Auto / allo-immune defense receptors for selective targeting of activated pathogenic t cells and nk cells

Abstract

To provide compositions and methods related to cells utilized for adoptive transfer to control pathogenic conditions due to immune activation.SOLUTION: The present invention provides engineered auto / allo-immune defense receptor (ADR)-expressing T cells that selectively target activated T cells, including pathogenic T cells, to incapacitate them. The chimeric receptors comprise moieties for targeting 4-1BB, OX40, and CD40L, for example, whose expression is indicative of activated T cells. In particular embodiments, there are methods of preventing or treating conditions associated with activated T cells using adoptive T-cell transfer of cells encoding the ADRs.SELECTED DRAWING: None

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application is a joint venture of U.S. Provisional Patent Application No. 62 / 662,817, filed April 26, 2018. This provisional patent application claims priority from US Provisional Patent Application No. 2005 / 0133991, which is incorporated herein by reference in its entirety. do.

[0002] [STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT] This invention was made possible by a P50 grant awarded by the National Institutes of Health and the National Cancer Institute. This invention was made with U.S. Government support under CA126752. The U.S. Government owns certain rights in this invention. To have an advantage.

[0003] Embodiments of the present disclosure include those directed to at least one of the fields of immunology, cell biology, molecular biology, and medicine. Includes: [Background technology]

[0004] In patients undergoing transplantation or receiving therapeutic cells from a third party, T cells Unwanted activation of mitochondrial and NK cells often promotes life-threatening alloimmune responses, which This may result in rejection of the transplanted organ / tissue or graft-versus-host disease (GVHD). Similarly, the absence of autoreactive T cells leads to the development of graft-v-host disease (GvHD). Extensive activation of the IL-1 receptors also contributes to the pathogenesis of devastating autoimmune diseases such as diabetes mellitus, autoimmune colitis, and multiple sclerosis. Currently, most of these diseases are managed by selectively eliminating pathogenic T cells. It is not curable because it cannot be eradicated. Instead, patients are treated with immunosuppressants. This is often done to render the patient immunocompromised, making them more susceptible to infections and malignancies. makes it easier for conversion to occur.

[0005] The present disclosure provides safe and Addressing a long-standing need in the art for effective tissue transplantation and adoptive cell transfer, By enhancing the ability of the transferred cells to control pathogenic conditions resulting from unwanted activation of the immune system. Provide a solution. Summary of the Invention

[0006] The present disclosure provides methods for the adoption of novel antibodies that can be used to control pathogenic conditions resulting from immune activation. The present invention is directed to compositions and methods relating to cells. The compositions and methods are directed to autologous and allogeneic cells. Some procedures involve the transplantation of allogeneic cells into the recipient individual. Although steps can be taken to reduce the reactivity of the The recipient's immune system (mainly T cells and NK cells) is activated, resulting in rejection and limiting the therapeutic benefit. cells) will be targeted.

[0007] The present disclosure relates to medical conditions associated with the presence of activated pathogenic T, NK-T and NK cells. Modifying adoptive therapy cells to target those cells to prevent or treat a condition. In certain embodiments, the compositions and methods overcome this problem by: Using cells expressing receptors that selectively target pathogenic T cells while sparing resting T cells In a specific embodiment, adoptive T cell transfer is utilized. Targeting pathogenic T cells that express specific target molecules whose presence on the surface is indicative of pathogenic T cells In certain embodiments, the present disclosure provides a method for the treatment of disease Auto / allo-immune defense receptors for selective targeting of inflammatory T cells Regarding ADR).

[0008] Certain embodiments of the present disclosure include the use of engineered allogeneic T cells to prevent elimination in a host individual. A method for protecting an individual from a disease by providing said individual with cells equipped with an ADR. Embodiments include, for example, the administration of cyclosporine in individuals undergoing tissue or organ transplantation. Methods for avoiding species immune responses are also included.

[0009] In certain embodiments, the cells encompassed by the present disclosure are those that are transplanted into an allogeneic recipient. Has been modified to enable it to survive in the recipient or has been so modified In a specific example, cells for adoptive cell therapy (T cells, NKT cells) (including the above) are suitable for "off-the-shelf" use. Here, the ready-made products are cells held in a repository, i.e., a bank. to individuals who need it for a specific purpose (with or without further modifications) In most cases, the individual is the original source of the cells. The cells used in this way are not pre-engineered to express ADR. However, in some cases, cells may be stored in a The banked cells are engineered to express CAR or DR. The cells obtained from the bank may or may not express a recombinant TCR. They may then be modified to express a CAR or a recombinant TCR. This requires the production of a patient-specific product each time it is needed without immune rejection by the host. This facilitates the use of therapeutic cells from a third party without the need for a separate cell line.

[0010] In particular embodiments, there is an isolated polynucleotide comprising a sequence encoding (1) OX40-specific ligand, 4-1BB-specific ligand, CD40L-specific ligand (2) a signaling molecule that promotes T cell activation; The polynucleotide is operably linked to a signaling domain of OX40. The ligand may include a CD40L specific ligand, a 4-1BB specific ligand, or a CD40L specific ligand. OX40-specific ligands include OX40L, antibodies targeting OX40, and OX40L- Fc fusions, or combinations thereof, or any other antibody capable of specific binding to OX40. The 4-1BB specific ligand can be any engineered protein. BL, antibodies targeting 4-1BB, 4-1BBL-Fc fusions, or combinations thereof or any other engineered protein that has specific binding ability to 4-1BB. The CD40L-specific ligand can be any of CD40, antibodies targeting CD40L, CD40-Fc fusion or any other engineered molecule that has specific binding ability to CD40L. In at least certain cases, the protein may be a protein that is specifically engineered, a protein that is specifically engineered, or a combination thereof. In this case, the polynucleotide is between (1) and (2), for example, 10 to 220 amino acids in length. The spacer further comprises a sequence encoding a spacer, which enables surface detection by the antibody. For example, the spacer may have a sequence that facilitates detection by anti-Fc Ab. The pacer may comprise an IgG Fc portion.

[0011] In certain embodiments, the polynucleotides of the present disclosure are directed to chimeric antigen receptors, T cell receptors, The polynucleotide may further encode a polypeptide, a polypeptide sequence, a polypeptide subunit, or both. For example, viral vectors (retroviral vectors, lentiviral vectors, Adenovirus vectors or adeno-associated virus vectors) or non-viral vectors They can take any form, including those present on vectors (plasmids, transposons, etc.). In certain cases, the polynucleotide may be expressed in a cell, including a eukaryotic cell or a bacterial cell. The cell may be an immune cell, such as a T cell. The cell may be engineered. The cells express one or more chimeric antigen receptors. r:CAR) and / or one or more engineered T cell receptors (T cell The antigen-binding domain may include a transcription factor receptor (TCR).

[0012] The polypeptides expressed by any polynucleotide encompassed by this disclosure are Specific embodiments include a polypeptide comprising: (1) O One or more of X40 specific ligand, 4-1BB specific ligand, and CD40 (2) operably linked to a signaling domain that promotes T cell activation. The signaling domain that promotes T cell activation is the CD3 zeta subunit, DAP1 2, an Fc receptor, or a combination thereof.

[0013] Any cell encompassed by this disclosure is part of this disclosure. Any of the chimeric receptor expressing cells may be used in combination with any of the polynucleotides contemplated herein. and / or cells containing any of the polypeptides contemplated herein. The cells may be engineered cells. The cells may be CAR-transduced cells. Such as T cells, including transduced T cells and / or T cell receptor (TCR)-transduced T cells. In a specific embodiment, the cell is an immune cell that expresses one or more genes endogenously. For example, to lack one or more of 4-1BB, OX40 and / or CD40L. The cells are engineered to lack the gene, CRISPR / Cas9, zinc finger transcription factor (FNG) and ribosomal protein kinase C (RIKFK). Engineering the enzyme using nucleases, TALE nucleases, or meganucleases Alternatively, specific proteins may be produced that are anchored, for example, in the endoplasmic reticulum or other intracellular compartments. Surface of the ADR ligand, such as by capturing the ADR ligand with an antibody or receptor. Cells may be engineered to prevent expression.

