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

Abstract

Embodiments disclosed herein relate to engineered T cells expressing autologous / allogeneic immune defense receptors (ADRs), which selectively target activated T cells including pathogenic T cells to disable them. Chimeric receptors include, for example, targeting 4-1BB, OX40 and CD40L parts, which express activated T cells. In a specific embodiment, there is a method for preventing or treating a disease associated with activated T cells using cells encoding ADRs for adoptive T cell transfer.

Description

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 662,817, filed on April 26, 2018, the entire contents of which are incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support from the National Institutes of Health and the National Cancer Institute under Grant P50 CA126752. The government has certain rights in this invention. Technical Field

[0004] Embodiments of the present disclosure include at least the fields of immunology, cell biology, molecular biology, and medicine. Background Art

[0005] In patients receiving transplants or third-party sourced therapeutic cells, unwanted activation of T cells and NK cells often 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 usually treated with immunosuppressive drugs, which make them immunodeficient and therefore susceptible to infection and malignant transformation.

[0006] 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 the use of 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

[0007] 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 reactions and limiting the therapeutic effect.

[0008] 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 medical conditions associated with their presence. In specific embodiments, the compositions and methods utilize adoptive T cells to transfer cells that express receptors that selectively target pathogenic T cells and leave resting T cells. In specific embodiments, the adoptive T cells used 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 specific embodiments, the present disclosure relates to autologous / allogeneic immune defense receptors (ADRs) for selectively targeting pathogenic T cells.

[0009] Specific embodiments of the present disclosure include methods of 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 of, for example, avoiding allogeneic immune responses in individuals receiving tissue or organ transplants.

[0010] In certain embodiments, the cells covered 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 (including T cells, NKT cells, etc.) for adoptive cell therapy are suitable for "standing" use, which refers to cells stored in a repository or a bank in this article, 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 reservoir and then modified to express ADR. The cells stored in the reservoir may also express or may not express CAR or recombinant TCR, or the cells obtained from the reservoir may be subsequently modified to express CAR or recombinant TCR. Such practice makes it easy to use therapeutic cells from third-party sources without being rejected by host immunity, and it is not necessary to prepare patient-specific products every time it is needed.

[0011] In a particular embodiment, there is an isolated polynucleotide comprising a sequence encoding: (1) one or more of an OX40-specific ligand, a 4-1BB-specific ligand, a CD40L-specific ligand, or a functional derivative thereof; which is operably 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) between (1) and (2). The spacer may have a sequence that facilitates surface detection with an antibody, e.g., the spacer may be detectable by an anti-Fc antibody. The spacer may comprise an IgG Fc portion.

[0012] In a specific embodiment, the polynucleotides of the present invention may further encode chimeric antigen receptors, T cell receptors or both. The polynucleotides may 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 certain cases, the polynucleotides are present in cells, including eukaryotic cells or bacterial cells. The cell may be an immune cell, such as a T cell. The cell may be engineered. The cell may include one or more chimeric antigen receptors (CAR) and / or one or more engineered T cell receptors (TCR).

[0013] The polypeptides expressed by any polynucleotides covered by the present disclosure are included as part of the present disclosure. In a specific embodiment, there is a polypeptide comprising: (1) one or more of OX40-specific ligands, 4-1BB-specific ligands, and CD40; which is operably 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.

[0014] Any cell covered by the present disclosure is part of the present disclosure. In a specific embodiment, any cell expressing a chimeric receptor is part of the present disclosure, including cells, which include any polynucleotides considered herein and / or any polypeptides considered herein. The cell can be an engineered cell. The cell can be an immune cell, such as a T cell, including a T cell transduced by CAR and / or a T cell receptor (TCR) transduced T cell. In a specific embodiment, the cell is engineered to lack endogenous expression of one or more genes, such as one or more of 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 surface expression of ADR ligands.

[0015] In one embodiment, there is a method for avoiding rejection of allogeneic cells, tissues or organs in an individual, comprising the step of delivering an effective amount of allogeneic immune cells expressing an engineered chimeric receptor to the individual, the engineered chimeric receptor comprising an extracellular domain and comprising CD3ζ, the extracellular domain targeting a compound selectively present on activated T cells, wherein the delivering step results in the individual: (1) inhibiting endogenous alloreactive T cells in the individual; and / or (2) suppressing NK cell activation in the individual. In a specific embodiment, the allogeneic cell is an allogeneic immune cell expressing the chimeric receptor. The allogeneic cell may express a chimeric antigen receptor and / or an engineered T cell receptor. The allogeneic immune cell may be delivered to the individual before, during and / or after tissue and / or organ transplantation in the individual. In a specific case, the activated T cell is a pathogenic T cell.

[0016] 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, wherein the chimeric receptor comprises: (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.

[0017] 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.

[0018] 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.

[0019] The features and technical advantages of the present invention have been outlined quite extensively above so that the following detailed description of the invention may be better understood. Additional features and advantages of the present invention that constitute the subject matter of the claims of the present invention will be described hereinafter. It should be understood by those skilled in the art that the disclosed concepts and specific embodiments can be easily used as the basis for modifying or designing other structures for achieving the same purpose of the present design. It should also be recognized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the present invention as set forth in the appended claims. When considered in conjunction with the accompanying drawings, the novel features that are considered to be features of the designs disclosed herein, with respect to their organization and methods of operation, and further objects and advantages will be better understood from the following description. However, it should be clearly understood that each of the accompanying drawings is provided for the purpose of illustration and description only and is not intended to be a definition of limitations of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] For a more complete understanding of the present disclosure, reference is now made to the following description in conjunction with the accompanying drawings, in which:

[0021] 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 ADR and its ligand on T cells is not required for the expansion or function of ADR-T cells.

[0022] Figure 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 are not cytotoxic to resting CD4+ and CD8+ T cells. Figure 2E )After 48 h of co-culture, ADR T cells eliminated activated CD4+ and CD8+ T cells.

[0023] 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 PBMCs at a ratio of 1:10 ADR T:PBMC. ( Figure 3B-Figure 3C ) The absolute counts of donor T cells and allogeneic T cells in PBMC during co-culture were Figure 3D-Figure 3F ) Virus-specific ADR T cells are identical.

[0024] 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 are protected from immune rejection by recipient allogeneic PBMCs. ( Figure 4B-Figure 4C ) Absolute counts of recipient T cells and donor VSTs at various time points during the MLR.

