to lumpectomy.

[0176] When removing part or all of cancerous cells, tissues or tumors, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with additional anticancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments may also be in various dosages. 5. Other agents

[0177] It is contemplated that other agents may be used in combination with certain aspects of the present embodiment to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that enhance the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing signaling between cells by increasing the number of gap junctions may enhance the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the present embodiment to improve the anti-hyperproliferative efficacy of the treatment. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that enhance the sensitivity of hyperproliferative cells to apoptosis (e.g., antibody c225) may be used in combination with certain aspects of the present embodiment to improve the efficacy of the treatment. V. Products or Kits

[0178] Also provided herein is an article of manufacture or kit that includes the immune cells. The article of manufacture or kit may further include a package insert that includes instructions for using the immune cells to treat or delay the progression of cancer in an individual or to enhance the immune function of an individual with cancer. Any of the antigen-specific immune cells described herein may be included in the article of manufacture or kit. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers may be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin) or metal alloys (e.g., stainless steel or Hastelloy). In some embodiments, the container holds the formulation and a label that is attached to or associated with the container, and the container may indicate how to use. The article of manufacture or kit may further include other materials that are desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts that include instructions for use. In some embodiments, the article of manufacture further includes one or more additional agents (e.g., chemotherapeutic and antineoplastic agents). Suitable containers for the one or more agents include, for example, bottles, vials, bags, and syringes. EXAMPLES

[0179] VI. Working Examples The following examples are included to demonstrate certain embodiments of the present disclosure. It should be recognized by those skilled in the art that the methods disclosed in the following examples are methods that the inventors have found to work well in implementing the method of the present disclosure, and therefore can be considered as specific forms for its implementation. However, those skilled in the art should recognize in light of the present disclosure that many changes can be made in the specific embodiments disclosed that still produce the same or similar results without departing from the spirit and scope of the present disclosure. Example 1 - Modular Vector System

[0180] The vector was generated as a gamma-retroviral transfer vector. The retroviral transfer vector pSFG4 had a backbone based on the pUC19 plasmid (large fragment [2.63 kb] between HindIII and EcoRI restriction enzyme sites) with viral components from Moloney Murine Leukemia Virus (MoMLV), including the 5'LTR, the psi packaging sequence, and the 3'LTR. LTRs are terminal repeat sequences found on either side of the provirus of retroviruses, and in the case of transfer vectors, flank the genetic cargo of interest, such as CAR and associated genetic components. The psi packaging sequence, which is the target site for packaging by the nucleocapsid, was also incorporated in cis between the 5'LTR and the CAR coding sequence. Thus, the basic structure of the pSFG4 transfer vector can be outlined as follows: pUC19 sequence-5'LTR-psi packaging sequence-genetic cargo of interest-3'LTR-pUC19 sequence.

[0181] Exemplary vectors are shown in Figures 2 and 3. In Figure 3, the vector DNA is circular and, by convention, position 1 (the 12 o'clock position at the top of the circle, with the remaining sequences oriented clockwise) is set to the start of the 5'LTR.