[0014] In one embodiment, the present invention relates to a method for avoiding rejection of allogeneic cells, tissues, or organs in an individual. The method comprises administering to the individual an effective amount of a compound that selectively targets activated T cells. The present invention relates to a method for the expression of an engineered chimeric receptor that contains an extracellular domain that includes CD3 zeta. and delivering allogeneic immune cells to said individual, said delivering step resulting in There are several ways to achieve this: (1) by inhibiting endogenous alloreactive T cells in the individual, and / or (2) by inhibiting endogenous alloreactive T cells in the individual. ) Suppression of NK cell activation in an individual. In a specific embodiment, the allogeneic cells are chimeric recipients. The allogeneic immune cells express chimeric antigen receptors and / or engineered antigen receptors. Allogeneic immune cells can express engineered T cell receptors in individuals. and / or may be delivered to an individual before, during and / or after organ transplantation. Thus, activated T cells are pathogenic T cells.

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

[0016] Certain embodiments include methods for preventing or treating a medical condition associated with activated T cells in an individual. The method of claim 1, further comprising administering to the individual an effective amount of an engineered antibody that selectively targets the activated T cells. delivering to the immune cells expressing an engineered chimeric receptor, However, there are methods that include the following: (1) targeting compounds that are selectively present on activated T cells; and (2) a signaling domain that promotes T cell activation. Conditions include autoimmune disorders such as transplant rejection, graft-versus-host disease, type I diabetes, multiple sclerosis, The disease may be an autoimmune disorder, such as autoimmune colitis, or a combination thereof.

[0017] In one embodiment, the method comprises administering to the individual a NK cell-mediated ... A method for avoiding host rejection comprising administering to an individual an effective amount of a marker selectively present on activated T cells. -cell signaling that promotes T cell activation and includes an extracellular domain that targets compounds that promote T cell activation The method includes providing an immune cell that expresses an engineered chimeric receptor that also includes a domain. Engineered immune cells expressing chimeric receptors may be useful in certain cases. , allogeneic T cells. Immune cells are those that express chimeric antigen receptors and / or engineered The engineered chimeric receptors that are provided to the individual may be capable of expressing T cell receptors. The amount of immune cells in 1ml 2 10 per 2 ~10 12 The range of chimeric receptors is The expressing cells can be provided systemically or locally to an individual. The immune cells are T cells. The immune cells can be delivered to an individual once, or they can be delivered more than once.

[0018] The above describes the features and techniques of the present disclosure in order that the detailed description that follows may be better understood. The above has outlined rather broadly the advantages of the present invention. Further advantages which form the subject matter of the claims of this application are set forth below. Features and advantages are described. The concepts and specific embodiments disclosed are intended to be consistent with the design and be readily utilized as a basis for modifying or designing other structures to carry out the objectives of the present invention; Those skilled in the art should recognize that such equivalent constructions are within the scope of the appended claims. It should also be understood by those skilled in the art that the present invention does not depart from the spirit and scope of the invention as described herein. The novel features believed to be unique to the design disclosed in the document include both the structure and the method of operation. The present invention, together with further objects and advantages thereof, will become apparent from the following description taken in conjunction with the accompanying drawings, in which: It will be better understood with reference to the accompanying drawings, each of which is for illustrative and explanatory purposes only. It should be expressly understood that these are provided for illustrative purposes only and are not intended to define the scope of the present disclosure. It should be. [Brief description of the drawings]

[0019] For a more complete understanding of the present disclosure, the following description should be taken in conjunction with the accompanying drawings, in which: sea ​​bream.

[0020] [Figure 1A-E] ADRs can be expressed on the cell surface of immune cells to enhance cytotoxicity against their respective targets. (Figure 1A) Schematic of ADR. GFP is optional. (Figure 1B) Expression of ADR on the cell surface. (Figure 1C) Proliferation of ADR T cells after transduction. (Figure 1D) Cytotoxicity of ADR T cells against target cells expressing the ADR ligand. (Figure 1E) Proliferation of wild-type and 4-1BB KO T cells expressing 4-1BB ADR and their cytotoxicity against 4-1BB+ targets. This shows that proliferation and cytotoxicity can be further enhanced by knocking out the ADR ligand on T cells, demonstrating that co-expression of ADR and its ligand in T cells is not required for ADR- T cell proliferation or function.

[0021] [Figure 2A-E] Selective expression of ADR ligands on activated T cells allows for their selective elimination by ADR T cells. (Figure 2A-C) Expression of ADR ligands on resting versus activated T cells after TCR stimulation. (Figure 2D) Lack of cytotoxicity of ADR T cells against resting CD4+ and CD8+ T cells. (Figure 2E) Elimination of activated CD4+ and CD8+ T cells by ADR T cells after 48 h of co-culture.

[0022] [Figure 3A-F]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 are protected from rejection after co-culture with allogeneic PBMCs at an ADR T:PBMC ratio of 1:10. (Figure 3B-C) Absolute numbers of donor and allogeneic T cells in co-culture. (Figure 3D-F) Same for virus-specific ADR T cells.

[0023] [Figure 4A-C] In in vitro mixed lymphocyte reactions, expression of ADR protects virus-specific allogeneic 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-C) Absolute numbers of recipient T cells and donor VSTs at various time points during the MLR.

[0024] [Diagram 5] ADR VST retains antiviral function. ADR VST was co-cultured with monocytes pulsed with a viral pepmix. Monocyte counts showed that ADR VST eliminated virus-infected cells as well as unmodified VST.

[0025] [Figure 6A-H] Activated NK cells upregulate ADR ligands and can be selectively targeted by ADR T cells. (Figure 6A-B) 4-1BB expression on resting versus activated NK cells. (Figure 6C) Residual numbers of resting versus activated NK cells after 24 h of co-culture with 4-1BB ADR T cells. (Figure 6D) MHC-depleted ADR T cells protect against immune rejection by allogeneic PBMCs by controlling NK cell proliferation. (Figure 6E) Absolute numbers of donor T cells and allogeneic NK cells in co-culture. (Figure 6F) Upon 48 h of co-culture at a 1:1 E:T ratio, MHC-depleted ADR T cells resist immune rejection by NK cells. (Figure 6G-H) ADR T cells control proliferation of alloreactive NK cells during MLR with PBMCs. (Figure 6H) Absolute numbers of NK cells are plotted.

[0026] [Figure 7A-E] ADR expression protects allogeneic T cells from immune rejection in vivo. (FIG. 7A) Schematic of a mouse model of immune rejection in which T cells from HLA-A2+ donors were given to mice after sublethal irradiation, followed by allogeneic HLA-A2- T cells 4 days later. (FIG. 7B-C) Control T cells from HLA-A2- donors were rejected by day 18, whereas ADR-expressing cells were protected. (FIG. 7C) Absolute numbers of T cells from HLA-A2+ and HLA-A2- donors at various time points. (FIG. 7D) Modified in vivo model in which mice were given whole PBMCs (containing both T and NK cells) from donor 1 instead of allogeneic T cells. (FIG. 7E) Representative flow plots showing that ADR T cells protected mice from immune rejection and from the rapid development of lethal GvHD.

[0027] [Figure 8A-E] Co-expression of CAR and ADR maintains the function of both receptors. (Figure 8A) Schematic of immune cells co-expressing ADR and CAR. (Figure 8B) Co-expression of CAR and ADR on the cell surface. (Figure 8C) Cytotoxicity of CAR-ADR T cells against NALM-6 (CD19+ CAR target). (Figure 8D) Cytotoxicity of CAR-ADR T cells against activated T cells (ADR target). (Figure 8E) Cytotoxic activity of CAR-ADR T cells against both targets when simultaneously co-cultured with both cell targets.