[0025] Figure 5ADR VSTs retain antiviral function. ADR VSTs were co-cultured with viral pepmix-pulsed monocytes, and monocyte counts showed that they cleared virus-infected cells equally well compared to unmodified VSTs.

[0026] 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 h of co-culture with 4-1BB ADR T cells. ( Fig.6D ) By controlling the expansion of NK cells, MHC-deficient ADR T cells are protected from immune rejection by allogeneic PBMCs. Fig. 6E ) Absolute counts of donor T cells and allogeneic NK cells during co-culture. Fig. 6F ) After 48 hours of co-culture at a 1:1 E:T ratio, ADR T cells lacking MHC 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.

[0027] Figures 7A-7E In vivo ADR expression protects allogeneic T cells from immune rejection. Fig. 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 and then allogeneic HLA-A2- T cells are administered 4 days later. ( Figure 7B-7C ) Control T cells from HLA-A2- donors were rejected at 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. ( Fig.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. Fig. 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.

[0028] Figures 8A-8E . Co-expression of CAR and ADR preserves the functionality of both receptors. ( Fig. 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 cells cytotoxicity against NALM-6 (CD19+CAR target). Fig.8D ) Cytotoxicity of CAR-ADR T cells against activated T cells (ADR targets). Fig. 8E ) After co-culture with the two cell targets, the cytotoxic activity of CAR-ADR T cells against the two targets.

[0029] Figures 9A-9E CAR-ADR T cells were protected from immune rejection and exerted potent anti-tumor activity. Fig. 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. ( Fig. 9B ) Kinetics of T cells from donor 2 in peripheral blood. Fig. 9C ) Kinetics of donor 1 T cells in the experimental groups. ( Fig.9D ) Leukemia burden in mice. Fig.9E ) Overall survival rate of mice.

[0030] Figures 10A-10C CAR-ADR T cells were protected from immune rejection and exerted potent antitumor activity in solid tumor models. ( Fig. 10A ) Schematic diagram of the mouse model. Mice received allogeneic T cells from donor 1 and b2mKO neuroblastoma cell line CHLA255 24 hours apart, followed by a single dose of CAR-ADR T cells from donor 2. ( Fig. 10B ) Donor 2GD2 CAR T cells were rejected on day 18, whereas CAR-ADR T cells resisted allogeneic rejection and persisted in peripheral blood. ( Fig. 10C ) Tumor burden in mice, * indicates xeno-GvHD-related death in the ATC+GD2 CAR T group.

[0031] Figures 11A-11E . TCR-knockout CAR-ADR T cells were protected from immune rejection and exerted effective anti-tumor activity. ( Fig.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. ( Fig. 11B ) Kinetics of T cells from donor 2 in peripheral blood. Fig. 11C ) Kinetics of donor 1 T cells in the experimental groups. ( Fig.11D ) Leukemia burden in mice. Fig.11E ) Overall survival rate of mice.

[0032] Figures 12A-12D . ADR T cells protect mice from lethal xenogeneic GvHD. ( Fig. 12A )Model schematic. Fig. 12B ) In vivo expansion of FFLuc-labeled ADR T cells. Fig. 12C ) Kinetics of weight gain / loss in mice. Fig.12D ) Overall survival rate of mice.

[0033] Figures 13A-13G . A second generation ADR with a CD28 intracellular signaling domain (ADR.28ζ). Fig.13A )The structure of ADR.28ζ. Figure 13B-Figure 13C ) In vitro cytotoxicity of ADR.28ζ against cell lines expressing the target. Figure 13D-13G )ADR.28ζ protects mice from xenogeneic GvHD. Fig.13D )Schematic diagram of the model. Fig.13E ) In vivo expansion of FFLuc-labeled ADR.28ζ T cells. Fig.13F ) Kinetics of weight gain / loss in mice. Figure 13G ) Overall survival rate of mice.

[0034] Fig.14 . Cytotoxicity of cells expressing ADRs in cancer. (Left) Cytotoxicity of T cells expressing 4-1BB ADR against HDLM-2 Hodgkin lymphoma cells, (Right) Cytotoxicity of T cells expressing 4-1BB ADR against K562 chronic myeloid leukemia (CML) cells. Absolute counts of tumor cells after 48 hours of co-culture at a 1:1 effector to target ratio are shown. DETAILED DESCRIPTION

[0035] As used herein, the words "a" and "an" when used with the word "comprising" in this specification (including in 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.

[0036] Throughout the specification, unless the context requires otherwise, the words "comprise", "comprising" and "containing" will be understood to imply the inclusion of the 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 anything in the phrase "consisting of". Thus, the phrase "consisting of" means that the listed elements are required or mandatory, and 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 effect specified for the listed elements in the present disclosure. Thus, the phrase "consisting essentially of" means that the listed elements are required or mandatory, but other elements are optional, and the presence or absence of other elements depends on whether they affect the activity or effect of the listed elements.

[0037] Throughout the specification, reference to "one embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "another embodiment," or "another embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the aforementioned phrases in various places throughout the 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.

[0038] As used herein, the term "subject" generally refers to an individual who needs to be treated for any kind of medical condition. The subject can be any kind of animal. The subject can be any biological or animal subject as 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, such as 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, thus including adults and juveniles (i.e., children) and infants, and including intrauterine individuals. The individuals may be of any race and gender. The term is not intended to imply a need for medical treatment, and thus, individuals may voluntarily or involuntarily become part of an experiment, whether clinical or in support of basic scientific research.

[0039] As used herein, the term "engineered" refers to a molecule that does not exist in nature and has been produced by man (eg, by genetic recombination techniques standard in the art).

[0040] In the context of the present disclosure, "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.

[0041] I. Autologous / allogeneic immune defense receptors and compositions and their uses

[0042] 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.

[0043] In certain embodiments, autologous / allogeneic immune defense receptor (ADR) includes the entity of the compound that targets one or more up-regulated 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 (relative to the individual receiving cells being allogeneic).In other cases, the ADR is expressed on autologous T cells, xenogeneic cells and / or synthetic cells.

[0044] A. Autologous / allogeneic immune defense receptor (ADR) molecules

[0045] ADR molecules are synthetic, non-natural and artificially produced, 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); it is operably linked to (2) a signaling domain that promotes T cell activation, including, for example, those derived from CD3ζ subunits, DAP12 and Fc receptors, 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 components of one or more type I transmembrane proteins and / or components of one or more type II transmembrane proteins.