[0182] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods and steps or steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while the same or similar results are achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. literature All publications mentioned below are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety. US Patent 5,844,905 US Patent 5,885,796 US Patent 6,207,156 US Patent 6,410,319 US Patent 6,451,995 US Patent 7,070,995 US Patent 7,109,304 US Patent 7,265,209 US Patent 7,354,762 US Patent 7,446,179 US Patent 7,446,190 US Patent 7,446,191 US Patent 8,324,353 US Patent 8,329,867 US Patent 8,354,509 US Patent 8,479,118 US Patent 8,735,553 US Patent 8,008,449 US Patent 8,017,114 US Patent 8,119,129 US Patent 8,252,592 US Patent 8,339,645 US Patent 8,398,282 US Patent Application US2002131960 US Patent Application US20110008369 U.S. Patent Application US2013287748 U.S. Patent Application US20130149337 U.S. Patent Application US20140294898 U.S. Patent Application US2014022021 WO1995001994 WO1998042752 WO199842752 WO2001014424 WO2000037504 WO200014257 WO200037504 WO200114424 WO2012129514 WO2013126726 WO2013166321 WO2013071154 WO2013123061 WO2014055668 WO2014031687 EP Patent Application EP2537416 Ausubel et al., 1996, (eds.) Current Protocols in Molecular Biology, John Wiley & Sons Inc Camacho et al. (2004) J Clin Oncology 22(145): Abstract No. 2505 Cohen et al. (2005) J Immunol. 175(9): 5799-5808 Chothia et al. (1988) EMBO J. 7(12):3745-55 Davila et al. (2013) Hematol Oncol Clin North Am. 27(2):341-53 Fedorov et al. (2013) Science Translational Medicine, Vol. 5, Issue 215, pp. 215ra172 Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17): 10067-10071 Jores et al. (1990) Proc Natl Acad Sci U S A. 87(23):9138-42 Lefranc et al. (2003) Dev Comp Immunol. 27(1):55-77 Li et al. (2005) Nature Biotechnology volume 23, pages 349-354 Lockey et al. (2008) Front Biosci. 1;13:5916-27 Mokyr et al. (1998) Cancer Res 58:5301-5304 Parkhurst et al. (2009) Clin Cancer Res. 15(1):169-80 Sadelain et al. (2013) Cancer Discov. 3(4):388-98 Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 1st ed., Cold Spring Harbor Laboratory Press Singh et al. (2008) Cancer Res.;68(8):2961-71 Singh et al. (2011) Cancer Res. 15;71(10):3516-27 Turtle et al. (2012) Curr Opin Immunol. 24(5):633-9. Varela-Rohena et al. (2008) Immunol Res. 42(1-3):166-81. Wu et al. (2012) Cancer J. 18(2):160-75

Claims

1. A polycistronic vector comprising at least three cistrons, each flanked by one or more restriction enzyme sites, at least one cistron encoding at least one antigen receptor, at least one of the cistrons on the vector comprising two or more modular components, each of the modular components within a cistron being flanked by one or more restriction enzyme sites, the antigen receptor being encoded by a cistron comprising two or more modular components.

2. 2. The vector of claim 1, wherein two, three, four or more of the cistrons are translatable into a single polypeptide, which is cleavable into separate polypeptides.

3. The vector of claim 2 , wherein four of the cistrons are translatable into a single polypeptide and cleavable into separate polypeptides.

4. The vector according to any one of claims 1 to 3, wherein adjacent cistrons on the vector are separated by a 2A self-cleavage site.

5. The vector of claim 1 , wherein each of the cistrons is configured to express a separate polypeptide from the vector.

6. The vector of claim 5 , wherein adjacent cistrons on the vector are separated by an IRES element.

7. The vector of claim 1 , wherein the cistron comprises three, four or five modular components.

8. 8. A vector according to claim 6 or 7, wherein the cistrons encode an antigen receptor with different portions of the receptor encoded by corresponding modular components.

9. The vector of claim 8 , wherein the first modular component of the cistron encodes the antigen-binding domain of the receptor.

10. The vector of claim 8 , wherein the second modular component of the cistron encodes the hinge region of the receptor.

11. The vector of claim 8 , wherein the third modular component of the cistron encodes the transmembrane domain of the receptor.

12. The vector of claim 8 , wherein the fourth modular component of the cistron encodes a first costimulatory domain.

13. The vector of claim 8 , wherein the fifth modular component of the cistron encodes a second costimulatory domain.

14. The vector of claim 8 , wherein the sixth modular component of the cistron encodes a signaling domain.

15. The vector of claim 1 , wherein two different cistrons on the vector each encode an antigen receptor.

16. The vector of claim 15, wherein both antigen receptors are encoded by a cistron comprising two or more modular components.

17. The vector of claim 1 , wherein the antigen receptor is a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR).

18. The vector of claim 1 , wherein the vector is a viral vector or a non-viral vector.

19. The vector of claim 18 , wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

20. 2. The vector of claim 1, wherein the vector comprises the 5'LTR, 3'LTR and psi packaging elements of Moloney murine leukemia virus (MMLV).

