Autologous / allogeneic immune defense receptors for selective targeting of activated pathogenic t cells and NK cells

Abstract

Embodiments of the present disclosure relate to engineered T cells expressing autologous / allogeneic immune defense receptors (ADRs) that selectively target activated T cells, including pathogenic T cells, to disable them. The chimeric receptors comprise moieties, e.g., targeting 4-1BB, OX40, and CD40L, whose expression is indicative of activated T cells. In particular embodiments, there are methods of using adoptive T cell transfer of ADR-encoding cells to prevent or treat conditions associated with activated T cells.

Description

This application is a divisional application of the invention patent application with application number 201980038227.6.

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 662,817, filed April 26, 2018, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant P50CA126752 from the National Institutes of Health and the National Cancer Institute. The government has certain rights in this invention. Technical Field

[0003] Embodiments of the present disclosure encompass at least the fields of immunology, cell biology, molecular biology, and medicine. Background Art

[0004] In patients receiving transplants or third-party derived therapeutic cells, unwanted activation of T cells and NK cells typically promotes life-threatening allogeneic immune responses, leading to rejection of transplanted organs / tissues or the development of graft-versus-host disease (GvHD). Similarly, unwanted activation of autoreactive T cells can lead to destructive autoimmune conditions such as diabetes, autoimmune colitis, and multiple sclerosis. Currently, most of these diseases cannot be cured due to the inability to selectively remove pathogenic T cells. Instead, patients are typically treated with immunosuppressive drugs, which make them immunodeficient and therefore susceptible to infection and malignant transformation.

[0005] The present disclosure provides a solution to a long-felt need in the field of safe and effective tissue transplantation and adoptive cell transfer, including utilizing off-the-shelf cells, by enhancing the ability of transferred cells to control pathogenic conditions resulting from unwanted activation of the immune system. Summary of the Invention

[0006] The present disclosure relates to compositions and methods related to cells for adoptive transfer to control pathogenic conditions caused by immune activation. The compositions and methods are applicable to autologous and allogeneic cells. Although some measures can be taken to reduce the reactivity of allogeneic cells in recipient individuals, such cells will still be targeted by the recipient's immune system (mainly T cells and NK cells), which will identify them as foreign substances, causing rejection and limiting therapeutic effects.

[0007] The present disclosure overcomes this problem by modifying adoptive therapy cells to target activated pathogenic T, NK-T and NK cells to prevent or treat the medical conditions associated with their presence. In a specific embodiment, the compositions and methods utilize adoptive T cell transfer to express cells that selectively target pathogenic T cells and leave receptors for resting T cells. In a specific embodiment, the adoptive T cells for transfer are engineered to express chimeric molecules targeting pathogenic T cells, and the pathogenic T cells express certain target molecules, and the presence of the target molecules on T cells indicates pathogenic T cells. In a specific embodiment, the present disclosure relates to autologous / allogeneic immune defense receptors (ADRs) for selectively targeting pathogenic T cells.

[0008] Certain embodiments of the present disclosure include methods for protecting engineered allogeneic T cells from being eliminated in a host individual by providing the individual with cells equipped with ADRs. Embodiments also include methods for avoiding allogeneic immune responses in individuals receiving tissue or organ transplants, for example.

[0009] In certain embodiments, the cells encompassed by the present disclosure have been modified or can be modified to allow them to survive in recipients (including allogeneic recipients). In certain cases, cells for adoptive cell therapy (including T cells, NKT cells, etc.) are suitable for "standing" use, and in this article, cells are stored in a repository (repository) or a bank (bank), and can be provided (with or without further modification) to individuals in need for specific purposes. In many cases, the individual is not an individual of the original source of the cell. The cells used in this way can be prepared in advance to express ADR, although in some cases, these cells are obtained from a bank and then modified to express ADR. The cells preserved in the bank may also express or may not express CAR or recombinant TCR, or the cells obtained from the bank may be subsequently modified to express CAR or recombinant TCR. Such practice makes it easy to use therapeutic cells from a third party source without being rejected by host immunity, and it is not necessary to prepare patient-specific products every time needed.

[0010] In a specific embodiment, there is an isolated polynucleotide comprising a sequence encoding one or more of: (1) an OX40-specific ligand, a 4-1BB-specific ligand, a CD40L-specific ligand, or a functional derivative thereof; operatively linked to (2) a signaling domain that promotes T cell activation. The polynucleotide may comprise an OX40-specific ligand, a 4-1BB-specific ligand, or a CD40L-specific ligand. The OX40-specific ligand may be OX40L, an antibody targeting OX40, an OX40L-Fc fusion, or a combination thereof, or any other engineered protein capable of specifically binding to OX40. The 4-1BB-specific ligand may be 4-1BBL, an antibody targeting 4-1BB, a 4-1BBL-Fc fusion, or a combination thereof, or any other engineered protein capable of specifically binding to 4-1BB. The CD40L-specific ligand may be CD40, an antibody targeting CD40L, a CD40-Fc fusion, or any other engineered protein capable of specifically binding to CD40L, or a combination thereof. In at least some cases, the polynucleotide further comprises a sequence encoding a spacer (e.g., between 10 and 220 amino acids in length) positioned between (1) and (2). The spacer may have a sequence that facilitates surface detection using an antibody, e.g., the spacer may be detectable by an anti-Fc antibody. The spacer may comprise an IgG Fc portion.

[0011] In a specific embodiment, the polynucleotides of the present invention can further encode chimeric antigen receptors, T cell receptors or both. The polynucleotides can be in any form, including being present on a vector, such as a viral vector (retroviral vector, lentiviral vector, adenoviral vector or adeno-associated viral vector) or a non-viral vector (plasmid, transposon, etc.). In particular cases, the polynucleotides are present in cells, including eukaryotic cells or bacterial cells. The cell can be an immune cell, such as a T cell. The cell can be engineered. The cell may include one or more chimeric antigen receptors (CAR) and / or one or more engineered T cell receptors (TCR).

[0012] The polypeptides expressed by any polynucleotides encompassed by the present disclosure are included as part of the present disclosure. In a particular embodiment, there is a polypeptide comprising: (1) one or more of an OX40-specific ligand, a 4-1BB-specific ligand, and CD40; which is operatively linked to (2) a signaling domain that promotes T cell activation. The signaling domain that promotes T cell activation can be from a CD3 ζ subunit, DAP12, an Fc receptor, or a combination thereof.

[0013] Any cell encompassed by the present disclosure is a part of the present disclosure. In a specific embodiment, any cell expressing a chimeric receptor is a part of the present disclosure, including cells comprising any polynucleotides considered herein and / or any polypeptide considered herein. The cell can be an engineered cell. The cell can be an immune cell, such as a T cell, including the T cells transduced by CAR and / or the T cells transduced by T cell receptor (TCR). In a specific embodiment, the cell is engineered to lack the endogenous expression of one or more genes, such as one or more lacking 4-1BB, OX40 and / or CD40L. CRISPR / Cas9, zinc finger nucleases, TALE nucleases or meganucleases can be used to engineer cells. Alternatively, ADR ligands can be captured by, for example, capturing ADR ligands with specific antibodies or receptors anchored in the endoplasmic reticulum or another intracellular compartment to engineer cells to prevent the surface expression of ADR ligands.

[0014] In one embodiment, there is a method for avoiding rejection of allogeneic cells, tissues, or organs in an individual, comprising the step of delivering to the individual an effective amount of allogeneic immune cells expressing an engineered chimeric receptor comprising an extracellular domain and comprising CD3ζ, wherein the extracellular domain targets a compound selectively present on activated T cells, wherein the delivering step results in the individual: (1) suppressing endogenous alloreactive T cells in the individual; and / or (2) suppressing NK cell activation in the individual. In specific embodiments, the allogeneic cells are allogeneic immune cells expressing the chimeric receptor. The allogeneic cells may express a chimeric antigen receptor and / or an engineered T cell receptor. The allogeneic immune cells may be delivered to the individual before, during, and / or after tissue and / or organ transplantation in the individual. In specific cases, the activated T cells are pathogenic T cells.

[0015] In one embodiment, there is a method for selectively targeting activated T cells in an individual, comprising providing to the individual an effective amount of cells expressing an engineered chimeric receptor comprising: (1) an extracellular domain that targets a compound that is selectively present on activated T cells; and (2) a signaling domain that promotes T cell activation. The signaling domain that promotes T cell activation can be derived from a CD3 zeta subunit, DAP12, an Fc receptor, any sequence containing ITAM, or a combination thereof. In certain instances, the activated T cells are pathogenic T cells.

[0016] In certain embodiments, there is a method for preventing or treating a medical condition associated with activated T cells in an individual, comprising the step of delivering to the individual an effective amount of immune cells expressing an engineered chimeric receptor that selectively targets the activated T cells, the chimeric receptor comprising: (1) an extracellular domain that targets a compound that is selectively present on activated T cells; and (2) a signaling domain that promotes T cell activation. The medical condition can be an autoimmune disorder, such as transplant rejection, graft-versus-host disease, type I diabetes, multiple sclerosis, autoimmune colitis, or a combination thereof.

[0017] In one embodiment, there is a method for avoiding NK cell-mediated host rejection of allogeneic T cells, tissues or organs in an individual, comprising the step of providing to the individual an effective amount of immune cells expressing an engineered chimeric receptor, the engineered chimeric receptor comprising an extracellular domain and further comprising a signaling domain that promotes T cell activation, the extracellular domain targeting a compound that is selectively present on activated T cells. In some cases, the immune cells expressing the engineered chimeric receptor are allogeneic T cells. The immune cells may express a chimeric antigen receptor and / or an engineered T cell receptor. The amount of immune cells expressing the engineered chimeric receptor provided to the individual is 10 per square meter. 2 -10 12 The cells expressing the chimeric receptor may be provided to the individual systemically or locally. The immune cells may be T cells. The immune cells may be delivered to the individual once or more than once.

[0018] The foregoing has outlined rather broadly the features and technical advantages of the present invention so that the following detailed description of the invention may be better understood. Additional features and advantages of the invention which form the subject of the claims of the present invention will be described hereinafter. It will be understood by those skilled in the art that the concepts and specific embodiments disclosed may readily be used as a basis for modifying or designing other structures for achieving the same purposes of the present design. It will be further recognized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the design disclosed herein, as to its organization and method of operation, and further objects and advantages will be better understood from the following description when considered in conjunction with the accompanying drawings. It will be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of limitations of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] For a more complete understanding of the present disclosure, reference is now made to the following description taken in conjunction with the accompanying drawings, in which:

[0020] Figures 1A-1E ADRs can be expressed on the cell surface of immune cells and promote cytotoxicity against the corresponding targets. Figure 1A ) Schematic diagram of ADR. GFP is optional. ( Figure 1B )ADR expression on the cell surface. Figure 1C ) Expansion of ADR T cells after transduction. ( Figure 1D ) Cytotoxicity of ADR T cells against target cells expressing ADR ligands. Figure 1E ) Expansion of wild-type and 4-1BB KO T cells expressing the 4-1BB ADR and their cytotoxicity against 4-1BB+ targets, indicating that knocking out the ADR ligand on T cells can further enhance expansion and cytotoxicity and demonstrating that co-expression of the ADR and its ligand on T cells is not required for the expansion or function of ADR-T cells.

[0021] Figures 2A-2E The selective expression of ADR ligands on activated T cells enables them to be selectively eliminated by ADR T cells. Figure 2A-2C ) Expression of ADR ligands on resting and activated T cells after TCR stimulation. Figure 2D )ADR T cells have no cytotoxicity against resting CD4+ and CD8+ T cells. ( Figure 2E ) After 48 h of co-culture, ADR T cells eliminated activated CD4+ and CD8+ T cells.

[0022] Figures 3A-3F . Expression of 4-1BB ADR protects T cells from immune rejection in the MLR model. ( Figure 3A ) Representative dot plots showing that TCRKO T cells co-expressing ADR were protected from immune rejection after co-culture with allogeneic PBMC at a ratio of 1:10 ADR T:PBMC. ( Figure 3B-3C ) The absolute counts of donor T cells and allogeneic T cells in PBMCs during co-culture were significant for ( Figure 3D-3F ) Virus-specific ADR T cells are identical.