[0046] In a specific embodiment, in the ADR molecule, the extracellular domain comprises a protein that selectively binds to an associated protein on activated T cells. For example, the ADR extracellular domain may comprise a ligand for a receptor on activated T cells, or the ADR extracellular domain may comprise a receptor for a ligand on activated T cells.

[0047] In a specific embodiment, in the ADR molecule, the extracellular domain comprises 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 activated T cells are targeted. For example, other activation markers (such as CD69, CD25, CD71, etc.) that are upregulated 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.

[0048] 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, OX40 ligands can be used in ADR to be able to target the activated T cells. In the case of using OX40 ligands in ADR, the OX40 ligand can be any suitable OX40 ligand, including at least OX40L, an antibody (or its functional fragment) bound to OX40, a fusion of Fc and OX40L, or its functional derivative or fragment thereof. OX40L may also be referred to as tumor necrosis factor (ligand) superfamily member 4 (tax transcriptionally activated glycoprotein 1 (tax-transcriptionally activated glycoprotein 1), 34kDa), OX40L, CD252, TNFSF4, TXGP1, OX-40L or gp34.

[0049] 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 any suitable ligand of 4-1BB, including at least 4-1BBL, antibodies (or their functional fragments) targeting 4-1BB, Fc and 4-1BBL fusions or their functional derivatives or fragments.

[0050] In some cases, in contrast to unactivated T cells, activated T cells with upregulated CD40L expression are targeted. In order to target these activated T cells, a receptor for CD40L can be used in the ADR to be able to target the activated T cells. In the case of using a receptor for CD40L in the ADR, the ADR can include 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).

[0051] In some cases, the ADR molecule includes two or more extracellular domains to promote the T cells of targeted activation.Such a combination can generally enhance the T cells of targeted activation or can allow certain subsets of specific targeted activated T cells.For example, the ADR can include OX40L and 4-1BBL as extracellular domains in the same ADR molecule at the same time, to allow the activated T cells of targeted expression OX40 or 4-1BB.The example of this combination will selectively target the activated T cells expressing OX40 or 4-1BB, regardless of whether those activated T cells also express CD40L.Similarly, ADR can include CD40 and OX40L at the same time to target the activated T cells expressing CD40L or OX40, regardless of whether those activated T cells also express 4-1BB.

[0052] 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 ITAM can include those derived from DAP12, Fc receptors, other CD3 subunits, etc. The signal transduction domain can be non-covalently connected to ADR through another domain.

[0053] 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, a spacer is not 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 inhibit. In a specific embodiment, the spacer includes a coded protein sequence that helps to detect cells expressing the ADR. For example, the spacer can encode an Fc region or a fragment thereof, which will allow surface detection of cells, such as 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.

[0054] 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 only as an example, when ADR includes 4-1BBL, the costimulatory domain of ADR may or may not be from 4-1BB.

[0055] In some embodiments, ADR will include a transmembrane domain, which can be any kind, as long as it allows the CD3ζ component of ADR to be located intracellularly and the extracellular domain of the compound that targets one or more up-regulated on activated T cells is 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, CD8α, CD27, CD28, 4-1BB, OX40, CD4, etc.

[0056] In the example of ADR polypeptides, the components may be in a specific N-terminal (N) to C-terminal (C) order. For general ADR, the receptor may include one of the following (only as an example), and wherein the extracellular domain includes a protein that selectively binds to a related protein on activated T cells:

[0057] N-ectodomain-signaling domain-C

[0058] N-ectodomain-CD3ζ-C

[0059] N-ectodomain-spacer-CD3ζ-C

[0060] N-extracellular domain-spacer-costimulatory domain-CD3ζ-C

[0061] N-extracellular domain-spacer-two co-stimulatory domains-CD3ζ-C

[0062] N-two extracellular domains-spacer-costimulatory domain-CD3ζ-C

[0063] N-two extracellular domains-spacer-two co-stimulatory domains-CD3ζ-C

[0064] In any case, the transmembrane domain may be located at the C-terminus relative to the spacer. A signal peptide at the N-terminus may 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).

[0065] 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, etc.

[0066] 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.

[0067] 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 (e.g., T cells, NK cells, or NKT cells). Such appropriate elements include promoters, etc.

[0068] 4-1BB ADR (SEQ ID NO: 1)

[0069] MEFGLSWLFLVAILKGVQCGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVAKAGVYYVFFQLELRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVT PEIPAGLPSPRSEESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPAYQ QGQNQLYNENLGRREEYDVLDKRRGRDRPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGGKGHDGLYQGLSTATKDTYDALHMQALPPRTSAAAGGGSGGGSGGGSGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0070] OX40 ADR(SEQ ID NO:2)

[0071] MEFGLSWLFLVAILKGVQCQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLESKYGPPCPPPCPGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGGKGHDGLYQGLSTATKDTYDALHMQALPPRTSAAAGGGSGGGSGGGSGMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0072] CD40L ADR(SEQ ID NO:3)

[0073] MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSAAAGGGGSGGGGSGGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0074] In certain embodiments, the extracellular domain of the targeted activated T cell 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 source or a recombinant source, and can be an immunoreactive portion of a complete immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. The antibody in the present invention can exist in a variety of 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).

[0075] 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 by antibody fragments.

[0076] 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).

[0077] B. Cells expressing ADR

[0078] 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 harmful reactions of the recipient.

[0079] The present disclosure includes cells modified to express synthetic ADR molecules and improved for allogeneic use. Therefore, the present disclosure includes cells having ADR (as polynucleotides and as expressed ADR polypeptides) on the cell surface. In certain cases, cells expressing ADR are produced for storage in a repository for regular use. Cells can be contained in a repository that has been configured to express ADR, or they can be contained in a repository and configured to express ADR after being taken out from the repository. Certain cells, such as bacterial cells, can be used to produce ADR molecules, while other cells with ADR, such as eukaryotic cells, can be used in the methods of the present invention, including targeted activated T cells. As shown herein, immune cells expressing ADR selectively remove activated T cells, and immune cells expressing ADR are protected from cell lysis by alloreactive T cells.