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

[0029] [Figure 10A-C] In solid tumor models, CAR-ADR T cells are protected from immune rejection and exert potent antitumor activity. (Figure 10A) Schematic of mouse model. Mice were given 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. (Figure 10B) GD2 CAR T cells from donor 2 were rejected by D18, whereas CAR-ADR T cells resisted allogeneic rejection and persisted in peripheral blood. (Figure 10C) Tumor burden in mice. * indicates xenogeneic GvHD-related death in the ATC+GD2 CAR T group.

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

[0031] [Figure 12A-D] ADR T cells protect mice from lethal xenogeneic GvHD. (Figure 12A) Schematic of the model. (Figure 12B) Expansion of FFluc-labeled ADR T cells in vivo. (Figure 12C) Kinetics of weight gain and loss in mice. (Figure 12D) Overall survival rate of mice.

[0032] [Figure 13A-G]Second generation ADR (ADR.28 zeta) with CD28 intracellular signaling domain. (Figure 13A) Structure of ADR.28 zeta. (Figures 13B-C) In vitro cytotoxicity of ADR.28 zeta against target expressing cell lines. (Figures 13D-G) ADR.28 zeta protected mice from xenogeneic GvHD. (Figure 13D) Schematic of the model. (Figure 13E) In vivo proliferation of FFLuc-labeled ADR.28 zeta T cells. (Figure 13F) Kinetics of mouse weight gain and loss. (Figure 13G) Overall survival of mice.

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

[0034] As used herein, the words "a" and "an" are inclusive. When used in conjunction with the word sing, the term singular refers to any word that is used in the claims. Some embodiments of the present invention may include one or more of the elements, methods, and / or components of the present invention. It comprises method steps and / or methods or comprises one or more elements, method steps and Any of the methods and compositions described herein may consist essentially of the methods of the present invention. It is contemplated that the present invention may be implemented with respect to any other method or composition described herein. do.

[0035] Throughout this specification, unless the context otherwise requires, the word "including" ( comprise, comprise and comprising are the The inclusion of a step or element or group of steps or elements is implied but does not include any other steps or It will be understood that no exclusion of any element, step or group of elements is implied. "consisting of" includes what follows the word "consisting of" Thus, the phrase "consisting of" means that the term is limited to the recited elements. It indicates that an element is necessary or essential, and that no other elements can be present. "consisting essentially of" means Any of the elements listed below may be used, including any of the elements specified in this disclosure with respect to the listed elements. It is meant to be limited to other elements that do not interfere with or contribute to the activity or action of the compound. The phrase "consisting essentially of" means that the recited elements are necessary or mandatory, but other The elements are not optional and depend on whether they affect the activity or action of the listed elements. to indicate whether it may or may not be present.

[0036] Throughout this specification, the terms "one embodiment," "embodiment," "particular embodiment," "related embodiment," "relevant embodiment," "particular embodiment," "related "embodiment," "particular embodiment," "additional embodiment" or "further embodiment" Reference to any particular feature, structure or combination thereof described in connection with that embodiment is intended to be illustrative, not restrictive. It is therefore intended that the features or characteristics be included in at least one embodiment of the present invention. The appearances of such phrases in various places in the specification do not necessarily all refer to the same embodiment. Moreover, any particular feature, structure or characteristic may be incorporated into one or more embodiments. may be combined in any suitable manner.

[0037] As used herein, the term "subject" refers generally to a person suffering from any type of medical condition. The subject may be any type of animal. A subject may be any living or animal subject that is the object of a method or substance. mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), Livestock (e.g., cattle, sheep, goats, pigs, turkeys, and chickens), house horses, Doberman pets (e.g. dogs, cats, and rodents), horses, and transgenic The subject may be, for example, a patient suffering from a disease (which may also be referred to as a medical condition), such as a 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 undergoing or have undergone antibiotic treatment. The subject may be asymptomatic. The subject may be a healthy individual. The term "individual" also refers to at least one In some cases, the terms "subject" and "individual" may be used interchangeably. may or may not be housed in a health care facility and may be treated as an outpatient in a health care facility. The individual may receive one or more pharmaceutical compositions via the internet. An individual may include humans or non-human animals of any age, and therefore includes both adults and juveniles. Individuals include animals (i.e. infants, children) and infants, including individuals in utero. Individuals may be of any breed and sex. The term is not intended to imply the need for medical treatment, so individuals may Voluntarily or involuntarily, for clinical trials or basic scientific research Any of these may be part of an experiment.

[0038] As used herein, the term "engineered" refers to a substance that is not naturally occurring. , artificially generated, for example by recombinant gene techniques standard in the art Refers to the molecule.

[0039] In the context of this disclosure, an "effective amount" or a "therapeutically effective amount" refers to an amount that, when administered to an individual, has an active and / or alleviating the signs and / or symptoms of a medical condition. The actual amount administered depends on the amount of cells that will restore functional immunity or prevent a medical condition. Either in vitro or in vivo, immune cells exhibit pharmacological activity for a medical condition. This can be determined based on testing performed.

[0040] I. Self / Alloimmune Defense Receptors and Compositions and Uses Thereof The present disclosure provides a method for the selective targeting of activated T cells, including pathogenic T cells, comprising administering to the subject a synthetic chimeric receptor agonist (TAG) that selectively targets activated T cells. These engineered molecules are synthetic molecules and are produced by recombinant techniques. These molecules can be produced by, for example, blocking activated T cells, including activated pathogenic T cells. It can be said to be a self / alloimmune defense receptor that targets with high specificity.

[0041] In certain embodiments, the autologous / alloimmune defense receptor (ADR) is an ADR on an activated T cell. The targeting entity includes one or more compounds that are upregulated on activated T cells. The upregulated compound may be any one or a combination of compounds, but in particular In embodiments, OX40, 4-1BB and CD40L are upregulated on activated T cells. These are the subjects that the ADRs are targeted to. In certain embodiments, the ADRs , present on allogeneic immune cells that are allogenic with respect to the individual receiving the cells. In this example, the ADRs are expressed on autologous T cells, on heterologous cells, and / or on synthetic cells. do.

[0042] (A. Autologous / Allologous Immune Defense Receptor (ADR) Molecules) ADR molecules are synthetic, non-naturally occurring molecules that are artificially produced and have at least (1) an activity and an extracellular domain (in a specific embodiment) that targets a compound that is selectively present on activated T cells. The extracellular domain targets one or more compounds that are upregulated on activated T cells. (a target protein or a functional fragment or derivative thereof), 2) signaling domains that promote T cell activation, such as the CD3 zeta subunit; DAP12, and those derived from Fc receptors or other ITAM-containing sequences. An ADR molecule may contain or be composed of elements (1) and (2). It may consist of or consist essentially of these elements. In examples, the ADR is one or more components of a type I transmembrane protein and / or a type II transmembrane protein. Contains one or more components of a transmembrane protein.

[0043] In a specific embodiment, the ADR molecule comprises an extracellular domain that binds to an associated IL-1 domain on activated T cells. Contains proteins that selectively bind to proteins. For example, the ADR extracellular domain binds to activating The ADR extracellular domain may contain a ligand for a receptor on T cells, or the ADR extracellular domain may be a ligand for an activated T cell. The polypeptide may comprise a receptor for the above ligand.

[0044] In a specific embodiment, the ADR molecule comprises an extracellular domain that is a ligator for OX40. In these particular examples, the ligand for 4-1BB and / or CD40 are included. The related proteins on activated T cells are OX40, 4-1BB, and CD4, respectively. OL. In alternative embodiments, other specific compositions are targeted on activated T cells. For example, other activation markers that are upregulated on the cell surface of T cells (e.g., CD69 , CD25, CD71, etc. can be targeted using a similar approach. In such cases, the corresponding ADR molecule is not a 4-1BB / OX40 specific ligand. or CD69 ligand, CD25 ligand, or CD71 ligand, respectively; It will have a targeting moiety derived from an antibody.