[0023] Figures 4A-4C In an in vitro mixed lymphocyte reaction, the expression of ADR protects allogeneic virus-specific T cells from immune rejection. ( Figure 4A ) Representative dot plots showing that ADR VSTs were protected from immune rejection by recipient allogeneic PBMCs. ( Figure 4B-4C ) Absolute counts of recipient T cells and donor VSTs at various time points during the MLR.

[0024] Figure 5The ADR VSTs retained their antiviral function. When the ADR VSTs were co-cultured with viral peptide mix-pulsed (pepmix-pulsed) monocytes, monocyte counts showed that they cleared virus-infected cells equally well compared to unmodified VSTs.

[0025] Figures 6A-6H Activated NK cells upregulate ADR ligands and can be selectively targeted by ADR T cells. ( Figure 6A-6B )4-1BB expression on resting and activated NK cells. ( Figure 6C ) Residual counts of resting and activated NK cells after 24 hours of co-culture with 4-1BB ADR T cells. ( Figure 6D By controlling the expansion of NK cells, MHC-deficient ADR T cells are protected from immune rejection by allogeneic PBMCs. Figure 6E ) Absolute counts of donor T cells and allogeneic NK cells during co-culture. Figure 6F ) After 48 hours of co-culture at a 1:1 E:T ratio, MHC-deficient ADR T cells resisted immune rejection by NK cells. ( Figure 6G-6H ) During MLR with PBMCs, ADR T cells control the expansion of alloreactive NK cells, and the absolute counts of NK cells are plotted in H.

[0026] Figures 7A-7E In vivo ADR expression protects allogeneic T cells from immune rejection. Figure 7A ) Schematic diagram of a mouse model of immune rejection in which mice are given T cells from an HLA-A2+ donor after sublethally irradiated, followed by administration of allogeneic HLA-A2- T cells 4 days later. ( Figures 7B-7C ) Control T cells from HLA-A2- donors were rejected on day 18, whereas cells expressing ADR were protected; ( Figure 7C ) Absolute counts of T cells from HLA-A2+ and HLA-A2- donors at various time points. ( Figure 7D ) modified in vivo model in which, instead of allogeneic T cells, mice received whole PBMCs (containing both T cells and NK cells) from donor 1. ( Figure 7E ) Representative flow cytometry plots showing that ADR T cells were protected from immune rejection and protected mice from the rapid onset of lethal GvHD.

[0027] Figures 8A-8E Co-expression of CAR and ADR preserves the functionality of both receptors. Figure 8A ) Schematic diagram of immune cells co-expressing ADR and CAR. ( Figure 8B ) Co-expression of CAR and ADR on the cell surface. ( Figure 8C ) CAR-ADR T cell cytotoxicity against NALM-6 (CD19+CAR target). ( Figure 8D ) Cytotoxicity of CAR-ADR T cells against activated T cells (ADR targets). ( Figure 8E ) Cytotoxic activity of CAR-ADR T cells against two cell targets after simultaneous co-culture with the two cell targets.

[0028] Figures 9A-9E CAR-ADR T cells were protected from immune rejection and exerted potent anti-tumor activity. ( Figure 9A Schematic diagram of the mouse model. Mice received allogeneic T cells from donor 1 and b2mKO NALM6 24 hours apart, followed by a single dose of CAR-ADR T cells from donor 2. ( Figure 9B ) Kinetics of T cells from donor 2 in peripheral blood. ( Figure 9C ) Kinetics of donor 1 T cells in the experimental group. ( Figure 9D ) Leukemia burden in mice. Figure 9E ) Overall survival rate of mice.

[0029] Figures 10A-10C CAR-ADR T cells were protected from immune rejection and exerted potent antitumor activity in solid tumor models. ( Figure 10A ) Schematic diagram of the mouse model. Mice received allogeneic T cells from donor 1 and the b2mKO neuroblastoma cell line CHLA255 24 hours apart, followed by a single dose of CAR-ADR T cells from donor 2. ( Figure 10B ) Donor 2GD2 CAR T cells were rejected on day 18, whereas CAR-ADR T cells resisted allogeneic rejection and persisted in peripheral blood. ( Figure 10C ) Tumor burden in mice, * indicates xeno-GvHD-related death in the ATC+GD2 CAR T group.

[0030] Figures 11A-11E TCR-knockout CAR-ADR T cells were protected from immune rejection and exerted effective anti-tumor activity. ( Figure 11A ) Schematic diagram of the mouse model. Mice received allogeneic T cells from donor 1 and b2mKO NALM6 24 hours apart, followed by a single dose of TCR-edited CAR-ADR T cells from donor 2. ( Figure 11B ) Kinetics of T cells from donor 2 in peripheral blood. ( Figure 11C ) Kinetics of donor 1 T cells in the experimental group. ( Figure 11D ) Leukemia burden in mice. Figure 11E ) Overall survival rate of mice.

[0031] Figures 12A-12D ADR T cells protect mice from lethal xenogeneic GvHD. Figure 12A )Model schematic. ( Figure 12B ) In vivo expansion of FFLuc-labeled ADR T cells. ( Figure 12C ) Kinetics of body weight gain / loss in mice. ( Figure 12D ) Overall survival rate of mice.

[0032] Figures 13A-13G . A second-generation ADR with a CD28 intracellular signaling domain (ADR.28ζ). Figure 13A )Structure of ADR.28ζ. ( Figure 13B-13C ) In vitro cytotoxicity of ADR.28ζ against target-expressing cell lines. ( Figure 13D-13G )ADR.28ζ protects mice from xenogeneic GvHD. ( Figure 13D )Schematic diagram of the model. Figure 13E ) In vivo expansion of FFLuc-labeled ADR.28ζ T cells. ( Figure 13F ) Kinetics of weight gain / loss in mice. Figure 13G ) Overall survival rate of mice.

[0033] Figure 14 Cytotoxicity of cells expressing ADRs in cancer. (Left) Cytotoxicity of T cells expressing the 4-1BB ADR against HDLM-2 Hodgkin lymphoma cells, (Right) Cytotoxicity of T cells expressing the 4-1BB ADR against K562 chronic myeloid leukemia (CML) cells. Absolute tumor cell counts after 48 hours of coculture at a 1:1 effector to target ratio are shown. DETAILED DESCRIPTION

[0034] As used herein, the words "a" and "an," when used with the word "comprising," in this specification (including the claims), mean "one or more." Some embodiments of the present invention may consist of or consist essentially of one or more elements, method steps, and / or methods of the present invention. It is contemplated that any method or composition described herein may be implemented with respect to any other method or composition described herein.

[0035] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprising," and "containing" will be understood to imply the inclusion of the recited steps or elements or groups of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements. "Consisting of" means including and limited to whatever is contained in the phrase "consisting of." Thus, the phrase "consisting of" means that the listed elements are required or mandatory, and that no other elements are present. "Consisting essentially of" means including any element listed in the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" means that the listed elements are required or mandatory, but other elements are optional, and their presence depends on whether they affect the activity or action of the listed elements.

[0036] Throughout this specification, reference to "one embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "another embodiment," or "a further embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the aforementioned phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0037] As used herein, the term "subject" generally refers to an individual who needs to be treated for any type of medical condition. The subject can be any type of animal. The subject can be any biological or animal subject that is the object of the method or material, including mammals, such as humans, laboratory animals (such as primates, rats, mice, rabbits), livestock (such as cattle, sheep, goats, pigs, turkeys and chickens), household pets (such as dogs, cats and rodents), horses and transgenic non-human animals. The subject can be a patient, for example, suffering from or suspected of having a disease (which can be referred to as a medical condition), such as one or more infectious diseases, one or more genetic disorders, one or more cancers or any combination thereof. The disease may be pathogenic. The subject may be receiving or has received antibiotic treatment. The subject may be asymptomatic. The subject may be a healthy individual. In at least some cases, the term "individual" can be used interchangeably. As used herein, "subject" or "individual" may or may not be admitted to a medical institution and may be treated as an outpatient of a medical institution. The individual may be receiving one or more medical compositions via the Internet. Individuals may include humans or non-human animals of any age, and thus include adults and adolescents (i.e., children) and infants, and include individuals in utero. The individuals may be of any race and gender. The term is not intended to imply a need for medical treatment, and thus, an individual may voluntarily or involuntarily become part of an experiment, whether clinical or in support of basic scientific research.

[0038] As used herein, the term "engineered" refers to a molecule that does not occur in nature and has been produced by man (eg, by genetic recombination techniques standard in the art).

[0039] In the context of the present disclosure, an "effective amount" or "therapeutically effective amount" refers to an amount of cells that, when administered to an individual, allows for the targeting of activated T cells and / or alleviation of signs and / or symptoms of a medical condition or prevention of a medical condition. The actual amount to be administered can be determined based on studies conducted in vitro or in vivo in which functional immune cells exhibit pharmacological activity against a medical condition. I. Autologous / Allogeneic Immune Defense Receptors and Compositions and Their Uses

[0040] The present disclosure includes synthetic chimeric receptor molecules that provide selective targeting of activated T cells, including pathogenic T cells. The engineered molecules are synthetic and can be produced by recombinant technology. The molecules can be referred to as autologous / allogeneic immune defense receptors that target activated T cells, including activated pathogenic T cells, including those with high specificity.

[0041] In certain embodiments, autologous / allogeneic immune defense receptor (ADR) includes the entity of the compound that targets one or more up-regulation on activated T cells.Although the compound up-regulated on activated T cells can be any one or its combination, in certain embodiments, OX40, 4-1BB and CD40L are up-regulated on activated T cells, and are the objects targeted by ADR.In certain embodiments, the ADR is present on allogeneic immune cells (being allogeneic relative to the individual receiving cells).In other cases, the ADR is expressed on autologous T cells, xenogeneic cells and / or synthetic cells. A. Autologous / allogeneic immune defense receptor (ADR) molecules

[0042] ADR molecules are synthetic, non-natural and artificially generated and comprise at least (1) an extracellular domain that targets a compound that is selectively present on activated T cells (in a specific embodiment, the extracellular domain is a protein or a functional fragment or derivative thereof that targets one or more compounds that are upregulated on activated T cells); which is operatively linked to (2) a signaling domain that promotes T cell activation, including, for example, those derived from the CD3ζ subunit, DAP12 and Fc receptor, or another sequence containing ITAM. The ADR molecule may comprise elements (1) and (2) or consist of elements (1) and (2) or consist essentially of elements (1) and (2). In at least some cases, the ADR comprises a component of one or more type I transmembrane proteins and / or a component of one or more type II transmembrane proteins.

[0043] In a specific embodiment, in an ADR molecule, the extracellular domain comprises a protein that selectively binds to a related protein on activated T cells. For example, the extracellular domain of the ADR may comprise a ligand for a receptor on activated T cells, or the extracellular domain of the ADR may comprise a receptor for a ligand on activated T cells.

[0044] In a specific embodiment, in the ADR molecule, the extracellular domain includes a ligand of OX40, a ligand of 4-1BB and / or CD40. These specific examples have associated proteins on activated T cells, which are OX40, 4-1BB and CD40L respectively. In an alternative embodiment, other specific compositions on the activated T cells are targeted. For example, other activation markers (such as CD69, CD25, CD71, etc.) that are raised on the cell surface of T cells can be targeted using a similar method. In this case, the corresponding ADR molecule will have respective CD69, CD25 or CD71 ligands, or antibody-derived targeting moieties, rather than 4-1BB / OX40 specific ligands.

[0045] In some cases, in contrast to unactivated T cells, activated T cells with upregulated OX40 expression are targeted. In order to target these activated T cells, a ligand of OX40 can be used in ADR to target the activated T cells. In the case of using a ligand of OX40 in ADR, the ligand of OX40 can be any suitable ligand of OX40, including at least OX40L, an antibody (or its functional fragment) that binds to OX40, a fusion of Fc and OX40L, or a functional derivative or fragment thereof. OX40L may also be referred to as tumor necrosis factor (ligand) superfamily member 4 (tax-transcriptionally activated glycoprotein 1, 34 kDa), OX40L, CD252, TNFSF4, TXGP1, OX-40L, or gp34.