21. 21. The vector of claim 20, wherein the psi packaging element is integrated between the 5'LTR and the antigen receptor coding sequence.

22. The vector of claim 1 , wherein the vector comprises a pUC19 sequence.

23. The vector of claim 1 , wherein at least one cistron encodes a cytokine, a chemokine, a cytokine receptor and / or a homing receptor.

24. 24. The vector of claim 23, wherein the cytokine is interleukin 15 (IL-15), IL-7, IL-12, IL-21, IL-18 or IL-2.

25. 5. The vector of claim 4, wherein the 2A self-cleavage site comprises a P2A, a T2A, an E2A and / or an F2A site.

26. The vector of claim 1 , wherein the cistron comprises a suicide gene.

27. The vector of claim 1 , wherein the cistron encodes a reporter gene product.

28. 2. The vector of claim 1, wherein the first cistron encodes a suicide gene, the second cistron encodes an antigen receptor, the third cistron encodes a reporter gene product, and the fourth cistron encodes a cytokine.

29. 29. The vector of claim 28, wherein different portions of the antigen receptor are encoded by corresponding modular components, a first component of the second cistron encodes an antigen-binding domain, a second component encodes a hinge and / or transmembrane domain, a third component encodes a costimulatory domain, and a fourth component encodes a signaling domain.

30. An immune cell comprising the vector of claim 1.

31. The immune cell of claim 30, wherein the immune cell is a T cell, a peripheral blood lymphocyte, a B cell, a NK cell, an invariant NK cell, a NKT cell, an iNKT cell, a macrophage, a stem cell or a mixture thereof.

32. The immune cell of claim 31 , wherein the stem cell is a mesenchymal stem cell (MSC) or an induced pluripotent stem (iPS) cell.

33. The immune cell of claim 30 , wherein the immune cell is derived from an iPS cell.

34. The immune cell of claim 31 , wherein the T cell is a CD8+ T cell, a CD4+ T cell, or a gamma-delta T cell.

35. The immune cell of claim 31 , wherein the T cell is a cytotoxic T lymphocyte (CTL).

36. 31. The immune cell of claim 30, wherein the immune cell is autologous or allogeneic to an individual.

37. The immune cell of claim 30 , wherein the immune cell is a human cell.

38. The immune cell of claim 30, wherein the immune cell is derived from umbilical cord blood, peripheral blood, bone marrow, CD34+ cells, or iPSCs.

39. The immune cell of claim 30 , wherein the immune cell is comprised in a cell population.

40. The immune cell of claim 30 , wherein the immune cell is contained in a pharma- ceutically acceptable carrier.

41. 1. A method for generating expanded immune cells, comprising: (a) obtaining a starting population of immune cells; (b) culturing the starting population of immune cells in the presence of artificial presentation cells (APCs); (c) introducing the vector according to any one of claims 1 to 29 into the immune cells; and (d) expanding the immune cells in the presence of APCs, thereby obtaining expanded immune cells. A method comprising:

42. 42. The method of claim 41, wherein the starting population of immune cells is obtained by isolating mononuclear cells using a Ficoll-Paque density gradient.

43. 42. The method of claim 41, wherein the APCs are gamma-irradiated APCs.

44. 42. The method of claim 41, further comprising the step of cryopreserving the expanded population of immune cells.

45. 31. A pharmaceutical composition comprising the population of immune cells of claim 30 and a pharma- ceutically acceptable carrier.

46. 31. A composition comprising an effective amount of the immune cells of claim 30 for use in treating a disease or disorder in an individual.

47. 47. The composition of claim 46, wherein the disease is cancer, an autoimmune disorder, graft-versus-host disease, allograft rejection or an inflammatory condition.

48. 1. A polycistronic vector comprising at least four cistrons, each flanked by one or more restriction enzyme sites, wherein at least one cistron encodes at least one antigen receptor, wherein at least one of the cistrons on the vector comprises two or more modular components, each of the modular components within a cistron being flanked by one or more restriction enzyme sites, and wherein the antigen receptor is encoded by a cistron comprising two or more modular components.

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