[0045] In some cases, OX40 expression was upregulated in contrast to non-activated T cells. Targeting activated T cells that are regulated. Targeting activated T cells that are regulated. To this end, ADR needs to bind OX40 ligands so that it can target activated T cells. A ligand for OX40 could be used in ADR. In some instances, the ligand for OX40 may be, for example, at least OX40L, OX40 an antibody (or a functional fragment thereof) that binds to Fc and OX40L; Any suitable ligand for OX40, such as a functional derivative or fragment thereof, may be used. OX40L is a tumor necrosis factor (ligand) superfamily member. 4(tax-transcriptionally activated glycoprotein ctivated glycoprotein)1, 34kDa), OX40L, CD2 52, also known as TNFSF4, TXGP1, OX-40L, or gp34.

[0046] In some cases, 4-1BB expression was upregulated in contrast to non-activated T cells. Targeting pre-regulated activated T cells. Targeting these activated T cells. To achieve this, ADRs must be designed to bind 4-1BB to target activated T cells. Ligands for 4-1BB may be useful in ADR. In the case where it is used, the ligand for 4-1BB is, for example, 4-1BBL, 4-1B Antibody (or functional fragment thereof) targeting B, Fc fusion with 4-1BBL or any suitable analogue to 4-1BB, such as a functional derivative or fragment thereof. may be an appropriate ligand.

[0047] In certain cases, CD40L expression is upregulated in contrast to non-activated T cells. To target these activated T cells, To this end, ADRs must bind to CD40L so that they can target activated T cells. The receptor for CD40L may be available in ADR. In the cases where ADR is used, CD40 (which is also Bp50, CDW40, TNFRSF 5, or p50), an antibody that targets CD40L (or its function The term may include a target polypeptide, a target polypeptide (e.g., a target polypeptide), or a functional derivative or fragment thereof.

[0048] In some cases, the ADR molecule is a 2-amino acid sequence that facilitates targeting of activated T cells. These combinations contain one or more extracellular domains, enhancing general targeting of activated T cells. or may allow for specific targeting of specific subsets of activated T cells. R allows targeting of activated T cells expressing either OX40 or 4-1BB To achieve this, we used the same ADR molecule with OX40L and 4-1BBL as extracellular domains. Examples of such combinations include either OX40 or 4-1BB alone. We identify activated T cells that express CD40L, regardless of whether those activated T cells also express CD40L. Similarly, ADRs may selectively target either CD40L or OX40. Activated T cells expressing either one of the two were examined to see whether they also expressed 4-1BB. For independent targeting, both CD40 and OX40L may be included.

[0049] In the ADR molecule, the extracellular domain is one or more One such component is capable of being operably linked to the above components during T cell activation. In certain embodiments, the ADR may be a protein that mediates downstream signaling. CD3 zeta (CD247, CD3-zeta, CD3H, CD3Q, CD3Z, IMD2 5, T3Z, or TCRZ) or a functional fragment or derivative thereof CD3 zeta mediates downstream ITAM-derived signaling during T cell activation. ITAM-containing signaling domains include those involved in DAP12, Fc receptors, and other CD3 subunits. Signaling domains can be derived from other domains. The ADR may be non-covalently linked via a domain.

[0050] In certain embodiments, the ADR is upregulated on CD3 zeta and activated T cells. The compound or compounds targeted by the antibody include a spacer between the extracellular protein and the compound or compounds targeted by the antibody. In other cases, no spacer is used. The spacer determines what function the ADR may have. Alternatively, the sequence may be inactive or contribute substantially little or nothing to the In other cases, the spacer may, for example, enhance the function of and / or detect the ADR. and / or ADR can be targeted for inhibition. In a specific embodiment, the spacer facilitates detection of cells expressing the ADR. The spacer may be used to encode a protein sequence, e.g., an anti-Fc Ab. The polypeptide may encode an Fc region or a fragment thereof that would allow for surface detection of the target cell. In certain embodiments, the spacer is, for example, a type II transmembrane protein (4-1BBL OX40L) and the type I ADR backbone (TM, signaling domain) To avoid potential steric hindrance caused by the ligand binding domain and the membrane The spacer may be any length, for example, about 10 to 220 amino acids. The length of the spacer may be 10 to 220, 10 to 200, or 10 to 1 50, 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, The range can be 150-200, 150-175, 175-200, etc. Approximately 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, 1 00, 105, 110, 120, 130, 140, 150, 160, 170, 180, 1 In other instances, the spacer may be 90, 200, 210 or 220 amino acids in length. It may be less than 10 amino acids or more than 200 amino acids.

[0051] In some cases, ADRs suppress cytokine production from cells expressing ADRs. The costimulatory domains include one, two, three or more costimulatory domains that enhance the For example, CD28, CD27, 4-1BB, OX40, ICOS, CD30, HVEM, C They may be derived from the intracellular signaling domains of costimulatory proteins such as D40. However, when the ADR contains 4-1BBL, the costimulatory domain of the ADR is 4-1BB It may or may not be from.

[0052] In some embodiments, the ADR is a polypeptide that is encoded by a CD3 zeta component of the ADR that is located intracellularly. and targeting one or more compounds that are upregulated on activated T cells. Any kind of extracellular domain may be used as long as it allows the extracellular location of the extracellular domain. In another example, the ADR may comprise a transmembrane domain that may act as a marker for the respective It can bind to its ligand and promote cytotoxicity by cross-linking the TCR. soluble proteins (e.g., ADR-CD3 T cell engager protein) that The extracellular domain is derived from a surface protein with a transmembrane domain (e.g., CD40). If so, the ADR may include a transmembrane domain from the corresponding endogenous molecule. In some cases, the R molecule contains one or more costimulatory domains. The nsmembrane domain (TM) is a co-stimulatory domain of the same endogenous factor. Examples of TMs include CD3, CD8α, CD27, CD2 8, 4-1BB, OX40, CD4, etc.

[0053] In one example of an ADR polypeptide, the components are arranged in the following order from the N-terminus (N) to the C-terminus (C): In the case of a common ADR, the receptor is one of the following (for example: and wherein the extracellular domain comprises an associated protein on activated T cells. Contains proteins that selectively bind to: N-extracellular domain-signaling domain-C N-extracellular domain-CD3 zeta-C N-extracellular domain-spacer-CD3zeta-C N-extracellular domain-spacer-costimulatory domain-CD3 zeta-C N-extracellular domain-spacer-two costimulatory domains-CD3zeta-C N-two extracellular domains-spacer-costimulatory domain-CD3 zeta-C N-2 extracellular domains-spacer-2 costimulatory domains-CD3 zeta-C

[0054] In each case, the transmembrane domain may be C-terminal to the spacer. Type I Transmembrane protein backbone (e.g. transmembrane domain, signaling domain, CD3 Promotes the expression of type II ligands (e.g., OX40L and 4-1BBL) on the . To achieve this, a signal peptide may be used at the N-terminus.

[0055] In some cases, the ADR may be one or more detectable markers, e.g., a colorimetric marker. , fluorescent markers and / or radioactive markers. Examples include green fluorescent protein (Green Fluorescent Protein). Examples of such proteins include blue fluorescent protein and blue fluorescent protein.

[0056] Although ADRs may be produced synthetically as proteins, ADRs may also be produced by polynucleotides or It may take the form of a polypeptide expressed by a polynucleotide. Recombinant techniques for producing otide and ADR polypeptides are known in the art. There are.

[0057] In certain cases, the ADR polynucleotide is in an expression construct or or part of an expression construct present on a vector, the vector being a viral vector. The vector may be a viral or non-viral vector. An example of a non-viral vector is a plasmid. Examples of viral vectors include lentiviral vectors and retroviral vectors. vectors, adenovirus vectors and adeno-associated virus vectors. Any vector expressing R can be used to target immune cells, such as T cells, NK cells, or NKT cells. It will have appropriate elements to allow expression in eukaryotic cells, including cells. Suitable elements include a promoter.