[0046] In some cases, in contrast to unactivated T cells, activated T cells with upregulated 4-1BB expression are targeted. In order to target these activated T cells, the ligand of 4-1BB can be used in ADR to be able to target the activated T cells. In the case of using the ligand of 4-1BB in ADR, the ligand of the 4-1BB can be the ligand of any suitable 4-1BB, including at least 4-1BBL, the antibody (or its functional fragment) targeting 4-1BB, the fusion of Fc and 4-1BBL or its functional derivative or fragment.

[0047] In some cases, activated T cells with upregulated CD40L expression are targeted, as opposed to unactivated T cells. To target these activated T cells, a receptor for CD40L can be used in an ADR to target the activated T cells. In the case of using a receptor for CD40L in an ADR, the ADR can comprise CD40 (which may also be referred to as Bp50, CDW40, TNFRSF5, or p50), an antibody targeting CD40L (or a functional fragment thereof, or a functional derivative or fragment thereof).

[0048] In some cases, the ADR molecule includes two or more extracellular domains to promote the T cells of target activation.Such a combination can generally enhance the T cells of target activation or can allow certain subsets of the T cells of specific target activation.For example, the ADR can include OX40L and 4-1BBL as extracellular domains in the same ADR molecule, to allow the activated T cells of target expression OX40 or 4-1BB.The example of this combination will selectively target the activated T cells of expression OX40 or 4-1BB, regardless of whether those activated T cells also express CD40L.Similarly, ADR can include CD40 and OX40L simultaneously to target the activated T cells of expression CD40L or OX40, regardless of whether those activated T cells also express 4-1BB.

[0049] In the ADR molecule, the extracellular domain can be operably connected to one or more components, including components that are part of the ADR molecule. One such component can be a protein that mediates downstream signal transduction during T cell activation. In a specific embodiment, the ADR comprises CD3ζ (also referred to as CD247, CD3-ζ, CD3H, CD3Q, CD3Z, IMD25, T3Z or TCRZ) or its functional fragment or derivative. CD3ζ mediates downstream ITAM-derived signal transduction during T cell activation. Other signal transduction domains comprising ITAMs may include those derived from DAP12, Fc receptors, other CD3 subunits, etc. The signal transduction domain can be non-covalently linked to the ADR through another domain.

[0050] In a specific embodiment, the ADR includes a spacer between CD3ζ and an extracellular protein, which targets one or more compounds that are upregulated on activated T cells. In other cases, no spacer is used. The spacer may include a sequence that is inert or substantially contributes little or no contribution relative to any function that the ADR may have, and in other cases, the spacer includes, for example, a sequence that enhances the function of the ADR and / or makes it detectable and / or targeted to suppress. In a specific embodiment, the spacer includes a coded protein sequence that helps detect cells expressing the ADR. For example, the spacer can encode an Fc region or a fragment thereof that will allow surface detection of cells, for example, using anti-Fc antibody detection. In a specific embodiment, the spacer provides a separation between the ligand binding domain and the membrane to avoid potential spatial obstacles, such as those caused by the engagement of type II transmembrane proteins (4-1BBL, OX40L) with type I ADR skeletons (TM, signaling domains). The spacer can have any suitable length, for example, including about 10-220 amino acids. The spacer length may be in the range of 10-220, 10-200, 10-150, 10-100, 10-50, 25-200, 25-150, 25-100, 25-75, 25-50, 50-200, 50-150, 50-125, 50-100, 50-75, 75-200, 75-150, 75-100, 100-200, 100-175, 100-150, 100-125, 125-200, 125-175, 125-150, 150-200, 150-175, 175-200, etc. The spacer length can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 220 amino acids. In other cases, the spacer is less than 10 amino acids or greater than 200 amino acids.

[0051] In some cases, ADR includes one, two, three or more costimulatory domains, which enhance the production of cytokines from cells expressing ADR. The costimulatory domain can be derived from the intracellular signaling domain of costimulatory proteins (including CD28, CD27, 4-1BB, OX40, ICOS, CD30, HVEM, CD40 etc.), and is only used as an example, when ADR includes 4-1BBL, the costimulatory domain of ADR may be from or may not be from 4-1BB.

[0052] In some embodiments, ADR will include a transmembrane domain, which can be any species, as long as it allows the CD3 ζ components of ADR to be located intracellularly and targeting the extracellular domain of the compound raised on one or more activated T cells to be located extracellularly. In other cases, ADR is a soluble protein (e.g., ADR-CD3 T cell engagement protein (engager protein)) that can be bound to each ligand on activated T cells and promote cytotoxicity by cross-linking TCR. In the case where the extracellular domain is from a surface protein (e.g., CD40) with a transmembrane domain, the ADR may include a transmembrane domain from the corresponding endogenous molecule. In some cases where the ADR molecule includes one or more costimulatory domains, the transmembrane domain (TM) may be from the same endogenous molecule with the costimulatory domain. The example of TM includes those from CD3, CD8 α, CD27, CD28, 4-1BB, OX40, CD4, etc.

[0053] In the example of an ADR polypeptide, the components may be in a specific N-terminal (N) to C-terminal (C) order. For a general ADR, the receptor may comprise one of the following (as an example only), and wherein the extracellular domain comprises a protein that selectively binds to a related protein on activated T cells: N-ectodomain-signaling domain-C N-ectodomain-CD3ζ-C N-ectodomain-spacer-CD3ζ-C N-extracellular domain-spacer-costimulatory domain-CD3ζ-C N-extracellular domain-spacer-two co-stimulatory domains-CD3ζ-C N-two extracellular domains-spacer-costimulatory domain-CD3ζ-C N-two extracellular domains-spacer-two co-stimulatory domains-CD3ζ-C

[0054] In any case, the transmembrane domain can be located at the C-terminus relative to the spacer. A signal peptide at the N-terminus can be used to promote expression of type II ligands (e.g., OX40L and 4-1BBL) on a type I transmembrane protein backbone (e.g., transmembrane domain, signaling domain, CD3ζ).

[0055] In some cases, the ADR comprises one or more detectable markers, such as colorimetric, fluorescent, and / or radioactive markers. Examples include green fluorescent protein, blue fluorescent protein, and the like.

[0056] The ADR may be in the form of a polynucleotide or a polypeptide expressed from a polynucleotide, although the ADR may be produced synthetically as a protein. Recombinant techniques for producing ADR polynucleotides and polypeptides are known in the art.

[0057] In some cases, the ADR polynucleotide is in an expression construct or is a part of an expression construct, and the expression construct is present on a vector, which can be a viral vector or a non-viral vector. Examples of non-viral vectors include plasmids. Examples of viral vectors include slow viruses, retroviruses, adenoviruses, and adeno-associated virus vectors. Any vector expressing ADR will have appropriate elements to allow expression in eukaryotic cells, including, for example, immune cells (such as T cells, NK cells, or NKT cells). Such appropriate elements include promoters, etc.

[0058] 4-1BB ADR (SEQ ID NO: 1) MEFGLSWLFLVAILKGVQCGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVAKAGVYYVFFQLELRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVT PEIPAGLPSPRSEESKYGPPCPPCGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPAYQ QGQNQLYNENLGRREEYDVLDKRRGRDRPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGGKGHDGLYQGLSTATKDTYDALHMQALPPRTSAAAGGGSGGGSGGGSGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0059] OX40 ADR(SEQ ID NO:2) MEFGLSWLFLVAILKGVQCQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLESKYGPPCPPCPGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGGKGHDGLYQGLSTATKDTYDALHMQALPPRTSAAAGGGSGGGSGGGSGMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0060] CD40L ADR(SEQ ID NO:3) MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSAAAGGGGSGGGGSGGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0061] In certain embodiments, the extracellular domain of the targeted activated T cells comprises an antibody or a functional fragment or derivative thereof. As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be a complete immunoglobulin derived from a natural or recombinant source, or an immunoreactive portion of a complete immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. The antibodies of the present invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, in: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, in: Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Bird et al., 1988, Science 242: 423-426).

[0062] In some cases, the extracellular domain of the ADR comprises an antibody fragment. The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0063] Synthetic antibodies can be used for ADR. The term "synthetic antibody" as used herein refers to an antibody produced using recombinant DNA technology, such as the phage-expressed antibodies described herein. The term should also be interpreted as meaning an antibody produced by synthesizing a DNA molecule encoding the antibody, and the DNA molecule expresses the antibody protein or specifies the amino acid sequence of the antibody, wherein the DNA or amino acid sequence has been obtained by using synthetic DNA or amino acid sequence technology (available and well known in the art). B. Cells expressing ADR

[0064] Due to the immune response of the recipient individual, allogeneic cells for adoptive transfer are prone to limited efficacy. Although in some cases, cells can be modified to remove endogenous TCR (e.g., using CRISPR), for example, to prevent graft-versus-host disease, alternatively, virus-specific T cells (complete or CAR / TCR modified) can be used to retain antiviral activity, which is useful in certain pathogenic conditions. Although such VSTs have very limited graft-versus-host activity because their TCRs are more confined to viral antigens, they are still susceptible to the harmful reactions of the recipient.

[0065] Included in the present disclosure are cells that have been modified to express synthetic ADR molecules and improved for allogeneic use. Thus, the present disclosure includes cells having ADRs (as polynucleotides and as expressed ADR polypeptides) on the cell surface. In certain cases, cells expressing ADRs are produced for storage in a repository for regular use. Cells can be housed in a repository that has been configured to express the ADRs, or they can be housed in a repository and configured to express the ADRs after being removed from the repository. Certain cells, such as bacterial cells, can be used to produce ADR molecules, while other cells with ADRs, such as eukaryotic cells, can be used in the methods of the present invention, including targeting activated T cells. As shown herein, immune cells expressing ADRs selectively eliminate activated T cells, and immune cells expressing ADRs are protected from cytolysis by alloreactive T cells.

[0066] The cells expressing ADR molecules can be of any kind, but in specific embodiments, they are immune cells, such as immune effector cells, such as T cells, NK cells, NKT cells or derived from the pedigree or engineered to have a cell line with cytotoxic activity, which has been modified to express ADR, and therefore does not exist in nature. The colonies of non-natural ADR-expressing cells are contemplated, including colonies in which at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% are ADR-expressing cells. For example, the cells can be produced by standard methods of transfecting or transducing synthetic ADR polynucleotides.

[0067] In some cases, the ADR molecules modified to express ADR molecules may have been engineered, or subsequently engineered to have another engineered non-natural molecule other than ADR.For example, cells expressing chimeric antigen receptors (CAR) or engineered T cell receptors (TCR) can protect such cells from host rejection and thus improve their therapeutic efficacy during this process. Cells expressing one or more CARs and / or one or more TCRs can be engineered to express one or more ADRs, or cells expressing one or more ADRs can be engineered to express one or more CARs and / or one or more TCRs. Therefore, in some cases, ADR and CAR and / or TCR are expressed on different carriers, while in other cases, ADR molecules and CAR and / or TCR are expressed on the same carrier. In the case where ADR and CAR are expressed on the same carrier (as an example), ADR and CAR expression can be directed by the same or different regulatory elements. In any case, ADR and CAR can be expressed as single polypeptides, with a cuttable element between them, such as 2A.

[0068] In the case where the cell expressing the ADR also expresses a CAR or TCR, the CAR or TCR can target any specific antigen. In the case of using a CAR, the CAR can be first generation, second generation, third generation, etc. In certain cases, the CAR can be bispecific.

[0069] In some cases, the cells expressing ADR molecules are engineered, such as engineered to lack the expression of one or more endogenous molecules.In certain cases, the cells are engineered to lack the expression of one or more endogenous genes (the gene will otherwise promote the killing of cells).In certain cases, for example, the cells expressing ADR molecules are engineered to lack the expression of 4-1BB or OX40.As an example only, cells can be engineered by CRISPR / Cas9. II. Methods Using Autologous / Allogeneic Immune Defense Receptors

[0070] Embodiments of the present disclosure include methods for providing an effective amount of cells expressing ADRs to an individual for any purpose. The method includes providing selectively targeting activated T cells in an individual for any purpose. Activated T cells are targeted by exposing the activated T cells to an effective amount of immune cells expressing ADRs (e.g., T cells expressing ADRs). This exposure can have, for example, one or more applications resulting therefrom.