[0080] The cells expressing ADR molecules can be of any kind, but in a specific embodiment, 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 colony of non-natural cells expressing ADR is considered, including at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the colony is a colony of cells expressing ADR. For example, the cell can be produced by the standard method of transfecting or transducing the synthesized ADR polynucleotide.

[0081] 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 in the 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 cuttable elements, such as 2A, between them.

[0082] In the case where the cell expressing ADR also expresses CAR or TCR, CAR or TCR can target any specific antigen. In the case of using CAR, CAR can be first generation, second generation, third generation, etc. In certain cases, CAR can be bispecific.

[0083] 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. Only as an example, cells can be engineered by CRISPR / Cas9.

[0084] II. Methods Using Autologous / Allogeneic Immune Defense Receptors

[0085] Embodiments of the present disclosure include methods for providing an effective amount of cells expressing ADR to an individual for any purpose. The method includes providing selectively targeting activated T cells in an individual for any purpose. The activated T cells are targeted by exposing the activated T cells to an effective amount of immune cells expressing ADR (e.g., T cells expressing ADR). This exposure can have, for example, one or more applications resulting therefrom.

[0086] 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 activation of innate immunity (e.g., macrophage activation syndrome, etc.). In other embodiments, ADR is used to specifically target malignant cells expressing their corresponding targets, including, for example, 4-1BB or OX40 or CD40L.

[0087] The scheme of providing an effective amount of cells expressing ADR to an individual can be known or determined by the individual who delivers the cells for treatment or prevention or regardless of its method of use. For example, in the case of prevention, the cells can be delivered before detecting one or more symptoms, or the cells can be delivered after detecting one or more symptoms but before further symptom development and / or deterioration. For treatment situations, an effective amount of cells can be provided to an individual after one, two or more symptom development and after clinical diagnosis.

[0088] In certain aspects of the method, the individual can be given a single dose of a therapeutically effective amount of cells, or the individual can be given 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 in a single regimen.

[0089] 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, bucally, transdermally, subcutaneously, etc. Cells may or may not be delivered as a bolus. In multiple administrations, the cells may or may not be provided to the individual by 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.

[0090] A. For standing implementation plans

[0091] The present disclosure includes cells for adoptive transfer that can be readily available, including being able to 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 stored in a repository, or they may be modified to express ADRs afterwards. 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 stored in a repository or after obtaining them from a repository.

[0092] 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 in vivo anti-cancer activity of "standing" T cells co-expressing CAR and ADR in a mouse model, while at the same time maintaining resistance to immune rejection from allogeneic T cells present in the same mouse. For example, in Fig.14 It was shown in the literature that 4-1BB ADR was effective against 4-1BB+ tumor cells, suggesting that ADR could be used as a therapeutic modality against malignancies expressing 4-1BB.

[0093] In certain embodiments, the "standing" therapeutic cells express ADRs to resist immune rejection and either retain endogenous TCR specificity (e.g., to a virus or tumor antigen) 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.

[0094] In a specific embodiment, the 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 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).

[0095] 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.

[0096] Although the cells contained in the repository can be allogeneic to the recipient individual, in alternative embodiments, the cells contained in the repository are autologous to the recipient individual. For example, an individual with cancer can store T cells expressing ADR in a repository for subsequent use, such as in the case of cancer remission. In other cases, autologous cells expressing ADR are contained in the repository for the treatment of autoimmune disorders.

[0097] B. For autoimmune diseases

[0098] 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, which affect the autoimmune disorder (or its potential development) by suppressing endogenous autoreactive T cells in the individual. 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.

[0099] In vivo activation of T cells with unwanted specificity may cause pathogenicity, and in specific 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.

[0100] In certain cases, T cells expressing ADRs 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 T cells expressing ADRs.

[0101] 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.

[0102] In some cases, one or more additional therapies are given to the individual of the T cell provided with expression of ADR to prevent or treat one or more autoimmune disorders. The individual may or may not be given one or more immunosuppressive drugs, such as glucocorticoids, cytostatic agents, antibodies and / or drugs acting on immunoaffinity proteins. Additionally or alternatively, one or more suitable vaccines may be given to the individual.

[0103] In some cases, an individual is at risk of an autoimmune disorder and is provided with an effective amount of cells expressing an ADR to prevent the onset of the autoimmune disorder or delay the onset and / or alleviate one or more symptoms, including, for example, severity and / or duration. An individual at risk of an autoimmune disorder is, for example, an individual with a personal or family history of the disorder, a woman of a certain ethnicity, etc. In some cases, an individual may suffer from an autoimmune disorder and wish to prevent or reduce its severity and / or duration or delay the onset of another autoimmune disorder.

[0104] 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 disease; Chagas disease (Chagas disease); disease); chronic inflammatory demyelinating polyneuropathy (CIDP); chronic relapsing multifocal osteomyelitis (CRMO); Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (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 Romberg syndrome; pars planitis (peripheral uveitis); Parsenager-Turner syndrome; pemphigus; peripheral neuropathy; peripheral encephalomyelitis; pernicious anemia (PA); POEMS syndrome; polyarteritis nodosa; polyglandular syndromes type 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-man 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; ulcerative colitis (UC); undifferentiated connective tissue disease (UCTD); uveitis; vasculitis; vitiligo; Vogt-Koyanagi-Harada disease; and Wegener's granulomatosis (or granulomatosis with polyangiitis (GPA)).

[0105] C. Used to eliminate NK cells

[0106] Immune cells expressing ADRs can be used to eliminate NK cells if necessary. As demonstrated herein, the presence of ADRs on certain immune cells provides specific cytotoxic activity against NK cells that are involved in mediating the expression of HLA low Therefore, in the need to maintain HLA low In the case of cells that are not compatible with or HLA-incompatible (e.g., adoptive transfer of allogeneic cells to certain individuals is possible), the use of cells expressing ADR avoids the activation of NK cells and the rejection of HLA-incompatible cells. Specifically, as shown herein, co-culture of T cells expressing ADR leads to the elimination of NK cells and thus counteracts NK cell-mediated host rejection of allogeneic T cells expressing ADR.