[0058] 4-1BB ADR (SEQ ID NO: 1) MEFGLSWLFLVAILKGVQCGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYV FFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARAR HAWQLTQGATVLGLFRVTPEIPAGLPSPRSEESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVV VGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSAAAGGGGSGGGGSGGGGSMVSKG EELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFF KSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVN FKTRHNIEDGSVQLADHYQQNTPIGDGGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0059] OX40 ADR MEFGLSWLFLVAILKGVQCQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQ EVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLESKYGPPC PPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLPGKKDPKFWLVVVGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPRTSAAAGGGGSGGGGSGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT LKFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRI ELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLST QSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0060] CD40L ADR (SEQ ID NO: 3) MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQH KYCDPNGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEK CHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVL VVVGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSAAAGGGGSGGGGSGGGGSMVS KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHD FFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0061] In certain embodiments, the extracellular domain that targets activated T cells is an antibody or As used herein, the term "antibody" includes functional fragments or derivatives of the antibody. The term refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be derived from natural sources or from tissues. The immunoglobulin may be an intact immunoglobulin derived from a recombinant source. Antibodies are typically composed of four groups of immunoglobulin molecules. The antibody in the present invention may be, for example, a polyclonal antibody, a monoclonal antibody, Fv, Fab and F(ab) 2 , as well as a variety of other antibodies, including single chain antibodies and humanized antibodies. (Harlow et al., 1999, "Using Anti bodies:A Laboratory Manual”,Cold Spring Harbor Laboratory Press, NY, Harlow et al. ,1989,“Antibodies:A Laboratory Manual”,C old Spring Harbor, NY, Houston et al.,19 88, Proc. Natl. Acad. Sci. USA 85:5879-5883, B ird et al., 1988, Science 242:423-426).

[0062] In some cases, the extracellular domain of the ADR comprises an antibody fragment. The term "fragment" refers to a portion of an intact antibody and does not include the antigen binding domain of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab' 2 and Fv fragments, linear antibodies, scFv antibodies, and antibody fragments. Examples of antibodies that can be used include, but are not limited to, multispecific antibodies formed.

[0063] Synthetic antibodies may be used in ADRs. As used herein, the term "synthetic antibodies" refers to The term also refers to antibodies produced using recombinant DNA techniques, such as antibodies produced using recombinant DNA techniques as described herein. The term refers to an antibody expressed by a bacteriophage. means an antibody produced by the synthesis of a molecule, the DNA molecule of which encodes the antibody protein or The amino acid sequence that specifies the antibody is expressed, and the DNA sequence or amino acid sequence is known in the art. The method may be performed using synthetic DNA or amino acid sequence techniques that are available and well known in the art. This should also be interpreted as having been

[0064] (B. Cells expressing ADR) Allogeneic cells used in adoptive transfer are limited in their efficacy due to immune responses by the recipient individual. In some cases, the cells may be involved in the pathogenesis of diseases such as graft-versus-host disease. Modifications to remove the endogenous TCR (e.g., using CRISPR) to prevent Alternatively, virus-specific T cells (IgE) may be added to maintain antiviral activity. Cells that are either intact or CAR / TCR engineered can also be used to treat certain diseases. Such VSTs are useful in physiological conditions because their TCRs are more strongly antiviral. Since the host is restricted by IgG1 antigens, its graft-versus-host activity is extremely limited, but the recipient Adverse reactions to nicotine remain a concern.

[0065] Improved for homologous use by being engineered to express a synthetic ADR molecule Cells are encompassed by the present disclosure. Accordingly, the present disclosure includes ADRs as polynucleotides. Also included are cells which possess the ADR polypeptide expressed on the surface of the cell. In certain cases, for off-the-shelf use ADR-expressing cells are produced for the purpose of being maintained in a repository. The repository may already be configured to express ADRs, or it may be Even if the database is configured to express ADR after recovery from the repository, Certain cells, such as bacterial cells, can be used to produce ADR molecules, but ADR Other cells, such as eukaryotic cells, that harbor the As shown herein, ADR-expressing immune cells can selectively activate T cells. and ADR-expressing immune cells are protected from cytolysis by alloreactive T cells.

[0066] The cells expressing the ADR molecule may be of any type, but in a specific embodiment, They have been engineered to express ADRs and therefore immunoglobulins not found in nature. immune cells, e.g., immune effector cells, e.g., T cells, NK cells, NKT cells, or the like Cell lines derived from lineages or cells engineered to have cytotoxic activity At least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 191, 200, 210, 220 Populations containing 94, 95, 96, 97, 98, 99, or 100% ADR-expressing cells In particular, a population of non-naturally occurring ADR expressing cells is envisaged. The cells may, for example, be a synthetic ADR polynucleotide. By standard methods of transfection or transduction of nucleotides. It can be generated.

[0067] In some cases, the ADR molecule that is modified to express an ADR molecule is has already been engineered to have another engineered non-natural molecule other than R Alternatively, the ADR may be engineered to have another non-naturally occurring molecule other than the ADR. For example, chimeric antigen receptors (CARs) or engineered Cells that express the T cell receptor (TCR) do so to protect them from host rejection. The combination of one or more CARs and one or more CARs can protect against infection and therefore increase their therapeutic potency. and / or engineered a cell expressing one or more TCRs to express one or more ADRs. The cells may be genetically engineered to express one or more ADRs, or the cells may be genetically engineered to express one or more CARs and and / or may be engineered to express one or more TCRs. In some cases, the ADR is expressed on a different vector than the CAR and / or TCR. In other cases, the ADR molecule is expressed on the same vector as the CAR and / or TCR. In cases where the ADR is expressed on the same vector as the CAR (for example), DR and CAR expression may be directed by the same or different regulatory elements. Even if ADR and CAR are not identical, they are a single polymer with a cleavable element such as 2A between them. It may be expressed as a peptide.

[0068] In cases where the ADR-expressing cells also express a CAR or TCR, the CAR or TCR Any specific antigen may be targeted. In instances where a CAR is used, the CAR may be a first generation, It can be second generation, third generation, etc. CARs can, in certain instances, be bispecific. Ugh.

[0069] In some cases, a cell expressing an ADR molecule may, for example, overexpress one or more endogenous molecules. In some cases, cells are engineered to lack a specific function. , one that would normally promote cell fratricide. These are engineered to lack expression of the endogenous genes listed above. Cells expressing the molecule can be engineered to lack expression of, for example, 4-1BB or OX40. As just one example, cell engineering can be performed using CRISPR / Cas9. Say.

[0070] [II. Methods using self / alloimmune defense receptors] Embodiments of the present disclosure include a method of providing an effective amount of ADR-expressing cells to an individual for any purpose. The methods include those that provide selective targeting of activated T cells in an individual for any purpose. The activated T cells include activating the activated T cells with an effective amount of ADR-expressing immune cells, e.g. For example, by exposure to ADR-expressing T cells. Such exposure can be, for example, For example, a given technology may have one or more resulting applications.

[0071] In some embodiments, the ADR is an activated immune cell other than an activated T cell, e.g. Used to selectively target activated B cells (which is a treatment for lupus, rheumatoid arthritis, etc.) may help to control useless B cells, as in the case of In another embodiment, the ADR also targets a disease, e.g., macrophage activation syndrome. For example, 4-1BB or OX40 or CD40L, It is used to specifically target malignant cells in which it is expressed.

[0072] The regimen for providing an individual with an effective amount of ADR-expressing cells can be used for treatment or prevention. or by the person or people delivering the cells, regardless of the method of use. For example, in the case of prevention, the cells may be detected or determined prior to detection of one or more symptoms. or after detecting one or more symptoms, but if further symptoms occur or In the case of treatment, an individual may receive one, two or more After the above symptoms have occurred, and optionally after clinical diagnosis, an effective amount of cells is provided. It is possible.