[0071] In some embodiments, ADR is used to selectively target activated immune cells other than activated T cells, such as B cells (which is useful for controlling unwanted B cell responses (e.g., lupus, rheumatoid arthritis, etc.)), and targeting the activation of innate immunity (e.g., macrophage activation syndrome, etc.). In other embodiments, ADR is used to specifically target malignant cells that express their corresponding targets, including, for example, 4-1BB or OX40 or CD40L.

[0072] The protocol for providing an effective amount of cells expressing an ADR to an individual can be known or determined by the individual delivering the cells for treatment or prevention or regardless of the method of use thereof. For example, in the case of prevention, the cells can be delivered before detecting one or more symptoms, or can be delivered after detecting one or more symptoms but before further symptom development and / or worsening. For therapeutic situations, an effective amount of cells can be provided to an individual after one, two or more symptoms develop and after clinical diagnosis.

[0073] In certain aspects of the methods, the individual can be administered a single dose of a therapeutically effective amount of cells, or the individual can be administered multiple doses of a therapeutically effective amount of cells, e.g., multiple deliveries spaced 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 1, 2, 3, 4, 5, or more years, or any range therebetween. The time intervals between doses may vary within a single regimen.

[0074] The administration of cells expressing ADR can reach the individual by any suitable route, including locally or systemically. In a specific embodiment, cells expressing ADR are delivered intravenously, orally, rectally, topically, intramuscularly, infused, intestinal, nasal, inhaled, sublingually, buccally, transdermally, subcutaneously, etc. Cells may or may not be delivered as boluses. In multiple administrations, the cells may or may not be provided to the individual with different delivery routes. When the cells are delivered to the individual, they may be delivered with a pharmaceutically acceptable carrier or excipient. Specific examples of dosages of cells expressing ADR include 10 4 cells / m2, 10 5 cells / m2, 10 6 cells / m2, 10 7 cells / m2, 10 8 cells / m2, 10 9 cells / m2, 10 10 cells / m2, 10 11 cells / m2 or 1012 cells / m2, and ranges in between. A. For standing implementation plans

[0075] The present disclosure includes cells for adoptive transfer that can be readily available, including cells that can be obtained from a repository for use in individuals that are not the original source of the cell. These cells may have expressed ADRs before being preserved in a repository, or may be modified to express ADRs thereafter. The cells may be any type of immune effector cells for adoptive transfer. For example, cells may be modified to express tumor-specific receptors (e.g., CAR or TCR) before being preserved in a repository or after obtaining them from a repository.

[0076] As shown elsewhere herein, cells expressing ADR selectively target activated T cells and NK cells, leaving behind resting subsets. In addition to protecting T cells from immune rejection in vivo, ADR also protects allogeneic T cells from immune rejection mediated by T cells and / or NK cells in vitro. ADR does this without interfering with the function of engineered anti-tumor receptors (e.g., CARs) because T cells co-expressing ADR and CAR can effectively eliminate tumors and activated T cells in vitro. The present disclosure further provides the use of "standing" T cells co-expressing CAR and ADR in mouse models to demonstrate anti-cancer activity in vivo, while at the same time maintaining resistance to immune rejection from allogeneic T cells present in the same mice. For example, in Figure 14 We show that 4-1BB ADR is effective against 4-1BB+ tumor cells, suggesting that ADR may be used as a therapeutic modality against malignancies expressing 4-1BB.

[0077] In certain embodiments, the "standing" therapeutic cells express ADRs to resist immune rejection and either retain endogenous TCR specificity (e.g., to viral or tumor antigens) or the endogenous TCR is replaced with an engineered anti-tumor receptor, such as one or more CARs and / or one or more recombinant TCRs.

[0078] In a specific embodiment, standing cells are contained in a repository and can be modified before or after being preserved in a repository for a specific purpose.For example, T cells expressing ADR can be contained in a repository and ready for use, such as after tissue or organ transplantation, to prevent transplant rejection. T cells expressing ADR can be contained in a repository and can be selected or engineered with natural or transgenic TCR, such as to resist viral infection or cancer. T cells expressing ADR can be contained in a repository and can be transduced with a CAR for cancer or pathogenic infection. Cells expressing ADR can be contained in a repository and can be transduced with one or more CARs and / or one or more TCRs (for specific cancer-associated antigens or neoantigens expressed by patient-specific tumors).

[0079] In some cases, stored allogeneic cells are used to prevent rejection of solid organ transplants by destroying rejecting host immune cells, particularly when the cells expressing the ADR are not themselves alloreactive.

[0080] Although the cells contained in the reservoir can be allogeneic to the recipient individual, in alternative embodiments, the cells contained in the reservoir are autologous to the recipient individual. For example, an individual suffering from cancer can store ADR-expressing T cells in a reservoir for subsequent use, such as in the case of cancer remission. In other cases, autologous ADR-expressing cells are contained in a reservoir for the treatment of autoimmune disorders. B. For autoimmune diseases

[0081] Unwanted activation of endogenous autoreactive T cells in an individual can lead to devastating autoimmune diseases in that individual, such as diabetes, autoimmune colitis, and multiple sclerosis. In certain embodiments, immune cells expressing ADRs are used in an individual to prevent or treat one or more autoimmune disorders by inhibiting endogenous autoreactive T cells in the individual to affect the autoimmune disorder (or its potential development). In certain embodiments, such use of cells expressing ADRs leaves quiescent non-pathogenic naive T cells in the individual. Therefore, certain methods of the present disclosure utilize specific cells modified to express ADRs, which are provided to an individual in sufficient amounts to target activated T cells (including pathogenic T cells), thereby triggering the destruction of activated T cells.

[0082] In vivo activation of T cells with unwanted specificity may cause pathogenicity, and in certain embodiments, cells expressing one or more ADRs target activated T cells. In some cases, cells expressing ADRs target pathogenic cells that are a subset of activated T cells.

[0083] In certain cases, ADR-expressing T cells can be used to prevent or reverse life-threatening and debilitating conditions driven by activated T cells (e.g., organ rejection, graft-versus-host disease, type I diabetes, multiple sclerosis, autoimmune colitis, lupus, rheumatoid arthritis) using adoptive T cell transfer of ADR-expressing T cells.

[0084] In some cases, the ADR-expressing T cells further comprise one or more compositions other than the ADR that promote the treatment or prevention of one or more autoimmune disorders.

[0085] In some cases, one or more additional therapies are given to the individuality of the T cell that is provided to express ADR, to prevent or treat one or more autoimmune disorders. The individuality may be given or may not be given one or more immunosuppressive drugs, such as glucocorticoids, cytostatic agents, antibodies and / or drugs that act on immunoaffinity proteins. Additionally or alternatively, the individuality may be given one or more suitable vaccines.

[0086] In some cases, an individual is at risk for an autoimmune disorder and is provided with an effective amount of cells expressing an ADR to prevent the onset of the autoimmune disorder or to delay the onset and / or alleviate one or more symptoms, including, for example, severity and / or duration. An individual at risk for an autoimmune disorder is, for example, an individual with a personal or family history of the disorder, a female of a certain ethnicity, etc. In some cases, an individual may have an autoimmune disorder and wish to prevent or reduce its severity and / or duration or delay the onset of another autoimmune disorder.

[0087] Examples of autoimmune disorders that can be prevented or treated with cells expressing ADRs include at least the following: Achalasia; Addison's disease; Adult Still's disease; Agammaglobulinemia; Alopecia areata; Amyloidosis; Ankylosing spondylitis; Anti-GBM / Anti-TBM nephritis; Antiphospholipid syndrome; Autoimmune angioedema; Autoimmune dysautonomia; Autoimmune encephalomyelitis; Autoimmune hepatitis; Autoimmune inner ear disease (AIED); Autoimmune myocarditis; Autoimmune oophoritis; Autoimmune orchitis; Autoimmune pancreatitis; Autoimmune retinopathy; Autoimmune urticaria; Axonal and neuronal neuropathy (AMAN); Baló disease; Behcet's disease; Benign mucous membrane pemphigoid; Bullous pemphigoid; Castleman disease (CD); Celiac purpura; Chagas disease. disease); chronic inflammatory demyelinating polyneuropathy (CIDP); chronic relapsing multifocal osteomyelitis (CRMO); Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis with pancreatic eosinophilia (EGPA); cicatricial pemphigoid; Cogan's syndrome; cold agglutinin disease; congenital heart block; coxsackie myocarditis; CREST syndrome; Crohn's disease; dermatitis herpetiformis; dermatomyositis; Divic disease (neuromyelitis optica); discoid lupus; Dressler's syndrome syndrome; endometriosis; eosinophilic esophagitis (EoE); eosinophilic fasciitis; erythema nodosum; essential mixed cryoglobulinemia; Evans syndrome; fibromyalgia; fibrosing alveolitis; giant cell arteritis (temporal arteritis); giant cell myocarditis; glomerulonephritis; Goodpasture's syndrome; granulomatosis with polyangiitis; Graves' disease; Guillain-Barré syndrome; Hashimoto's thyroiditis; hemolytic anemia; Henoch-Schonlein purpura purpura (HSP); herpes gestationis or pemphigoid gestationis (PG); hidradenitis suppurativa (HS) (acne inversa); hypogammaglobulinemia; IgA nephropathy; IgG4-related sclerosing disease; immune thrombocytopenic purpura (ITP); inclusion body myositis (IBM); interstitial cystitis (IC); juvenile arthritis; juvenile diabetes mellitus (type 1 diabetes); juvenile myositis (JM); Kawasaki disease; Lambert-Eaton syndrome; leukocytoclastic vasculitis; lichen planus; lichen sclerosus; woody conjunctivitis; linear IgA disease (LAD); lupus; chronic Lyme disease; Meniere's disease; microscopic polyangiitis (MPA); mixed connective tissue disease (MCTD); corneal erosion (Mooren's ulcer); Mucha-Habermann disease;Multifocal motor neuropathy (MMN) or MMNCB; multiple sclerosis; myasthenia gravis; myositis; narcolepsy; neonatal lupus; neuromyelitis optica; neutropenia; ocular cicatricial pemphigus; optic neuritis; relapsing rheumatic disease (PR); PANDAS; paraneoplastic cerebellar degeneration (PCD); paroxysmal nocturnal hemoglobinuria (PNH); Parry's disease Romberg syndrome; pars planitis (peripheral uveitis); Parsenager-Turner syndrome; pemphigus; peripheral neuropathy; peripheral encephalomyelitis; pernicious anemia (PA); POEMS syndrome; polyarteritis nodosa; polyglandular syndromes types I, II, and III; polymyalgia rheumatica; polymyositis; postmyocardial infarction syndrome; postpericardiotomy syndrome; primary biliary cirrhosis; primary sclerosing cholangitis; progesterone dermatitis; psoriasis; psoriatic arthritis; pure red cell aplasia (PRCA); pyoderma gangrenosum; Raynaud's phenomenon; reactive arthritis; reflex sympathetic dystrophy; relapsing polychondritis; restless legs syndrome (RLS); retroperitoneal fibrosis; rheumatic fever; rheumatoid arthritis; sarcoidosis; Schmidt's syndrome; scleritis; scleroderma; Sjögren's syndrome (; syndrome; sperm and testicular autoimmunity; stiff-person syndrome (SPS); subacute bacterial endocarditis (SBE); Susac's syndrome; sympathetic ophthalmia (SO); Takayasu's arteritis; temporal arteritis / giant cell arteritis; thrombocytopenic purpura (TTP); Tolosa-Hunt syndrome (THS); transverse myelitis; type 1 diabetes mellitus; ulcerative colitis (UC); undifferentiated connective tissue disease (UCTD); uveitis; vasculitis; vitiligo; Vogt-Koyanagi-Harada disease; and Wegener's granulomatosis (or granulomatosis with polyangiitis (GPA)). C. Used to eliminate NK cells

[0088] Immune cells expressing ADRs can be used to eliminate NK cells if desired. As demonstrated herein, the presence of ADRs on certain immune cells provides specific cytotoxic activity against NK cells involved in mediating HLA low Therefore, in the case of the need to maintain HLA low In the case of HLA-incompatible or HLA-incompatible cells (e.g., adoptive transfer of allogeneic cells to certain individuals is possible), the use of cells expressing ADRs avoids NK cell activation and rejection of HLA-incompatible cells. Specifically, as shown herein, co-culture of ADR-expressing T cells leads to the elimination of NK cells and thus counteracts NK cell-mediated host rejection of ADR-expressing allogeneic T cells. D. To promote the engraftment of allogeneic cells / tissues / organs

[0089] In certain aspects of avoiding the activation of alloreactive T cells, rejection of allogeneic cells, tissues or organs in the recipient can be avoided (which is primarily mediated by an alloreactive T cell population from the recipient). For example, if no measures are taken to avoid such rejection, the activation of the recipient's alloreactive T cells can lead to transplant failure. Thus, in certain embodiments, methods of transplanting cells, tissues or organs into an individual utilize delivery of an effective amount of cells expressing an ADR before, during and / or after the respective transplantation of cells, tissues or organs. In some cases, the cells expressing the ADR are not themselves part of the cells, tissues or organs of the transplanted subject, while in other cases, the cells expressing the ADR are part of the respective cells, tissues or organs.