[0107] D. To promote the engraftment of allogeneic cells / tissues / organs

[0108] In particular aspects of avoiding activation of alloreactive T cells, rejection of allogeneic cells, tissues or organs in individuals receiving transplants (which is primarily mediated by alloreactive T cell populations from the recipient) can be avoided. For example, if measures are not taken to avoid such rejection, activation of alloreactive T cells in the recipient can lead to transplant failure. Therefore, in specific embodiments, methods of transplanting cells, tissues or organs into individuals utilize cells expressing ADRs delivered before, during and / or after each transplant of cells, tissues or organs. In some cases, the cells expressing ADRs themselves are not part of the cells, tissues or organs of the transplanted subject, while in other cases, the cells expressing ADRs are part of each cell, tissue or organ.

[0109] 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.

[0110] In a specific embodiment, cells expressing ADR enhance the use of allogeneic cells in an individual in a two-pronged approach: (1) they suppress endogenous alloreactive T cells in an individual; and (2) they suppress NK cell-mediated rejection in an individual. In this way, the ADR molecules can enhance the persistence and activity of any type of third-party-derived 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.).

[0111] E. For the prevention or treatment of graft-versus-host disease (GvHD) during allogeneic cell / tissue / organ transplantation

[0112] In another specific aspect of avoiding the activation of alloreactive T cells, life-threatening alloreactive immune responses in individuals receiving alloreactive cells, tissues or organs 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 a specific embodiment, the method of transplanting cells, tissues or organs into an individual utilizes cells expressing ADRs delivered before, during and / or after each transplant of cells, tissues or organs. In some cases, the cells expressing ADRs themselves are not part of the cells, tissues or organs of the transplanted subject, while in other cases, the cells expressing ADRs are part of each cell, tissue or organ.

[0113] 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.

[0114] III. Generation of ADR-Expressing Cells

[0115] 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.

[0116] Origin of AT cells

[0117] Prior to amplifying and genetically modifying the ADR-expressing T cells of the present disclosure, a source of T cells may 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 the manipulation thereof may be performed by a party other than the party providing the ADR-expressing T cells to the individual. T cells may 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 may be used. In certain embodiments, any number of techniques known to those skilled in the art may be used, such as Ficoll. 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 generally contains lymphocytes, for example, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and platelets. In one embodiment, cells collected by apheresis can be washed to remove plasma fractions and the cells are placed in appropriate buffer or culture medium for subsequent processing steps. In one embodiment, cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the washing solution lacks calcium, and magnesium may be lacking, or may lack many (if not all) divalent cations. As will be readily understood by those of ordinary skill 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.

[0118] In another embodiment, the erythrocytes are lysed and the monocytes are removed (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.

[0119] Enrichment of T cell populations by negative selection can be achieved by combining antibodies against surface markers specific to 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 +For cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich or positively select for cells that normally 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.

[0120] In order to separate the desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles, such as beads) can vary. In certain embodiments, it may be desirable to significantly reduce the volume (i.e., increase the concentration of cells) that beads and cells are mixed together to ensure the maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In another embodiment, greater than 100 million cells / ml is used. In another embodiment, the cell concentration used is 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 yields, 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.

[0121] The T cells for stimulation can also be frozen after the washing steps. It is hoped that without being bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing the granulocytes and (to some extent) monocytes in the cell population. 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 allowed to stand at room temperature for one hour before using the method of the present invention to activate.

[0122] It is also contemplated in the context of the present disclosure that a blood sample or a single blood component product may be collected from a subject in a time period before the cells that may be expanded as described herein are needed. In this way, the source of cells to be expanded 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 therapy (for any number of diseases or conditions, which will benefit from T cell therapy, 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, samples are collected from patients 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 as, 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 patients. 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, e.g., Rituxan).

[0123] Activation and expansion of BT cells

[0124] Whether before or after the T cells are genetically modified to express ADR, the 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 of a ligand attached with a reagent that stimulates CD3 / TCR complex-related signals and a co-stimulatory molecule on the surface of the stimulatory T cell. Such processes are known in the art. In other cases, T cells can be modified to express ADR without prior activation.

[0125] C. Generation of ADR molecules

[0126] Looking at the polynucleotides encoding ADR 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 directly from cells and tissues containing the gene (using standard techniques). Alternatively, the target ADR polynucleotide can be produced synthetically rather than cloned.

[0127] In brief overview, the expression of synthetic polynucleotides encoding ADRs is generally achieved by operably linking a nucleic acid encoding an ADR polypeptide or portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration of eukaryotic organisms. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating expression of the desired nucleic acid sequence.

[0128] 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.

[0129] 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 viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. Generally, suitable vectors are included in at least one biologically active replication origin, promoter sequence, convenient restriction endonuclease site and one or more selection markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent number 6,326,193).

[0130] 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 in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a 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.

[0131] Other promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, they are located in the region of 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 spacing between promoter elements is usually flexible, so that when elements are reversed or moved relative to each other, promoter function is retained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50bp before the activity begins to decline. Depending on the promoter, it seems that a single element can work collaboratively or independently to activate transcription.

[0132] 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 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 a polynucleotide sequence operably linked to it when such expression is needed, or turn off the expression when expression is not needed. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0133] In order to assess the expression of ADR polypeptide or its part, the expression vector to be introduced into the cell can also include selection marker gene or reporter gene or both, to promote identification and selection of expression cells from the cell colony transfected or infected by viral vector. In other aspects, selection marker can be carried on a separate DNA fragment and used for cotransfection step. Selection marker and reporter gene can be flanked by suitable regulatory sequences, so that expression can be achieved in host cells. Useful selection markers include, for example, antibiotic resistance genes, such as neo etc.

[0134] 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 a recipient organism or tissue and encodes a polypeptide whose expression is manifested by some easily detectable properties (e.g., enzyme 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 may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or a 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 or commercially available using known techniques. 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.

[0135] Methods for introducing and expressing ADR polynucleotides into cells are known in the art. In the case of expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method in the art. For example, the expression vector can be transferred into the host cell by physical, chemical or biological means.

[0136] Physical methods for introducing ADR polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. 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.

[0137] 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 slow viruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0138] 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).

[0139] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is considered that lipid formulations are used to introduce nucleic acids into host cells (in vitro, in vitro or in vivo). On the other hand, nucleic acids can be connected to lipids. Nucleic acids connected to lipids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, attached to liposomes by connecting molecules that are uniformly connected to liposomes and oligonucleotides, wrapped in liposomes, compounded with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles or compounded with micelles, or connected to lipids. The composition related to lipids, lipid / DNA or lipid / expression vectors is not limited to any specific structure in the solution. For example, they can exist with a double-layer structure, exist as micelles or exist with a "collapse" structure. They can also be simply dispersed in a solution, and may form aggregates of uneven size or shape. Lipid is a fatty substance, which can be a 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.