[0073] In a specific embodiment of the method, the individual is administered a single administration of a therapeutically effective amount of cells, or may be delivered multiple times, e.g., on 1, 2, 3, 4, 5, 6, 7 days, or on 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, if or 12 months, or 1, 2, 3, 4, 5 or more years, or any period therebetween Multiple deliveries at intervals ranging from 100 to 200 mg / kg may be used. The time between doses may vary even within a single regimen. It can move.

[0074] Administration of the ADR-expressing cells can be by any suitable method to an individual, including local or systemic administration. In a specific embodiment, the ADR-expressing cells can be administered by, for example, intravenous delivery, oral delivery, rectal delivery, topical application, intramuscular delivery, delivery by injection, enteral delivery, nasal delivery The drug may be delivered by inhalation, sublingually, buccally, transdermally, or subcutaneously. The cells may or may not be delivered as a bolus. The cells may or may not be provided to the individual by different delivery routes. When delivered, they may be delivered in a pharma- ceutically acceptable carrier or excipient. Specific examples of doses of ADR-expressing cells include 10 4 cells / m 2 , 10 5 cells / m 2 , 10 6 cells / m 2 , 10 7 cells / m 2 , 10 8 cells / m 2 , 10 9 cells / m 2 , 10 10 cells / m 2 , 10 11 cells / m 2 , or 10 12 cells / m 2 and the range between them Some examples include:

[0075] (A. Implementation of use for off-the-shelf products state) The present disclosure relates to a method for producing a cell that is available as a ready-made product, for example, a cell that is different from the individual from which the cell was originally obtained. Adoptive transfer samples that can be obtained from the repository for use in different individuals. The cells may already express the ADR prior to being deposited in the repository. The cells may be cultured in vitro and subsequently modified to express the ADR. The cells or the repositories may be immune effector cells of the present invention. After being obtained from the library, the cells may express, for example, a tumor-specific receptor (e.g., CAR or TCR). The above-mentioned configuration may be modified to reflect the above-mentioned changes.

[0076] As shown elsewhere herein, cells expressing ADR can be used to protect against T cell proliferation while sparing resting T cells. ADR selectively targets activated T cells and NK cells. protect allogeneic T cells from immune rejection mediated by mitochondrial and / or NK cells, , protecting T cells from immune rejection in vivo. In so doing, ADRs can be engineered Does not interfere with the function of the expressed anti-tumor receptor (e.g. CAR). Co-expression of ADR and CAR T cells capable of efficiently eliminating both tumors and activated T cells in vitro The present disclosure further provides a method for the treatment of cancer using "off-the-shelf" T cells that co-express a CAR and an ADR. This allows the mouse to grow while retaining resistance to immune rejection from allogeneic T cells present in the same mouse. The in vivo anticancer activity in the model is shown. B ADR is effective against 4-1BB+ tumor cells, and ADR is effective against 4-1BB-expressing malignant tumors. It has been shown that it can be used as a treatment modality for tumors.

[0077] In certain embodiments, the "off-the-shelf" therapeutic cells contain ADRs to resist immune rejection. express and retain endogenous TCR specificity (e.g., specificity for viral or tumor antigens) or an anti-tumor receptor in which the endogenous TCR has been engineered, e.g., one or more Replaced with a CAR and / or one or more recombinant TCRs.

[0078] In a specific embodiment, the pre-fabricated cells are housed in a repository and are pre-fabricated prior to deposit in the repository. Alternatively, after deposit, the ADR-expressing T cells may be modified for a specific purpose. to prevent graft rejection, for example after tissue or organ transplantation. ADR-expressing T cells can be stored in a repository and used to treat, for example, viruses. Selected or native TCR or transgenic mice can be used to combat infection or cancer. ADR-expressing T cells can be engineered with transgenic TCRs. ADR-expressing cells can be transduced with CARs directed against cancer or pathogenic infections. The repository contains specific cancer-associated antigens or antigens expressed by a patient's specific tumor. transduction of one or more CARs and / or one or more TCRs directed against neo-antigens Acceptable.

[0079] In some cases, especially when the ADR-expressing cells are not themselves alloreactive, the host To prevent solid organ transplant rejection by destroying the immune cells that reject the organs. The transplanted allogeneic cells are utilized.

[0080] Cells housed in repositories may be allogeneic with respect to the recipient individual, but alternative In embodiments, the cells contained in the repository are autologous to the recipient individual. For example, an individual with cancer may be given a 20-mg dose of the drug for later use, for example, if the cancer goes out of remission. In other cases, the patient may have ADR-expressing T cells deposited in a repository. The cells are contained in the repository for treating autoimmune disorders.

[0081] B. Use in Autoimmune Disorders Unwanted activation of endogenous autoreactive T cells in an individual can lead to diseases such as diabetes mellitus, autoimmune This can lead to devastating autoimmune diseases for the individual, such as chronic colitis and multiple sclerosis. In certain embodiments, autoimmune diseases are prevented by inhibiting endogenous autoreactive T cells in an individual. ADR-expressing immune cells in an individual that affect autoimmune disorders (or their potential development) The use prevents or treats one or more autoimmune disorders. In the present invention, the use of such ADR-expressing cells can be used to generate dormant, non-pathogenic Thus, certain methods of the present disclosure preserve naive and memory T cells. The method utilizes specific cells that have been modified to express ADR, and the specific cells are pathogenic T cells. sufficient to target activated T cells, including The amount is provided to the individual.

[0082] In vivo activation of T cells with unwanted specificities may contribute to pathogenesis and may be a cause of specific In some embodiments, cells expressing one or more ADRs are targeted to activated T cells. In some cases, ADR-expressing cells are a subset of activated T cells that produce pathogenic cells. To target.

[0083] In certain cases, debilitating, life-threatening conditions driven by activated T cells (e.g., organ dysfunction) can occur. Rejection, Graft-versus-host disease, Type I diabetes, Multiple sclerosis, Autoimmune colitis, Lupus, Joint To prevent or reverse diseases such as rheumatoid arthritis using adoptive T cell transfer of ADR-expressing T cells To achieve this, ADR-expressing T cells can be used.

[0084] In some cases, the ADR-expressing T cells are used to treat one or more autoimmune disorders or It also contains one or more components other than ADRs that promote prevention.

[0085] In some cases, the individual receiving the ADR-expressing T cells will have one or more autologous immune responses. One or more additional therapies are administered to prevent or treat the epidemic. Immunosuppressants such as glucocorticoids, cytostatics, antibodies and / or inhibitors Drugs that act on munofilin may or may not be given. Alternatively, the individual may be given one or more suitable vaccines.

[0086] In some cases, an individual is at risk for an autoimmune disorder and is at increased risk of developing an autoimmune disorder. to prevent or delay the onset and / or reduce, for example, the severity and / or An effective amount of ADR-expressing cells is provided to relieve one or more symptoms, such as duration of pain, Individuals at risk for an autoimmune disorder include, for example, those with a personal or family history of an autoimmune disorder. , women of certain ethnicities, etc. An individual may have an autoimmune disorder, In some cases, preventing or reducing the severity and / or duration of another autoimmune disorder. or hoping to delay its onset.