[0090] The tissue for transplantation can be of any kind, including, for example, at least skin, cornea, bone, tendon, heart valve, vein or artery. The organ for transplantation can be of any kind, including, for example, heart, kidney, liver, lung, pancreas, intestine and thymus.

[0091] In certain embodiments, cells expressing ADRs enhance the use of allogeneic cells in an individual in a two-pronged approach: (1) they suppress endogenous alloreactive T cells in the individual; and (2) they suppress NK cell-mediated rejection in the individual. Thus, the ADR molecules can enhance the persistence and activity of any type of third-party source therapeutic cells in the individual, including, for example, allogeneic therapeutic cells, including T cells, NK cells, NK-T cells, mucosal-associated invariant T cells (MAIT) and other cytotoxic cells, including cells expressing engineered constructs (e.g., chimeric antigen receptors (CARs), transgenic TCRs, etc.). E. For the prevention or treatment of graft-versus-host disease (GvHD) during allogeneic cell / tissue / organ transplantation

[0092] In another specific aspect of avoiding the activation of alloreactive T cells, life-threatening alloimmune reactions in individuals receiving allogeneic cell, tissue or organ transplants can be avoided. For example, such transplants will contain alloreactive T cells of the donor, which can cause the development of graft-versus-host disease (GvHD) if measures are not taken to avoid their activation. Therefore, in specific embodiments, methods for transplanting cells, tissues or organs into individuals utilize cells expressing an effective amount of an ADR before, during and / or after the respective transplantation of cells, tissues or organs. In some cases, the cells expressing the ADR are not themselves part of the cells, tissues or organs of the transplanted subject, while in other cases, the cells expressing the ADR are part of the respective cells, tissues or organs.

[0093] The tissue for transplantation can be of any kind, including, for example, at least skin, cornea, bone, tendon, heart valve, vein or artery. The organ for transplantation can be of any kind, including, for example, heart, kidney, liver, lung, pancreas, intestine and thymus. III. Generation of ADR-Expressing Cells

[0094] Cells expressing ADR molecules can be produced in a variety of ways, all of which are routine in the art. The production method can include obtaining a cell to be modified to express an ADR molecule and producing the ADR molecule. Origin of AT cells

[0095] Before the T cells expressing the ADR of the present disclosure are amplified and genetically modified, a source of T cells can be obtained from a subject. Such an acquisition step may or may not be part of the method. In some cases, obtaining the T cells to be modified and their manipulation can be performed by a party other than the party providing the T cells expressing the ADR to the individual. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present disclosure, any number of T cell lines available in the art can be used. In certain embodiments, any number of techniques known to those skilled in the art, such as Ficoll, can be used. TMSeparation, T cells are obtained from a unit of blood collected from a subject. In one embodiment, cells from individual circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, for example, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and the cells are placed in an appropriate buffer or culture medium for subsequent processing steps. In one embodiment, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the washing solution lacks calcium and may lack magnesium, or may lack many (if not all) divalent cations. As will be readily understood by those skilled in the art, the washing step can be accomplished by methods known to those skilled in the art, for example, by using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate or Haemonetics Cell Saver 5), used in accordance with the manufacturer's instructions. After washing, cells can be resuspended in a variety of biocompatible buffers, such as Ca-free 2+ Mg-free 2+ Alternatively, the apheresis sample can be freed of undesirable components and the cells resuspended directly in culture medium.

[0096] In another embodiment, the monocytes are removed by lysing the red blood cells and removing the monocytes (e.g., by PERCOLL TM T cells are separated from peripheral blood lymphocytes by gradient centrifugation or by counterflow centrifugal elutriation. Specific subsets of T cells, such as CD3 + 、CD28 + 、CD4 + 、CD8 + 、CD45RA + and CD45RO + T cells.

[0097] The enrichment of T cell populations by negative selection can be achieved by combining antibodies against surface markers specific to the negatively selected cells. One method is cell sorting and / or selection (by negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies against cell surface markers present on negatively selected cells). For example, to enrich CD4 T cells by negative selection, the cells are separated and / or selected. +For cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be necessary to enrich or positively select cells that typically express CD4 + 、CD25 + 、CD62L hi , GITR + and FoxP3 + Alternatively, in certain embodiments, regulatory T cells are removed by anti-C25 conjugated beads or other similar selection methods.

[0098] In order to separate the desired cell mass by positive or negative selection, the concentration of cells and surfaces (for example, particles, such as beads) can vary. In certain embodiments, it may be desirable to significantly reduce the volume (that is, increase the concentration of cells) that beads and cells mix together to ensure the maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml was used. In one embodiment, a concentration of 1 billion cells / ml was used. In another embodiment, greater than 100 million cells / ml was used. In another embodiment, the cell concentration used was 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million or 50 million cells / ml. In another embodiment, the cell concentration used is selected from 75 million, 80 million, 85 million, 90 million, 95 million or 100 million cells / ml. In a further embodiment, a concentration of 125 million or 150 million cells / ml can be used. Use of high concentrations can result in increased cell yield, cell activation, and cell expansion. In another embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and a surface (e.g., particles, such as beads), the interaction between the particles and the cells is minimized.

[0099] The T cells for stimulation can also be frozen after the washing steps. It is hoped that, without being bound by theory, freezing and subsequent thawing steps provide a more uniform product by removing the granulocytes and (to some extent) monocytes in the cell mass. After the washing steps of removing plasma and platelets, the cells can be suspended in a freezing solution. Many freezing solutions and parameters are known in the art. In certain embodiments, the frozen cells are thawed and washed, as described herein, and before using the method of the present invention to activate, they are allowed to stand at room temperature for one hour.

[0100] It is also contemplated in the context of the present disclosure that a blood sample or a single blood component method product may be collected from a subject in the time period before the cells for amplification as described herein may be needed. In this way, the source of cells to be amplified can be collected at any necessary time point, and the required cells (e.g., T cells) are separated and frozen for use in subsequent T cell therapies (for any number of diseases or conditions, which will benefit from T cell therapies, such as those described herein). In one embodiment, a blood sample or a single blood component is obtained from a generally healthy subject. In certain embodiments, a blood sample or a single blood component is obtained from a generally healthy subject, and the generally healthy subject has the risk of developing a disease but has not yet developed a disease, and the target cells are separated and frozen for subsequent use. In certain embodiments, T cells can be expanded, frozen, and used at a later time. In certain embodiments, a sample is collected from a patient shortly after diagnosing a specific disease as described herein but before any treatment is performed. In further embodiments, cells are isolated from a blood sample or apheresis of a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants, e.g., cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunoablative agents, e.g., CAMPATH, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (which is important for growth factor-induced signaling) (rapamycin) (Liu et al., Cell 66: 807-815, 1991; Henderson et al., Immun. 73: 316-321, 1991; Bierer et al., Curr. Opin. Immun. 5: 763-773, 1993). In a further embodiment, cells are isolated and frozen for later use in combination with (e.g., before, at the same time, or after) bone marrow or stem cell transplantation, T cell ablative therapy (using chemotherapeutic agents such as fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH). In another embodiment, cells are previously isolated and can be frozen for later therapeutic use (following B cell ablative therapy such as an agent reactive with CD20, eg, Rituxan). Activation and expansion of BT cells

[0101] Whether before or after genetically modifying the T cells to express the ADR, T cells can generally be activated and expanded using methods such as those described in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041 and U.S. Patent Application Publication No. 20060121005. Typically, T cells of the present disclosure are expanded by contacting with a surface to which are attached reagents that stimulate CD3 / TCR complex-related signals and ligands that stimulate co-stimulatory molecules on the T cell surface. Such processes are known in the art. In other cases, T cells can be modified to express ADRs without prior activation. C. Generation of ADR molecules

[0102] Looking at polynucleotides encoding ADRs as a whole, nucleic acid sequences encoding ADR molecules can be obtained using recombinant methods known in the art, for example, by screening libraries from cells expressing the gene, by deriving the gene from a vector known to contain the gene, or by isolating the gene directly from cells and tissues containing the gene (using standard techniques). Alternatively, the target ADR polynucleotide can be produced synthetically rather than cloned.

[0103] In brief overview, expression of a synthetic polynucleotide encoding an ADR is typically achieved by operatively linking a nucleic acid encoding an ADR polypeptide or portion thereof to a promoter and incorporating the construct into an expression vector. The vector can be suitable for replication and integration in eukaryotic organisms. A typical cloning vector contains transcriptional and translational terminators, an initiation sequence, and a promoter for regulating expression of the desired nucleic acid sequence.

[0104] ADR polynucleotides can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Specific target vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0105] In addition, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. Generally, suitable vectors are included in at least one biological replication origin, promoter sequence, convenient restriction endonuclease site and one or more selection markers (for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent number 6,326,193).

[0106] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0107] Other promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, they are located in the region 30-110bp upstream of the start site, although recently many promoters have been shown to also contain functional elements downstream of the start site. The interval between promoter elements is normally flexible, so that when elements are reversed or moved relative to each other, promoter function is retained. In the thymidine kinase (tk) promoter, the interval between promoter elements can be increased to 50bp before activity begins to decline. Depending on the promoter, it seems that a single element can work collaboratively or independently to activate transcription.

[0108] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation growth factor-1 alpha (EF-1alpha). However, other constitutive promoter sequences may also be used, including but not limited to: simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of inducible promoters provides a molecular switch that can turn on the expression of the polynucleotide sequence operably linked to it when such expression is desired, or turn off such expression when such expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0109] In order to assess the expression of ADR polypeptide or its part, the expression vector to be introduced into the cell can also comprise selection marker gene or reporter gene or both, to promote from by viral vector transfection or infected cell colony identification and selection expression cell.In other aspects, selection marker can be carried on independent DNA fragmentation and be used for cotransfection step.Selection marker and reporter gene can both be flanked by suitable regulatory sequence, so that can be expressed in host cell.Useful selection marker comprises for example antibiotic resistance gene, for example neo etc.

[0110] Reporter genes are used to identify potentially transfected cells and to assess the function of regulatory sequences. Typically, a reporter gene is a gene that is not present or expressed in the recipient organism or tissue and encodes a polypeptide whose expression is demonstrated by some easily detectable properties (e.g., enzymatic activity). The expression of the reporter gene is measured at a suitable time after the DNA is introduced into the recipient cell. Suitable reporter genes can include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Typically, a construct with a minimal 5' flanking region that shows the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be connected to a reporter gene and used to assess the ability of an agent to regulate promoter-driven transcription.