[0140] 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 Avanti Polar 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 monolayer and multilayer lipid vehicles formed by producing closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicle structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. 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 that have 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 contemplated.

[0141] In some cases, the ADR molecule can be integrated into the endogenous nucleic acid of the cell. It is possible to have a target site for homologous recombination, wherein it is desired to integrate the construct at a specific site. For example, materials and methods known in the art for homologous recombination 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 specific sites in the genome to promote homologous recombination.

[0142] Exemplary T cells that have been engineered to include constructs expressing ADRs can be grown in culture under selective conditions, and cells selected to have the constructs can then be expanded and further analyzed, e.g., using polymerase chain reaction for determining the presence of the constructs in host cells. Once the engineered host cells are identified, they can then be used as planned, e.g., expanded in culture or introduced into a host organism.

[0143] 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 particular embodiment, the cells can be introduced into a tumor site, although in alternative embodiments, the cells can home to cancer or be modified to home to infected tissues. The number of cells used will depend on a variety of circumstances, the purpose of the introduction, the life span 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, usually injected at or near the target site. The cells can be in a physiologically acceptable culture medium.

[0144] 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, wherein 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.

[0145] Example

[0146] 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.

[0147] Example 1

[0148] Selective targeting of autologous / allogeneic immune defense receptors on pathogenic T cells

[0149] This article discloses a novel method of 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).

[0150] 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. The expression of 4-1BB, OX40, and / or CD40L is maintained only when the T cells have activated cytotoxicity, and the expression is gradually downregulated within 4-5 days after TCR signaling ceases. Notably, activated CD8 + T cells showed higher 4-1BB expression levels, 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 some other non-critical and 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.

[0151] In order to explore the feasibility of targeting these activated T cells, the autologous / allogeneic immune defense receptor (ADR) is designed to contain 4-1BB- or OX40-specific ligands, or CD40L-specific receptors, which are directly connected to the CD3ζ chain (encoded in the γ-retroviral vector SFG) by a spacer. Combined with the spacer region: a) enable type II proteins 4-1BBL and OX40L to be integrated into the type I skeleton of ADR; b) promote the detection of ADR on the cell surface by FACS staining. Transducing 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 power to cells expressing 4-1BB, OX40 and CD40L, and 90-99% of target cells are eliminated within 48 hours. These results demonstrate the feasibility of generating functional 4-1BB, OX40 and CD40L-specific ADR T cells.

[0152] 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 a CD7-specific CAR. In this case, using CRISPR / Cas9, the inventors were able to knock out 4-1BB expression in ~70% of ADR T cells, with 4-1BB knocking out 4-1BB in ~70% of ADR T cells. + This consistently increased ADR T cell expansion >2-fold 48 h after target cell co-culture without affecting cytotoxicity.

[0153] 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 live CD4 + and CD8 + T cells. After 72 h of co-culture with T cells expressing 4-1BB-, OX40-, or CD40L-specific ADRs, there was 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 + T cells. Incubation with OX40 ADR T cells resulted in activated CD4 + Corresponding high levels of removal and activation of CD8 T cells + Moderate depletion of T cells. CD40L ADRT cells are activated CD4 + T cells produced moderate cytotoxic effects, but CD8 + No effect was observed on T cells. OX40, CD40L and 4-1BB ADR T cells target 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.

[0154] We evaluated whether ADR-expressing virus-specific T cells (VSTs) could protect against allogeneic rejection in an in vitro mixed lymphocyte reaction (MLR) assay. CMV-specific T cells were generated from HLA-A2-negative donors, and control non-transduced or ADR-transduced VSTs were co-cultured with alloreactive HLA-A2-negative VSTs 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. Altogether, these results demonstrate the feasibility and selectivity of targeting activated T cells using a newly developed ADR platform implementation.

[0155] Example 2

[0156] Autologous / allogeneic immune defense receptors for selective targeting of NK cells

[0157] ADR showed specific cytotoxic activity against NK cells, which mediate the expression of HLA low or HLA-incompatible cells.

[0158] As part of anti-tumor and anti-viral immune surveillance, NK cells can recognize HLA-incompatible cells or cells with low HLA expression. Therefore, adoptive transfer of allogeneic cells to 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 removal of NK cells, thereby offsetting the rejection of NK cell-mediated hosts to 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.

[0159] Example 3

[0160] T cells expressing ADR eliminate target cells

[0161] 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 1E We 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 co-expression of the ADR and its ligand on T cells is not required for the expansion or function of ADR-T cells ( Figure 1E ).

[0162] 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 ( Figure 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 ).

[0163] 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 PBMCs at a ratio of 1:10 ADR T:PBMCs ( Figure 3A Absolute counts of donor T cells and allogeneic T cells in PBMCs during co-culture ( Figure 3B-3C ) for virus-specific ADR T cells ( Figure 3D-3F ) are the same.

[0164] In an in vitro mixed lymphocyte reaction, expression of ADR 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 .

[0165] exist Figure 5 In the study, the ADR VSTs retained 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.

[0166] Activated NK cells upregulate ADR ligands and can be selectively targeted by ADR T cells. Expression of 4-1BB on resting and activated NK cells was confirmed ( Figure 6A-6B The 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 Fig.6D In this study, MHC-deficient ADR T cells were protected from immune rejection by allogeneic PBMCs by controlling the expansion of NK cells. Fig. 6E Absolute counts of donor T cells and allogeneic NK cells during coculture were determined in . MHC-deficient ADR T cells resisted immune rejection of NK cells after 48 h of coculture at a 1:1 E:T ratio ( Fig. 6F During MLR with 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.

[0167] ADR expression protects allogeneic T cells from immune rejection in vivo. Fig. 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, and then allogeneic HLA-A2- T cells were administered 4 days later. Control T cells from an HLA-A2- donor were rejected at day 18, while 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 ). Fig.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. Fig. 7E Representative dot plots in show that ADR T cells were protected from immune rejection and also protected mice from the rapid onset of lethal GvHD.