[0087] Examples of autoimmune disorders that may be prevented or treated with ADR-expressing cells include at least the following: Examples include: achalasia; Addison's disease; adult Still's disease; agammaglobulinemia; alopecia. Alopecia; amyloidosis; ankylosing spondylitis; anti-GBM / anti-TBM nephritis; antiphospholipid syndrome ;Autoimmune angioedema;Autoimmune autonomic neuropathy;Autoimmune encephalomyelitis;Autoimmune hepatopathy inflammation; autoimmune inner ear disease (AIED); autoimmune myocardium autoimmune oophoritis; autoimmune orchitis; autoimmune pancreatitis; autoimmune retinopathy; autoimmune Urticaria; Axonal and Neuronal Neuropathy :AMAN);Barro's disease;Behcet's disease;Benign mucous membrane pemphigoid;Bullous pemphigoid;Ca Castleman disease (CD); Celiac disease; Chagas disease; Chronic inflammatory proliferative disorders Chronic inflammatory demyelinating polyneuropathy (CI) DP); chronic recurrent multifocal osteomyelitis (CR) MO); Churg-Strauss Syndrome (CSS) or Eosinophilic Eosinophilic granulomatosis (EGPA); cicatricial pemphigoid; Cogan disease syndrome;cold agglutinin disease;congenital heart block;coxsackie myocarditis;crest syndrome;clo Dermatitis herpetiformis; Dermatomyositis; Devic's disease (neuromyelitis optica); Discoid lupus; Dress Larsen syndrome; endometriosis; eosinophilic esophagitis (EoE); eosinophilic Bulbar fasciitis; erythema nodosum; essential mixed cryoglobulinemia; Evans syndrome; fibromyalgia fibrosing alveolitis;giant cell arteritis (temporal arteritis);giant cell myocarditis;glomerulonephritis; Adpasture's syndrome; granulomatosis with polyangiitis; Graves' disease; Guillain-Barre syndrome; Hashimoto's thyroiditis; Hemolytic anemia; Henoch-Schonlein purpura ra:HSP); herpes gestationis or pemphigoid gestationis (PG); Hidradenitis Suppurativa (HS) (inverse acne); hypogammaglobulinemia IgA nephropathy; IgG4-related sclerosing disease; immune thrombocytopenic purpura cytopenic purpura (ITP); inclusion body myositis (IBM); interstitial Interstitial cystitis (IC); juvenile arthritis; juvenile diabetes (type 1 diabetes) );Juvenile myositis (JM);Kawasaki disease;Lambert-Eaton syndrome; Leukocytoclastic vasculitis; lichen planus; lichen sclerosus; ligneous conjunctivitis; linear IgA disease disease:LAD); Lupus; Chronic Lyme disease; Meniere's disease; Microscopic polyangiitis (Mi croscopic polyangiitis (MPA); Mixed connective tissue diseases e:MCTD; Mooren's ulcer; Mucha-Habermann disease; Multifocal motor neuropathy (M ultifocal motor neuropathy (MMN) or MMNCB; multiple sclerosis; myasthenia gravis ;myositis;narcolepsy;neonatal lupus;neuromyelitis optica;neutropenia;ocular cicatricial keratoid Smallpox; Optic neuritis; Palindromic rheumatism (PR); PANDAS; Tumors Paraneoplastic cerebellar degeneration (PCD); paroxysmal nocturnal syndrome Hemoglobinuria (Paroxysmal nocturnal hemoglobinuria: PNH); Parry-Lombe Luk's syndrome; Parsplanitis (peripheral uveitis); Parsonage-Turner syndrome (Pa rsonnage-Turner syndrome); pemphigus; peripheral neuropathy; perivenous encephalomyelitis; malignant Pernicious anemia (PA); POEMS syndrome; polyarteritis nodosa; polyglandularism Syndrome types I, II, and III; polymyalgia rheumatica; polymyositis; post-myocardial 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; Sjogren's syndrome Group;Sperm&testicular autoimmunity;Stiff per son syndrome (SPS); Subacute bacterial endocarditis SBE); Suzak syndrome; Sympathetic ophthalmia (SO); Takayasu arteritis ;Temporal arteritis / Giant cell arteritis;Thrombocytopenic purpura (TT) P); Tolosa-Hunt syndrome (THS) ;Transverse myelitis;Type 1 diabetes;Ulcerative colitis (UC);Anaplastic junctions Undifferentiated connective tissue disease (UCTD); uveitis; hematologic vascular inflammation; vitiligo; Vogt-Koyanagi-Harada disease; and Wegener's granulomatosis (or polyangiitis) Granulomatosis with Polyangiitis (GPA)).

[0088] (C. Use to eliminate NK cells) ADR-expressing immune cells are able to suppress NK cells in cases where it is desirable to eliminate them. As demonstrated herein, the A on specific immune cells can be used to eliminate The presence of DR is related to HLA low Involved in mediating rapid rejection of immune cells or HLA-incompatible cells This results in specific cytotoxic activity against NK cells. HLA to allow adoptive transfer of seed cells low Cells or HLA-mismatched cells In situations where it is desirable to maintain the NK cell population, the use of ADR-expressing cells can enhance NK cell activation. and avoids the rejection of HLA-incompatible cells. Thus, co-culture of ADR-expressing T cells leads to the elimination of NK cells and thus to the development of AD. Counteracts NK cell-mediated host rejection of R-expressing allogeneic T cells.

[0089] D. Use to Promote Engraftment of Allogeneic Cell / Tissue / Organ Transplants In a specific embodiment of the avoidance of activation of alloreactive T cells, the alloreactive T cells are mainly derived from the recipient. Allogeneic cells in the individual receiving the transplant, mediated by a population of reactive T cells Rejection of tissue or allogeneic organs may be avoided. Activation of alloreactive T cells in the recipient may If measures are not taken to avoid such rejection, it could lead to, for example, graft failure. Therefore, in certain embodiments, the method of transplanting cells, tissues or organs into an individual comprises: before, during and / or after transplantation of the cells, tissue or organ, Delivery of ADR-expressing cells is utilized. In some cases, the ADR-expressing cells themselves In other cases, ADRs may occur because the cells, tissues, or organs that are the subject of transplantation are not part of the transplanted tissue. The present cells are part of each cell, tissue or organ.

[0090] Tissues for transplantation include at least skin, cornea, bone, tendon, heart valve, vein, or artery. Organs for transplantation may be of any type, such as the heart, kidney, liver, lung, pancreas, intestine, etc. The thymus gland may be of any type, such as the thymus gland, ... or the thymus gland.

[0091] In certain embodiments, ADR-expressing cells are administered to an individual as a two-pronged approach: Enhance the use of allogeneic cells in the treatment of cancer: (1) inhibiting endogenous alloreactive T cells in individuals and (2) inhibit NK cell-mediated rejection in an individual. In an individual, e.g., T cells, NK cells, NK-T cells, mucosal-associated invariant T cells (mucosal associated invariant T cells:MAIT) and other cytotoxic cells. Allogeneic therapeutic cells, such as chimeric antigen receptors (CARs), transgenic TCRs, Any type of recombinant gene can be used, including those expressing engineered constructs such as Therapeutic cells derived from the three sources can have enhanced survival and activity in an individual.

[0092] (E. For the prevention or treatment of graft-versus-host disease (GvHD) during allogeneic cell / tissue / organ transplantation (Use in In another embodiment that avoids activation of alloreactive T cells, allogeneic cells, allogeneic tissue or to avoid life-threatening alloimmune responses in individuals receiving allogeneic organ transplants. Such transplants contain donor alloreactive T cells and Unless steps are taken to avoid their activation, the cells can cause diseases such as graft-versus-host disease (GVHD). Therefore, in certain embodiments, the cells, tissues or In the method of transplanting the cells, tissue or organ into an individual, the method further comprises administering the cells, tissue or organ before, during or after transplantation, respectively. and / or post-transplantation, delivery of an effective amount of ADR-expressing cells. In this study, the ADR-expressing cells themselves are part of each cell, tissue or organ to be transplanted. In other cases, ADR-expressing cells are part of an individual cell, tissue or organ, whereas in other cases, ADR-expressing cells are part of an individual cell, tissue or organ.

[0093] Tissues for transplantation include at least skin, cornea, bone, tendon, heart valve, vein, or artery. Organs for transplantation may be of any type, such as the heart, kidney, liver, lung, pancreas, intestine, etc. The thymus gland may be of any type, such as the thymus gland, ... or the thymus gland.

[0094] III. Production of ADR-Expressing Cells Cells expressing ADR molecules can be cultured using a variety of methods, all of which may be routine in the art. The method of production can be achieved by obtaining cells that are modified to express the ADR molecule. The method of production may also include generating an ADR molecule.