[0111] Methods for introducing and expressing ADR polynucleotides into cells are known in the art. In the case of expression vectors, the vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0112] Physical methods for introducing ADR polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0113] Biological methods for introducing target ADR polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used methods for inserting genes into mammals (e.g., human cells). Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0114] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0115] In the case of using non-viral delivery system, exemplary delivery vehicle is liposome. Consider lipid preparation for nucleic acid being introduced into host cell (in vitro, in vitro or in vivo). On the other hand, nucleic acid can be connected with lipid. The nucleic acid being connected with lipid can be encapsulated in the aqueous interior of liposome, be dispersed in the lipid bilayer of liposome, be attached to liposome by the connecting molecule being connected with liposome and oligonucleotide, be wrapped in liposome, be compounded with liposome, be dispersed in the solution containing lipid, mix with lipid, be combined with lipid, be included in lipid as suspension, be included in micelle or be compounded with micelle, or be connected with lipid. The composition that lipid, lipid / DNA or lipid / expression vector are relevant is not limited to any particular structure in solution. For example, they can exist with double-layer structure, exist as micelle or exist with " collapse " structure. They can also be simply dispersed in solution, may form the aggregate of size or shape inhomogeneity. Lipid is fatty substance, and it can be naturally occurring or synthetic lipid. For example, lipids include fat droplets naturally present in the cytoplasm as well as a class of compounds comprising long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0116] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, New York); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristoylphosphatidylglycerol ("DMPG") and other lipids can be obtained from AvantiPolar Lipids, Inc. (Birmingham, Alabama). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it is more volatile than methanol. "Liposome" is a general term that includes various unilamellar and multilamellar lipid vehicles formed by creating closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicle structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess of aqueous solution, they form spontaneously. The lipid components undergo self-rearrangement before forming a closed structure and entrain water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions having structures that differ from normal vesicle structures in solution are also included. For example, lipids can adopt micellar structures or exist only as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0117] In some cases, ADR molecules can be integrated into the endogenous nucleic acid of the cell. There can be a target site for homologous recombination, wherein it is desired to integrate the construct at a specific site. For example, materials and methods for homologous recombination known in the art can be used to knock out endogenous genes and replace the endogenous genes (at the same locus or other positions) encoded by the construct. For homologous recombination, OMEGA or O-vectors can be used. CRISPR / Cas9, zinc finger nucleases, TALE nucleases, meganucleases and other site-directed nucleases can be used for targeting and cutting the specific site in the genome to promote homologous recombination.

[0118] Exemplary T cells that have been engineered to include a construct expressing an ADR can be grown in culture under selective conditions, and cells selected to have the construct can then be expanded and further analyzed, for example, using a polymerase chain reaction to determine the presence of the construct in the host cell. Once the engineered host cells are identified, they can then be used as planned, for example, by being expanded in culture or introduced into a host organism.

[0119] Depending on the nature of the cells, the cells can be introduced into a host organism, such as a mammal, in a variety of ways. In a specific embodiment, the cells can be introduced into a tumor site, although in alternative embodiments, the cells can home to the cancer or be modified to home to infected tissues. The number of cells used will depend on a variety of circumstances, the purpose of introduction, the lifespan of the cells, the regimen used, for example, the number of administrations, the ability of the cells to proliferate, the stability of the recombinant construct, etc. The cells can be administered in the form of a dispersion, typically injected at or near the target site. The cells can be in a physiologically acceptable culture medium.

[0120] DNA introduction does not necessarily lead to integration in every case. In some cases, it may be sufficient to maintain the introduced DNA for a short period of time. In this way, a short-term effect can be achieved, where cells can be introduced into a host and then started after a predetermined time, for example, after the cells have been able to home to a specific site. Example

[0121] The following examples are presented in order to more fully illustrate specific embodiments of the present disclosure. However, they should in no way be construed as limiting the broad scope of the present disclosure. Example 1 Selectively targeting autologous / allogeneic immune defense receptors on pathogenic T cells

[0122] This article discloses a novel method for specifically targeting pathogenic T cells using autologous / allogeneic immune defense receptors (ADRs) expressed on normal T cells. T cells expressing ADRs only find and eliminate activated T cells, leaving behind resting non-pathogenic naive and memory T cells (which make up the majority of circulating lymphocytes).

[0123] The concept of ADR-mediated targeting is based on the observation that within 24 hours of activation, T cells transiently upregulate the co-stimulatory genes 4-1BB, OX40, and / or CD40L on their cell surface. Expression of 4-1BB, OX40, and / or CD40L is maintained only when the T cells are activated for cytotoxicity, and the expression is gradually downregulated within 4-5 days after TCR signaling ceases. Notably, activated CD8 + T cells showed higher expression levels of 4-1BB, while CD4 + T cells preferentially express OX40 and / or CD40L. In addition to activated T cells, ADR ligands are only expressed on activated NK cells and a few other non-critical, replenishable cell subsets. The expression patterns of 4-1BB, OX40, and CD40L make these genes attractive targets for highly specific targeting of activated cells while avoiding permanent destruction of critical immune and non-immune tissues.

[0124] To explore the feasibility of targeting these activated T cells, the autologous / allogeneic immune defense receptor (ADR) was designed to include a 4-1BB- or OX40-specific ligand, or a CD40L-specific receptor, which was directly connected to the CD3ζ chain (encoded in the γ-retroviral vector SFG) via a spacer. The spacer region is combined: a) to enable type II proteins 4-1BBL and OX40L to be integrated into the type I skeleton of the ADR; b) to facilitate detection of ADR on the cell surface by FACS staining. Transduction of T cells with this construct effectively forces ADR to be expressed on the cell surface. These ADR T cells have a strong and robust killing ability against cells expressing 4-1BB, OX40, and CD40L, clearing 90-99% of target cells within 48 hours. These results demonstrate the feasibility of generating functional 4-1BB, OX40, and CD40L-specific ADR T cells.

[0125] Because ADR signaling in T cells leads to upregulation of 4-1BB, OX40, and CD40L, thereby promoting killing and preventing effector cell expansion, the effect of CRISPR / Cas9 genomic disruption of ADR target genes in effector T cells was explored. The inventors have previously shown that this CRISPR / Cas9 approach can prevent killing of primary human T cells expressing CD7-specific CARs. In this case, using CRISPR / Cas9, the inventors were able to knock out 4-1BB expression in ~70% of ADR T cells, and in the presence of 4-1BB, the expression of 4-1BB was significantly reduced. + This consistently increased ADR T cell expansion >2-fold 48 h after target cell co-culture without affecting cytotoxicity.

[0126] Next, the ability of ADR T cells to selectively eliminate activated T cells was tested. 4-1BB, OX40, and CD40LADR T cells were co-cultured with fluorescently labeled resting or CD3 / CD28 activated T cells. Residual viable CD4 + and CD8 + T cells. Co-culture with T cells expressing 4-1BB-, OX40-, or CD40L-specific ADRs for 72 hours resulted in no reactivity to resting autologous T cells ( Figure 2B In contrast, co-culturing 4-1BB ADR T cells with CD3 / CD28 activated T cells eliminated most CD8 + and some CD4 + Incubation with OX40 ADR T cells resulted in activated CD4 + The corresponding high levels of depletion and activation of CD8 T cells + Moderate depletion of T cells. CD40L ADRT cells are activated CD4 + T cells produced moderate cytotoxicity, but CD8 + No effect was observed on T cells. OX40, CD40L and 4-1BB ADR T cells targeted activated CD4 + and CD8 + The different targeting spectra of T cells are related to the magnitude and kinetic differences of OX40, CD40L and 4-1BB expression on each T cell subset observed. This characteristic of ADR can be used to preferentially target one or two subsets of allogeneic or autoreactive T cells (as needed). Therefore, ADR expression enables T cells to specifically target activated (pathogenic) T cells, but leaves resting cells, indicating their clinical use.

[0127] In an in vitro mixed lymphocyte reaction (MLR) assay, we evaluated whether virus-specific T cells (VSTs) expressing ADRs could protect against allogeneic rejection. CMV-specific T cells were generated from HLA-A2-negative donors, and control non-transduced or ADR-transduced VSTs were co-transfected with alloreactive HLA-A2-positive CMV-specific T cells at a 1:2 cell:cell ratio. + The inventors then cultured the cells for 12 days. At the end of the co-culture, the control VST was almost completely HLA-A2 + PBMCs were completely depleted, whereas VSTs expressing either the 4-1BB ADR or the OX40 ADR resisted rejection. Taken together, these results demonstrate the feasibility and selectivity of targeting activated T cells using a newly developed ADR platform implementation. Example 2 Autologous / allogeneic immune defense receptors for selective targeting of NK cells

[0128] ADR showed specific cytotoxic activity against NK cells, which mediate the expression of HLA low or HLA-incompatible cells.

[0129] As part of anti-tumor and antiviral immune surveillance, NK cells are able to recognize HLA-incompatible cells or cells with low HLA expression. Therefore, adoptive transfer of allogeneic cells into immune-sufficient (immunoreplete) patients will result in NK cell activation and rejection of HLA-incompatible cells. Here, it is shown that co-cultured T cells expressing 4-1BB and OX40-specific autologous / allogeneic immune defense receptors (ADR) lead to the clearance of NK cells, thereby offsetting the NK cell-mediated host rejection of allogeneic T cells equipped with ADR. Therefore, ADR not only suppresses alloreactive T cell responses, but also suppresses NK cell-mediated rejection reactions, further supporting the application of ADR to enhance the persistence and activity of "standing" therapeutic T cells. Example 3 T cells expressing ADR eliminate target cells

[0130] ADRs can be expressed on the cell surface of immune cells and promote cytotoxicity against the corresponding targets. Figure 1A An example of a schematic diagram of ADR is shown (a marker such as GFP is optional). Expression of ADR on the surface of T cells was confirmed ( Figure 1B ), and cells expanded commensurately with controls ( Figure 1C ). ( Figure 1D ) T cells expressing ADR are cytotoxic to target cells expressing the corresponding ADR ligand ( Figure 1D ). Figure 1EWe demonstrate the expansion of wild-type and 4-1BB KO T cells expressing the 4-1BB ADR and their cytotoxicity against 4-1BB+ targets. Knocking out the ADR ligand on T cells further enhances expansion and cytotoxicity, and the expansion or function of ADR-T cells does not require co-expression of the ADR and its ligand on T cells ( Figure 1E ).

[0131] The selective expression of ADR ligands on activated T cells enables their selective elimination by ADR T cells. The expression of ADR ligands on resting T cells and activated T cells after TCR stimulation was determined ( Figures 2A-2C ). ADR T cells to resting CD4 + and CD8 + T cells have no cytotoxicity ( Figure 2D ), but after 48 hours of co-culture, ADR T cells eliminated activated CD4 + and CD8 + T cells ( Figure 2E ).

[0132] As an example, expression of 4-1BB ADR protected T cells from immune rejection in the MLR model. Representative dot plots show that TCR KO T cells co-expressing ADR were protected from rejection after co-culture with allogeneic PBMC at a ratio of 1:10 ADR T:PBMC ( Figure 3A Absolute counts of donor T cells and allogeneic T cells in PBMCs during co-culture ( Figures 3B-3C ) for virus-specific ADR T cells ( Figures 3D-3F ) are the same.

[0133] In an in vitro mixed lymphocyte reaction, ADR expression protected allogeneic virus-specific T cells from immune rejection. Representative dot plots show that ADR VSTs were protected from immune rejection by recipient allogeneic PBMCs ( Figure 4A ).exist Figure 4B and Figure 4C Absolute counts of recipient T cells and donor VSTs at various time points during the MLR are provided in .

[0134] exist Figure 5 In a study published in the journal Cell Biology, the ADR VSTs retained their antiviral function. When the ADR VSTs were co-cultured with monocytes pulsed with a mixture of viral peptides, monocyte counts showed that they cleared virus-infected cells equally well as unmodified VSTs.

[0135] Activated NK cells upregulate ADR ligands and can be selectively targeted by ADR T cells. 4-1BB expression on resting and activated NK cells was confirmed ( Figures 6A-6BThe residual counts of resting and activated NK cells were determined after 24 h of co-culture with 4-1BB ADR T cells ( Figure 6C ).exist Figure 6D In this study, MHC-deficient ADR T cells were protected from immune rejection by allogeneic PBMCs by controlling the expansion of NK cells. Figure 6E Absolute counts of donor T cells and allogeneic NK cells during coculture were determined in the . MHC-deficient ADR T cells resisted immune rejection of NK cells after 48 h of coculture at a 1:1 E:T ratio ( Figure 6F During MLR using PBMCs, ADR T cells control the expansion of alloreactive NK cells ( Figure 6G ), where the absolute counts of NK cells are plotted on Figure 6H middle.