[0168] Co-expression of CAR and ADR preserves the functionality of both receptors. Fig. 8A Representative examples of immune cells co-expressing ADR and CAR are shown. Co-expression of CAR and ADR on the cell surface was confirmed ( Figure 8B ).exist Figure 8C In, the cytotoxicity of CAR-ADR T cells against NALM-6 (CD19+CAR target) as an example of a target is shown. Fig.8D The cytotoxicity of CAR-ADR T cells against activated T cells (ADR targets) was also determined in Fig. 8EFigure 3 shows the cytotoxic activity of CAR-ADR T cells against two cell targets when they were co-cultured with the two targets simultaneously.

[0169] CAR-ADR T cells are protected from immune rejection and exert potent antitumor activity. Examples of mouse models and protocols are available in Fig. 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. Fig. 9B The kinetics of T cells from donor 2 in peripheral blood are provided. Fig. 9C The kinetics of donor 1 T cells in the experimental groups are provided. Fig.9D The leukemic burden in mice was shown and the overall survival of the mice was determined ( Fig.9E ).

[0170] 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 in Fig. 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 ( Fig. 10B The tumor burden of mice is shown in Fig. 13C , where * indicates xeno-GvHD-related death in the ATC+GD2 CAR T group.

[0171] TCR knockout CAR-ADR T cells are protected from immune rejection and exert potent antitumor 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 ( Fig.11A The kinetics of T cells from donor 2 in peripheral blood are provided ( Fig. 11B ). The kinetics of donor 1 T cells in the experimental group are shown ( Fig. 11C ). Leukemia burden in mice is provided ( Fig.11D ) and the overall survival rate of mice ( Fig.11E ).

[0172] ADR T cells protect mice from lethal xenogeneic GvHD. Fig. 12AA schematic diagram of the model is provided in , and the expansion of FFLuc-labeled ADR T cells in vivo is demonstrated ( Fig. 12B The kinetics of weight gain / loss in mice were determined ( Fig. 12C ). The overall survival rate of mice is depicted ( Fig.12D ).

[0173] 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 ( Fig.13A The in vitro cytotoxicity of ADR.28ζ against cells expressing the target was determined ( Fig. 13B and Fig. 13C ADR.28ζ protected mice from xeno-GvHD ( Fig. 13B ). A schematic diagram of the model is shown ( Fig.13D ). The expansion of FFLuc-labeled ADR.28ζT cells in vivo was confirmed ( Fig.13E The kinetics of weight gain / loss in mice were determined ( Fig.13F ), and the overall survival rate of mice ( Figure 13G ).

[0174] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and modifications can be made herein without departing from the spirit and scope of the design defined by the appended claims. Moreover, the scope of the present 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 easily understood by those of ordinary skill in the art from the present disclosure, the processes, machines, manufactures, material compositions, means, methods or steps that currently exist or will be developed later to perform functions substantially the same as the corresponding embodiments described herein or to achieve results substantially the same as the corresponding embodiments described herein can be utilized according to the present disclosure. Thus, the appended claims are intended to include such processes, machines, manufactures, material compositions, means, methods or steps within their scope.

Claims

1. An isolated polynucleotide comprising a sequence encoding a polypeptide, wherein the polypeptide comprises: (1) an extracellular domain, wherein the extracellular domain comprises one or more of an OX40-specific ligand, a 4-1BB-specific ligand, or a CD40L-specific ligand; the extracellular domain is operably linked to (2) Signal transduction domain that promotes T cell activation, wherein the OX40-specific ligand is OX40L, an antibody targeting OX40, an OX40L-Fc fusion or a combination thereof, wherein the 4-1BB-specific ligand is 4-1BBL, an antibody targeting 4-1BB, a 4-1BBL-Fc fusion or a combination thereof, and The CD40L-specific ligand is CD40, an antibody targeting CD40L, a CD40-Fc fusion or a combination thereof.

2. The polynucleotide of claim 1, wherein the polypeptide comprises an OX40-specific ligand.

3. The polynucleotide of claim 1, wherein the polypeptide comprises a 4-1BB-specific ligand.

4. The polynucleotide of claim 1, wherein the polypeptide comprises a CD40L-specific ligand.

5. The polynucleotide of claim 1, wherein the extracellular domain comprises an OX40-specific ligand, and further wherein the OX40-specific ligand is OX40L, an antibody targeting OX40, an OX40L-Fc fusion, or a combination thereof.

6. The polynucleotide of claim 1, wherein the extracellular domain comprises a 4-1BB-specific ligand, and further, 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 claim 1, wherein the extracellular domain comprises a CD40L-specific ligand, and further wherein the CD40L-specific ligand is CD40, an antibody targeting CD40L, a CD40-Fc fusion, or a combination thereof.

8. The polynucleotide of claim 1, wherein the polypeptide further comprises one, two or more co-stimulatory domains.

9. The polynucleotide according to claim 1, wherein the polynucleotide further comprises a sequence encoding a spacer located between (1) and (2).

10. The polynucleotide of claim 9, wherein the spacer has a length of 10 to 220 amino acids.

11. The polynucleotide of claim 10, wherein the spacer has a sequence that facilitates surface detection with an antibody.

12. The polynucleotide of claim 11, wherein the spacer is detectable with an anti-Fc antibody.

13. The polynucleotide of claim 12, wherein the spacer comprises an IgG Fc portion.

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

15. The polynucleotide according to claim 14, wherein a 2A element or an IRES element is present on the polynucleotide encoding the polypeptide according to claim 1 and the polynucleotide encoding the chimeric antigen receptor, the T cell receptor, or both.

16. The polynucleotide of claim 15, wherein the chimeric antigen receptor comprises 1, 2 or more co-stimulatory domains.

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

18. The polynucleotide of claim 17, wherein the vector is a viral vector or a non-viral vector.

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

21. The polynucleotide of claim 20, wherein the cell is a eukaryotic cell or a bacterial cell.

22. The polynucleotide of claim 20, wherein the cell is an immune cell.

23. The polynucleotide of claim 20, wherein the cell is engineered.

24. The polynucleotide of claim 22, wherein the immune cell is a T cell.

25. The polynucleotide of claim 24, wherein the T cell comprises one or more chimeric antigen receptors.

26. The polynucleotide of claim 25, wherein the chimeric antigen receptor comprises 1, 2 or more co-stimulatory domains.

27. The polynucleotide of claim 24, wherein the T cell comprises one or more engineered T cell receptors (TCRs).

28. A polypeptide expressed by a polynucleotide according to any one of claims 1-27.

29. A polypeptide comprising: (1) an extracellular domain, wherein the extracellular domain comprises one or more of an OX40-specific ligand, a 4-1BB-specific ligand, and a CD40L-specific ligand; the extracellular domain is operably linked to (2) Signal transduction domain that promotes T cell activation, wherein the OX40-specific ligand is OX40L, an antibody targeting OX40, an OX40L-Fc fusion or a combination thereof, wherein the 4-1BB-specific ligand is 4-1BBL, an antibody targeting 4-1BB, a 4-1BBL-Fc fusion or a combination thereof, and The CD40L-specific ligand is CD40, an antibody targeting CD40L, a CD40-Fc fusion or a combination thereof.