[0136] ADR expression protects allogeneic T cells from immune rejection in vivo. Figure 7A , an example of a mouse model of immune rejection is shown, in which mice were given T cells from an HLA-A2+ donor after sublethal irradiation, followed by administration of allogeneic HLA-A2- T cells 4 days later. Control T cells from an HLA-A2- donor were rejected at day 18, whereas cells expressing ADR were protected ( Figure 7B The absolute counts of T cells from HLA-A2+ and HLA-A2- donors at different time points were determined ( Figure 7C ). Figure 7D The modified in vivo model in depicts that instead of allogeneic T cells, mice received whole PBMCs (containing both T cells and NK cells) from donor 1. Figure 7E Representative dot plots in Figure 5 show that ADR T cells protected against immune rejection and also protected mice from the rapid onset of lethal GvHD.

[0137] Co-expression of CAR and ADR preserves the functionality of both receptors. Figure 8A An example of a representative immune cell that co-expresses ADR and CAR is shown. Co-expression of CAR and ADR on the cell surface was confirmed ( Figure 8B ).exist Figure 8C In Figure 2, the cytotoxicity of CAR-ADR T cells against NALM-6 (CD19+CAR target) as an example of a target is shown. Figure 8D The cytotoxicity of CAR-ADR T cells against activated T cells (ADR targets) was also determined in Figure 8E Figure 5 shows the cytotoxic activity of CAR-ADR T cells against two cellular targets when they were co-cultured with the two targets.

[0138] CAR-ADR T cells are protected from immune rejection and exert potent anti-tumor activity. Examples of mouse models and protocols are available in Figure 9A As an example of a regimen, mice received allogeneic T cells from donor 1 and b2mKO NALM6 24 hours apart, followed by a single dose of CAR-ADR T cells from donor 2. Figure 9B The kinetics of T cells from donor 2 in peripheral blood are provided. Figure 9C The kinetics of donor 1 T cells in the experimental groups are provided. Figure 9D Leukemic burden in mice was shown, and overall survival of mice was determined ( Figure 9E ).

[0139] Figures 13A-13C In solid tumor models, CAR-ADR T cells were protected from immune rejection and exerted potent antitumor activity. Schematic diagram of mouse models and treatment examples are provided in Figure 10A , where mice received allogeneic T cells from donor 1 and the b2mKO neuroblastoma cell line CHLA255 24 hours apart, followed by a single dose of CAR-ADR T cells from donor 2. Donor 2 GD2 CAR T cells were rejected on day 18, whereas CAR-ADR T cells resisted allogeneic rejection and persisted in the peripheral blood ( Figure 10B The tumor burden of mice is shown in Figure 13C , where * indicates death related to xeno-GvHD in the ATC+GD2 CAR T group.

[0140] TCR knockout CAR-ADR T cells were protected from immune rejection and exerted potent anti-tumor activity. A schematic diagram of the mouse model is provided, in which mice received allogeneic T cells from donor 1 and b2mKONALM6 24 hours apart, and then received a single dose of TCR-edited CAR-ADR T cells from donor 2 ( Figure 11A The kinetics of T cells from donor 2 in peripheral blood are provided ( Figure 11B ). Shows the kinetics of donor 1 T cells in the experimental group ( Figure 11C ). Leukemia burden in mice is provided ( Figure 11D ) and the overall survival rate of mice ( Figure 11E ).

[0141] ADR T cells protect mice from lethal xenogeneic GvHD. Figure 12A A schematic diagram of the model is provided in

[15] , and the expansion of FFLuc-labeled ADR T cells in vivo is demonstrated ( Figure 12B The kinetics of weight gain / loss in mice were determined ( Figure 12CThe overall survival rate of mice is depicted ( Figure 12D ).

[0142] A second generation ADR with a CD28 intracellular signaling domain ("ADR.28ζ") was utilized (as an example). An example of the structure of ADR.28ζ is depicted ( Figure 13A The in vitro cytotoxicity of ADR.28ζ against cells expressing the target was determined ( Figure 13B and Figure 13C ADR.28ζ protected mice from xeno-GvHD ( Figure 13B ). A schematic diagram of the model is shown ( Figure 13D ). The expansion of FFLuc-labeled ADR.28ζT cells in vivo was confirmed ( Figure 13E The kinetics of weight gain / loss in mice were determined ( Figure 13F ), and the overall survival rate of mice ( Figure 13G ).

[0143] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and modifications may be made herein without departing from the spirit and scope of the design defined by the appended claims. Moreover, the scope of this application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, material compositions, means, methods and steps described in the specification. As will be readily understood by those skilled in the art from this disclosure, processes, machines, manufactures, material compositions, means, methods or steps that currently exist or will be developed in the future for performing functions substantially the same as those of the corresponding embodiments described herein or for achieving results substantially the same as those of the corresponding embodiments described herein may be utilized according to this disclosure. Thus, the appended claims are intended to include such processes, machines, manufactures, material compositions, means, methods or steps within their scope. This application provides the following implementation schemes: 1. An isolated polynucleotide comprising a sequence encoding a polypeptide, wherein the polypeptide comprises: (1) one or more of OX40-specific ligand, 4-1BB-specific ligand, CD40L-specific ligand or functional derivatives thereof; operatively linked to (2) Signal transduction domain that promotes T cell activation. 2. The polynucleotide of embodiment 1, wherein the polypeptide comprises an OX40-specific ligand. 3. The polynucleotide of embodiment 1, wherein the polypeptide comprises a 4-1BB-specific ligand. 4. The polynucleotide of embodiment 1, wherein the polypeptide comprises a CD40L-specific ligand. 5. The polynucleotide of any one of embodiments 1-2, wherein the OX40-specific ligand is OX40L, an antibody targeting OX40, an OX40L-Fc fusion, or a combination thereof. 6. The polynucleotide of any one of embodiments 1 and 3, wherein the 4-1BB-specific ligand is 4-1BBL, an antibody targeting 4-1BB, a 4-1BBL-Fc fusion, or a combination thereof. 7. The polynucleotide of any one of embodiments 1 and 4, wherein the CD40L-specific ligand is CD40, an antibody targeting CD40L, a CD40-Fc fusion, or any other engineered protein capable of specifically binding to CD40L. 8. The polynucleotide of any one of embodiments 1-7, wherein the polypeptide further comprises 1, 2 or more costimulatory domains. 9. The polynucleotide of any one of embodiments 1 to 8, wherein the polynucleotide further comprises a sequence encoding a spacer located between (1) and (2). 10. The polynucleotide of embodiment 9, wherein the spacer has a length of 10 to 220 amino acids. 11. The polynucleotide of embodiment 10, wherein the spacer has a sequence that facilitates surface detection using an antibody. 12. The polynucleotide of embodiment 11, wherein the spacer is detectable with an anti-Fc antibody. 13. The polynucleotide of embodiment 12, wherein the spacer comprises an IgG Fc portion. 14. The polynucleotide of any one of embodiments 1-13, wherein the polynucleotide further encodes a chimeric antigen receptor, a T cell receptor, or both. 15. The polynucleotide of embodiment 14, wherein a 2A element or an IRES element is present on the polynucleotide encoding the polypeptide of embodiment 1 and the polynucleotide encoding the chimeric antigen receptor, T cell receptor, or both. 16. The polynucleotide of embodiment 15, wherein the chimeric antigen receptor comprises one, two or more co-stimulatory domains. 17. The polynucleotide of any one of embodiments 1-16, wherein the polynucleotide is present on a vector. 18. The polynucleotide of embodiment 17, wherein the vector is a viral vector or a non-viral vector. 19. The polynucleotide of embodiment 18, wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector. 20. The polynucleotide of any one of embodiments 1-19, wherein the polynucleotide is present in a cell. 21. The polynucleotide of embodiment 20, wherein the cell is a eukaryotic cell or a bacterial cell. 22. The polynucleotide of embodiment 20 or 21, wherein the cell is an immune cell. 23. The polynucleotide of any one of embodiments 20-22, wherein the cell is engineered. 24. The polynucleotide of embodiment 22, wherein the immune cell is a T cell. 25. The polynucleotide of embodiment 24, wherein the T cell comprises one or more chimeric antigen receptors. 26. The polynucleotide of embodiment 25, wherein the chimeric antigen receptor comprises 1, 2 or more co-stimulatory domains. 27. The polynucleotide of embodiment 24, wherein the T cell comprises one or more engineered T cell receptors (TCRs). 28. A polypeptide expressed by the polynucleotide of any one of embodiments 1-27. 29. A polypeptide comprising: (1) one or more of OX40-specific ligand, 4-1BB-specific ligand, and CD40; operatively linked to (2) Signal transduction domain that promotes T cell activation. 30. A polypeptide according to embodiment 29, wherein the signaling domain that promotes T cell activation is from the CD3ζ subunit, DAP12, Fc receptor or a combination thereof. 31. The polypeptide of embodiment 29 or 30, wherein the polypeptide further comprises 1, 2 or more co-stimulatory domains. 32. A cell expressing a chimeric receptor comprising the polynucleotide of any one of embodiments 1-27 or the polypeptide of any one of embodiments 29-31. 33. The cell of embodiment 32, wherein the cell is an immune cell. 34. The cell of embodiment 32 or 33, wherein the cell is engineered. 35. The cell of embodiment 33, wherein the immune cell is a T cell. 36. A cell according to embodiment 35, wherein the T cell is a CAR-transduced T cell. 37. The cell of embodiment 35, wherein the T cell is a T cell receptor (TCR)-transduced T cell. 38. The cell of any one of embodiments 32-37, wherein the cell is engineered to lack endogenous expression of one or more genes. 39. The cell of embodiment 38, wherein the cell is engineered to lack endogenous expression of 4-1BB, OX40 and / or CD40L. 40. The cell of embodiment 38 or 39, wherein the cell is engineered using CRISPR / Cas9, zinc finger nuclease, TALE nuclease, or meganuclease. 41. The cell of any one of embodiments 32 to 40, wherein the cell is housed in a cell storage bank. 42. A method of preparing cells for cell therapy comprising the step of transfecting immune effector cells with the polynucleotide of any one of embodiments 1-27. 43. The method according to embodiment 42 further comprises the step of storing the cells in a cell storage bank. 44. The method of embodiment 42 or 43, further comprising the step of modifying the cell to express one or more chimeric antigen receptors and / or one or more recombinant T cell receptors. 45. The method of any one of embodiments 42-44, further comprising the step of providing an effective amount of the cells to an individual in need thereof. 46. The method of embodiment 45, wherein the cells are allogeneic with respect to the individual. 47. A method for treating a medical condition in an individual with allogeneic therapeutic cells, comprising the step of providing to the individual a therapeutically effective amount of allogeneic cells, wherein the cells express a polypeptide comprising: (1) one or more of an OX40-specific ligand, a 4-1BB-specific ligand, a CD40L-specific ligand, or a functional derivative thereof; operably linked to (2) a signaling domain that promotes T cell activation. 48. A method according to embodiment 47, wherein the cells are obtained from a cell storage bank. 49. The method of embodiment 47 or 48, wherein the cell expresses one or more chimeric antigen receptors and / or one or more recombinant T cell receptors. 50. A method of preventing rejection of allogeneic cells, tissues or organs in an individual comprising the steps of: delivering to the individual an effective amount of allogeneic immune cells expressing an engineered chimeric receptor comprising an extracellular domain that targets a compound selectively present on activated T cells and comprising CD3ζ, wherein the delivering step produces the following results in the individual: (1) suppressing endogenous alloreactive T cells in the individual; and / or (2) inhibiting NK cell activation in the individual. 51. The method of embodiment 50, wherein the allogeneic cell is an allogeneic immune cell expressing the chimeric receptor. 52. The method of embodiment 50 or 51, wherein the allogeneic cells express a chimeric antigen receptor or an engineered T cell receptor. 53. A method according to embodiment 50, wherein the allogeneic immune cells are delivered to the individual before, during and / or after tissue and / or organ transplantation in the individual. 54. The method of any one of embodiments 50-53, wherein the activated T cells are pathogenic T cells. 55. A method for selectively targeting activated T cells in an individual, comprising the step of providing to the individual an effective amount of immune cells expressing an engineered chimeric receptor comprising: (1) targeting the extracellular domain of a compound that is selectively present on activated T cells; and (2) Signal transduction domain that promotes T cell activation. 56. A method according to embodiment 55, wherein the signaling domain that promotes T cell activation is derived from the CD3ζ subunit, DAP12, Fc receptor or a combination thereof. 57. A method according to embodiment 55 or 56, wherein the activated T cells are pathogenic T cells. 58. A method of preventing or treating a medical condition associated with activated T cells in an individual, comprising the step of delivering to the individual an effective amount of immune cells expressing an engineered chimeric receptor that selectively targets the activated T cells, the chimeric receptor comprising: (1) targeting the extracellular domain of a compound that is selectively present on activated T cells; and (2) Signal transduction domain that promotes T cell activation. 59. A method according to embodiment 58, wherein the chimeric receptor further comprises 1, 2 or more co-stimulatory domains. 60. The method of embodiment 58 or 59, wherein the medical condition is an autoimmune disease. 61. The method of any one of embodiments 58-60, wherein the medical condition comprises transplant rejection, graft-versus-host disease, type I diabetes, multiple sclerosis, autoimmune colitis, or a combination thereof. 62. A method for avoiding NK cell-mediated host rejection of allogeneic T cells, tissues or organs in an individual, comprising the step of providing to the individual an effective amount of immune cells expressing an engineered chimeric receptor, wherein the engineered chimeric receptor comprises an extracellular domain and further comprises a signaling domain that promotes T cell activation, wherein the extracellular domain targets a compound that is selectively present on activated T cells. 63. A method according to embodiment 62, wherein the immune cell expressing the engineered chimeric receptor is an allogeneic T cell. 64. A method according to embodiment 62 or 63, wherein the immune cell expresses a chimeric antigen receptor or an engineered T cell receptor. 65. The method of any one of embodiments 62-64, wherein the amount of immune cells expressing the engineered chimeric receptor provided to the individual is between 10 2 -10 12 within the range of . 66. The method of any one of embodiments 47-65, wherein cells expressing the chimeric receptor are provided to the individual systemically or locally. 67. The method of any one of embodiments 47-66, wherein the immune cell is a T cell. 68. The method of any one of embodiments 47-67, wherein the immune cells are delivered to the individual once or more than once.