30. The polypeptide of claim 29, wherein the signaling domain that promotes T cell activation is from a CD3 zeta subunit, DAP12, an Fc receptor, or a combination thereof.

31. The polypeptide of claim 29, wherein the polypeptide further comprises 1, 2 or more co-stimulatory domains.

32. A cell expressing a chimeric receptor comprising a polynucleotide according to any one of claims 1-27 or a polypeptide according to any one of claims 29-31.

33. The cell of claim 32, wherein the cell is an immune cell.

34. The cell of claim 32, wherein the cell is engineered.

35. The cell of claim 33, wherein the immune cell is a T cell.

36. The cell of claim 35, wherein the T cell is a CAR-transduced T cell.

37. The cell of claim 35, wherein the T cell is a T cell receptor (TCR) transduced T cell.

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

39. The cell of claim 38, wherein the cell is engineered to lack endogenous expression of 4-1BB, OX40 and / or CD40L.

40. The cell of claim 38, wherein the cell is engineered using CRISPR / Cas9, zinc finger nucleases, TALE nucleases, or meganucleases.

41. The cell of claim 32, wherein the cell is contained in a cell storage bank.

42. A method for preparing cells for cell therapy, comprising the step of transfecting immune effector cells with a polynucleotide according to any one of claims 1-27.

43. The method of claim 42, further comprising the step of storing the cells in a cell storage bank.

44. The method of claim 42, 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.

45. 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 one or more of an OX40-specific ligand, a 4-1BB-specific ligand or a CD40L-specific ligand; the extracellular domain is operably linked to (2) a signaling domain that promotes T cell activation; wherein the OX40-specific ligand is OX40L, an antibody targeting OX40, an OX40L-Fc fusion or a combination thereof, wherein the 4-1BB-specific ligand is 4-1BBL, an antibody targeting 4-1BB, a 4-1BBL-Fc fusion or a combination thereof, and The CD40L-specific ligand is CD40, an antibody targeting CD40L, a CD40-Fc fusion or a combination thereof.

46. ​​The use according to claim 45, wherein the engineered allogeneic cells are obtained from a cell bank.

47. The use according to claim 45, wherein the engineered allogeneic cells express one or more chimeric antigen receptors and / or one or more recombinant T cell receptors.

48. 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 operably linked to (2) a CD3ζ signaling domain, wherein the extracellular domain comprises one or more of an OX40-specific ligand, a 4-1BB-specific ligand or a CD40L-specific ligand, wherein the OX40-specific ligand is OX40L, an antibody targeting OX40, an OX40L-Fc fusion or a combination thereof, wherein the 4-1BB-specific ligand is 4-1BBL, an antibody targeting 4-1BB, a 4-1BBL-Fc fusion or a combination thereof, and The CD40L-specific ligand is CD40, an antibody targeting CD40L, a CD40-Fc fusion or a combination thereof.

49. The use according to claim 48, wherein the allogeneic cell is an allogeneic immune cell expressing the chimeric receptor.

50. The use of claim 48, wherein the allogeneic cells express a chimeric antigen receptor or an engineered T cell receptor.

51. The use of claim 48, wherein the allogeneic immune cells are formulated to be delivered to the individual before, during and / or after tissue and / or organ transplantation in the individual.

52. The use according to claim 48, wherein the activated T cells are pathogenic T cells.

53. 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; the extracellular domain is operably linked to (2) Signal transduction domain that promotes T cell activation, wherein the extracellular domain comprises one or more of an OX40-specific ligand, a 4-1BB-specific ligand or a CD40L-specific ligand, wherein the OX40-specific ligand is OX40L, an antibody targeting OX40, an OX40L-Fc fusion or a combination thereof, wherein the 4-1BB-specific ligand is 4-1BBL, an antibody targeting 4-1BB, a 4-1BBL-Fc fusion or a combination thereof, and The CD40L-specific ligand is CD40, an antibody targeting CD40L, a CD40-Fc fusion or a combination thereof.

54. The use according to claim 53, wherein the signaling domain promoting T cell activation is derived from CD3 zeta subunit, DAP12, Fc receptor or a combination thereof.

55. The use according to claim 53, wherein the activated T cells are pathogenic T cells.

56. The use of claim 53, wherein the chimeric receptor further comprises 1, 2 or more co-stimulatory domains.

57. The use according to claim 53, wherein the individual's rejection of allogeneic cells, tissues or organs comprises transplant rejection, graft-versus-host disease or a combination thereof.

58. 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 operably linked to (2) a signaling domain that promotes T cell activation, wherein the extracellular domain comprises one or more of an OX40-specific ligand, a 4-1BB-specific ligand or a CD40L-specific ligand, wherein the OX40-specific ligand is OX40L, an antibody targeting OX40, an OX40L-Fc fusion or a combination thereof, wherein the 4-1BB-specific ligand is 4-1BBL, an antibody targeting 4-1BB, a 4-1BBL-Fc fusion or a combination thereof, and The CD40L-specific ligand is CD40, an antibody targeting CD40L, a CD40-Fc fusion or a combination thereof.

59. The use according to claim 58, wherein the immune cell expressing the engineered chimeric receptor is an allogeneic T cell.

60. The use according to claim 58, wherein the immune cell expresses a chimeric antigen receptor or an engineered T cell receptor.

61. The use according to claim 58, wherein the amount of immune cells expressing the engineered chimeric receptor provided to the individual is 10 2 -10 12 within the range of .

62. The use of any one of claims 45-61, wherein the cells are formulated for systemic or local delivery to the subject.

63. The use according to any one of claims 45-61, wherein the cell is a T cell.

64. The use according to any one of claims 45-61, wherein the cells are delivered to the individual once or more than once.

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