Claims

1. A recombinant polynucleotide comprising a sequence encoding a polypeptide, wherein the polypeptide comprises: (1) an extracellular domain comprising: (i) a 4-1BB-specific ligand, and (ii) an OX40-specific ligand, a CD40L-specific ligand, a CD69-specific ligand, a CD25-specific ligand, or a CD71-specific ligand; the extracellular domain being operatively linked to (2) signaling domains that promote T cell activation; The 4-1BB-specific ligand comprises 4-1BBL, an antibody or fragment thereof targeting 4-1BB, a 4-1BBL-Fc fusion, or a combination thereof.

2. The polynucleotide of claim 1, wherein the polypeptide further comprises one, two or more costimulatory domains.

3. The polynucleotide according to claim 1, wherein the polynucleotide further comprises a sequence encoding a spacer located between (1) and (2). The polynucleotide of claim 3 , wherein the spacer is 10 to 220 amino acids in length.

5. The polynucleotide of claim 4, wherein the spacer has a sequence that facilitates surface detection using an antibody. The polynucleotide of claim 5 , wherein the spacer is detectable with an anti-Fc antibody.

7. The polynucleotide of claim 6, wherein the spacer comprises an IgG Fc portion.

8. The polynucleotide of claim 1, wherein the polynucleotide further encodes a chimeric antigen receptor, a T cell receptor, or both.

9. The polynucleotide of claim 8, wherein a 2A element or an IRES element is present on the polynucleotide encoding the polypeptide of claim 1 and the polynucleotide encoding the chimeric antigen receptor, T cell receptor, or both.

10. The polynucleotide of claim 9, wherein the chimeric antigen receptor comprises one, two or more costimulatory domains.

11. The polynucleotide of claim 1, wherein the polynucleotide is present on a vector.

12. The polynucleotide of claim 11, wherein the vector is a viral vector or a non-viral vector.

13. The polynucleotide of claim 12, wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

14. The polynucleotide of claim 1, wherein the polynucleotide is present in a cell.

15. The polynucleotide of claim 14, wherein the cell is a eukaryotic cell or a bacterial cell.

16. The polynucleotide of claim 14, wherein the cell is an immune cell.

17. The polynucleotide of claim 14, wherein the cell is engineered.

18. The polynucleotide of claim 16, wherein the immune cell is a T cell.

19. The polynucleotide of claim 18, wherein the T cell comprises one or more chimeric antigen receptors.

20. The polynucleotide of claim 19, wherein the chimeric antigen receptor comprises one, two or more costimulatory domains.

21. The polynucleotide of claim 18, wherein the T cell comprises one or more engineered T cell receptors (TCRs).

22. A polypeptide encoded by the polynucleotide according to any one of claims 1 to 21.

23. A polypeptide comprising: (1) an extracellular domain comprising: (i) a 4-1BB-specific ligand, and (ii) an OX40-specific ligand, a CD40L-specific ligand, a CD69-specific ligand, a CD25-specific ligand, or a CD71-specific ligand; the extracellular domain being operatively linked to (2) signaling domains that promote T cell activation; The 4-1BB-specific ligand comprises 4-1BBL, an antibody or fragment thereof targeting 4-1BB, a 4-1BBL-Fc fusion, or a combination thereof.

24. The polypeptide of claim 23, wherein the signaling domain that promotes T cell activation is from a CD3 zeta subunit, DAP12, an Fc receptor, or a combination thereof.

25. The polypeptide of claim 23, wherein the polypeptide further comprises one, two or more costimulatory domains.

26. A cell comprising the polynucleotide of any one of claims 1-21 or the polypeptide of any one of claims 23-25.

27. The cell of claim 26, wherein the cell is an immune cell.

28. The cell of claim 26, wherein the cell is engineered.

29. The cell of claim 27, wherein the immune cell is a T cell.

30. The cell of claim 29, wherein the T cell is a CAR-transduced T cell.

31. The cell of claim 29, wherein the T cell is a T cell receptor (TCR)-transduced T cell.

32. The cell of claim 26, wherein the cell is engineered to lack endogenous expression of one or more genes.

33. The cell of claim 32, wherein the cell is engineered to lack endogenous expression of 4-1BB, OX40 and / or CD40L.

34. The cell of claim 32, wherein the cell is engineered using CRISPR / Cas9, zinc finger nucleases, TALE nucleases, or meganucleases.

35. The cell of claim 26, wherein the cell targets activated T cells having activation markers comprising 4-1BB, OX40, CD40L, CD69, CD25, or CD71.

36. A method for preparing cells for cell therapy, comprising the step of transfecting immune effector cells with the polynucleotide according to any one of claims 1 to 21.

37. The method of claim 36, further comprising the step of depositing the cells in a cell bank.

38. The method of claim 36, further comprising the step of modifying the cells to express one or more chimeric antigen receptors and / or one or more recombinant T cell receptors.

39. Use of an engineered allogeneic cell in the preparation of a medicament for treating rejection of an allogeneic cell, tissue, or organ in an individual, wherein the engineered allogeneic cell expresses a polypeptide comprising: (1) an extracellular domain comprising: (i) a 4-1BB-specific ligand, and (ii) an OX40-specific ligand, a CD40L-specific ligand, a CD69-specific ligand, a CD25-specific ligand, or a CD71-specific ligand; the extracellular domain being operably linked to (2) a signaling domain that promotes T cell activation; The 4-1BB-specific ligand comprises 4-1BBL, an antibody or fragment thereof targeting 4-1BB, a 4-1BBL-Fc fusion, or a combination thereof.

40. The use according to claim 39, wherein the engineered allogeneic cells are obtained from a cell bank.

41. The use of claim 39, wherein the engineered allogeneic cells express one or more chimeric antigen receptors and / or one or more recombinant T cell receptors.

42. Use of an allogeneic immune cell in the preparation of a medicament for preventing or treating rejection of an allogeneic cell, tissue, or organ in an individual, wherein the allogeneic immune cell expresses an engineered chimeric receptor comprising: (1) an extracellular domain that targets a compound that is selectively present on activated T cells, the extracellular domain being operatively linked to (2) a CD3ζ signaling domain, wherein the extracellular domain comprises: (i) a 4-1BB-specific ligand, and (ii) an OX40-specific ligand, a CD40L-specific ligand, a CD69-specific ligand, a CD25-specific ligand, or a CD71-specific ligand, The 4-1BB-specific ligand comprises 4-1BBL, an antibody or fragment thereof targeting 4-1BB, a 4-1BBL-Fc fusion, or a combination thereof.

43. The use according to claim 42, wherein the allogeneic cell is an allogeneic immune cell expressing the chimeric receptor.

44. The use of claim 42, wherein the allogeneic cells express a chimeric antigen receptor or an engineered T cell receptor.

45. The use of claim 42, wherein the allogeneic immune cells are delivered to the individual before, during and / or after tissue and / or organ transplantation in the individual.

46. The use according to claim 42, wherein the activated T cells are pathogenic T cells.

47. Use of an immune cell expressing an engineered chimeric receptor in the preparation of a medicament for treating rejection of allogeneic cells, tissues or organs in an individual, wherein the immune cell is effective to selectively target activated T cells in the individual, and wherein the chimeric receptor comprises: (1) targeting an extracellular domain of a compound selectively present on activated T cells; said extracellular domain being operatively linked to (2) Signaling domains that promote T cell activation, wherein the extracellular domain comprises: (i) a 4-1BB-specific ligand, and (ii) an OX40-specific ligand, a CD40L-specific ligand, a CD69-specific ligand, a CD25-specific ligand, or a CD71-specific ligand, The 4-1BB-specific ligand comprises 4-1BBL, an antibody or fragment thereof targeting 4-1BB, a 4-1BBL-Fc fusion, or a combination thereof.

48. The use according to claim 47, wherein the signaling domain that promotes T cell activation is derived from a CD3 zeta subunit, DAP12, an Fc receptor, or a combination thereof.

49. The use according to claim 47, wherein the activated T cells are pathogenic T cells.

50. The use of claim 47, wherein the chimeric receptor further comprises one, two or more costimulatory domains.

51. The use of claim 47, wherein the subject's rejection of allogeneic cells, tissues, or organs comprises transplant rejection, graft-versus-host disease, or a combination thereof.

52. Use of an immune cell expressing an engineered chimeric receptor in the preparation of a medicament for preventing or treating NK cell-mediated host rejection in an individual, wherein the immune cell comprises: (1) an extracellular domain that targets a compound that is selectively present on activated T cells, the extracellular domain being operatively linked to (2) a signaling domain that promotes T cell activation, wherein the extracellular domain comprises: (i) a 4-1BB-specific ligand, and (ii) an OX40-specific ligand, a CD40L-specific ligand, a CD69-specific ligand, a CD25-specific ligand, or a CD71-specific ligand, The 4-1BB-specific ligand comprises 4-1BBL, an antibody or fragment thereof targeting 4-1BB, a 4-1BBL-Fc fusion, or a combination thereof.

53. The use of claim 52, wherein the immune cell expressing the engineered chimeric receptor is an allogeneic cell.

54. The use of claim 52, wherein the immune cell expresses a chimeric antigen receptor or an engineered T cell receptor.

55. The use according to claim 52, wherein the amount of immune cells expressing the engineered chimeric receptor provided to the individual is between 10 2 -10 12 within the range of .

56. The use of any one of claims 39-55, wherein the cells are formulated for systemic or local delivery.

57. The use according to any one of claims 39-55, wherein the immune cell is a T cell.

58. The use of any one of claims 39-55, wherein the immune cells are delivered to the individual in one or more than one dose.

59. A recombinant polynucleotide comprising a sequence encoding a polypeptide, wherein the polypeptide comprises: (1) an extracellular domain comprising: a CD69-specific ligand, a CD25-specific ligand, or a CD71-specific ligand, wherein the extracellular domain is operatively linked to (2) Signal transduction domain that promotes T cell activation.

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