[0095] (Source of AT cells) Prior to expansion and genetic modification of the ADR-expressing T cells of the present disclosure, the source of T cells is targeted. Such a obtaining step may be part of the method or may be a part of the method. In some cases, obtaining the T cells to be engineered and manipulating them may involve: This can be done by an entity different from the entity that provides the ADR-expressing T cells to the individual. Peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infected sites, ascites, breast It can be obtained from a number of sources, including water, spleen tissue and tumors. In certain embodiments, many T cell lines available in the art may be used. In one embodiment, T cells are derived from a unit of blood collected from a subject using techniques known in the art. These can be obtained using a number of techniques known in the art, such as Ficoll™ separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains, for example, lymphocytes, including T cells, monocytes, granulocytes, In one embodiment, the apoptotic cell population comprises B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by erythrosis are then purified by removing the plasma fraction and storing the cells for subsequent processing steps. In one embodiment, the cells are washed to obtain a suitable buffer or medium. The cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the washing solution is calcium carbonate. It may lack calcium, may lack magnesium, or may lack many, if not all, divalent calcium. As will be readily understood by those of ordinary skill in the art, However, the washing steps may be carried out by methods known to those skilled in the art, for example, by a semi-automated "flow-through" centrifuge. Cell processing machines (e.g., Cobe 2991 cell processing machine, Baxter CytoMate, Haemonetics Cell Saver 5) was used according to the manufacturer's instructions. After washing, the cells can be incubated with, for example, Ca 2+ Free Mg 2+ Non Contains PBS, PlasmaLyte A, or with or without buffer The apheresis may be performed in a variety of biocompatible buffers, including other saline solutions. Undesirable components of the cis sample may be removed and the cells resuspended directly in culture medium.

[0096] In another embodiment, T cells are derived from peripheral blood lymphocytes by lysing red blood cells and lysing monocytes, e.g. By centrifugation on a PERCOLL™ gradient or by counterflow centrifugal elutriation Specific subpopulations of T cells, such as CD3 + , CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO + T cells These can then be further isolated by positive or negative selection methods. This can be done.

[0097] Enrichment of T cell populations by negative selection is achieved by selectively enriching the T cell population. This can be achieved using a combination of antibodies directed against surface markers unique to the cells. One method is directed to a cell surface marker present on the cells to be negatively selected. Negative magnetic immunoadhesion or negative free antibody assay using a cocktail of monoclonal antibodies Cell sorting and / or cell selection by flow cytometry. For example, negative By selection, CD4 +To enrich for cells, a cocktail of monoclonal antibodies is used. Typically, CD14, CD20, CD11b, CD16, HLA-DR, and CD8 In certain embodiments, typically CD4 + , CD25 + , CD62 L hi , G.I.T.R. + , and FoxP3 + Enrich or positively identify regulatory T cells expressing Alternatively, in certain embodiments, it may be desirable to subselect against the anti-C25 antibody. Regulatory T cells are depleted by conjugated beads or other similar selection methods.

[0098] Regarding isolation of desired cell populations by positive or negative selection, cells The concentration and surface (e.g., particles such as beads) of the agent can vary. In an embodiment, the beads and cells are mixed together to ensure maximum contact between the cells and the beads. It would be desirable to significantly reduce the combined volume (i.e., increase the cell concentration). For example, in one embodiment, a concentration of 2 billion cells / ml is used. A concentration of 0 billion cells / ml is used. In a further embodiment, a concentration of more than 100 million cells / ml is used. In a further embodiment, 10 million, 15 million, 20 million, 25 million, 30, 35, 40, 45, or 50 million cells / ml In yet another embodiment, concentrations of 75 million, 80 million, 85 million, Cell concentrations of 10, 90, 95, or 100 million cells / ml are used. In embodiments, a concentration of 125 million or 150 million cells / ml may be used. The use of high concentrations has been shown to result in increased cell yield, cell activation and cell proliferation. In another embodiment, it may be desirable to use a lower cell concentration. By significantly diluting the mixture of T cells and the surface (e.g., particles such as beads), the particles Interaction between the cells and the nuclei is minimized.

[0099] Stimulatory T cells can also be frozen after a washing step. However, the freezing and subsequent thawing process removes granulocytes and, to a lesser extent, monocytes from the cell population. This provides a more uniform product. The cells may then be suspended in a freezing solution. Many freezing solutions and freezing parameters are known in the art. In certain embodiments, cryopreserved cells are thawed as described herein. , washed and allowed to rest at room temperature for 1 hour before activation using the method of the present invention.

[0100] The expanded cells described herein may be used in a subject for a period of time prior to the time at which they may be needed. Collecting a blood sample or apheresis product from a blood sample is also contemplated in the context of the present disclosure. In this way, a source of cells to be expanded can be harvested at any desired time point. In one embodiment, desired cells, such as T cells, can be isolated and subsequently used to treat a number of diseases for which T cell therapy would be beneficial. For use in T cell therapy of a disease or condition, such as those described herein, In one embodiment, the blood sample or apheresis can be used to determine the overall health status of the patient. In certain embodiments, blood samples or apheresis are taken from normal subjects. Obtained from generally healthy subjects who are at risk of developing disease but have not yet developed the disease and the cells of interest are isolated and frozen for later use. In certain embodiments, the T cells may be expanded, frozen, and used at a later time. A sample is collected from the patient shortly after diagnosis of the particular disease in question, but prior to any treatment. In a further embodiment, the cells are modified with a polypeptide such as, but not limited to, natalizumab, Agents such as efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants, e.g. Cyclosporine, azathioprine, methotrexate, mycophenolate and FK50 6. Antibodies or other immune ablative agents, e.g., CAMPATH, anti-CD3 antibodies, Itoxan, Fludarabine, Cyclosporine, FK506, Rapamycin, Mycophenone Many related treatments have been reported, including treatment with benzodiazepines, steroids, FR901228, and radiation. isolated from a blood sample or apheresis from a subject prior to treatment modality These drugs inhibit calcineurin, a calcium-dependent phosphatase. or (cyclosporine and FK506), which are important for growth factor-induced signaling. Inhibits 70S6 kinase (rapamycin) (Liu et al, Cell 66:807-815, 1991, He Anderson et al, Immun. 73:316-321, 1991, Bierer et al, Curr.Opin.Immun. 5:763-773 In a further embodiment, the cells are isolated for a patient and subsequently transplanted into bone marrow or other tissue. Treatment with chemotherapy, such as fludarabine, or external beam radiation therapy (EBRT) is also available. rnal-beam radiation therapy (XRT), cyclophosphamide, or OKT3 or together with antibody-based T cell ablation therapy such as CAMPATH (e.g., In another embodiment, the cells are frozen for subsequent use (at the same time, or at a later time). The B cell antibody is isolated before and then administered with an agent that reacts with CD20, such as Rituxan. The cells can be frozen for use in subsequent treatments following ionization therapy.

[0101] (Activation and proliferation of BT cells) Either before or after genetic modification of T cells to express ADRs, T cells are generally See, for example, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905, No. 680, No. 6,692,964, No. 5,858,358, No. 6,887,4 No. 66, No. 6,905,681, No. 7,144,575, No. 7,067,31 No. 8, No. 7,172,869, No. 7,232,566, No. 7,175,843 No. 5,883,223, No. 6,905,874, No. 6,797,514 , 6,867,041, and U.S. Patent Application Publication No. 20060121005. They can be activated and expanded using the methods described above. Agents that stimulate fusion-associated signals and ligands that stimulate costimulatory molecules on the T cell surface The T cells of the present disclosure are expanded by contact with a surface having attached thereto. Processes known in the art. In another example, ADRs are expressed without prior activation. The T cells can be engineered to

[0102] (C. Generation of ADR molecules) Next, the polynucleotide encoding ADR will be briefly described. The nucleic acid sequence can be expressed, for example, by recombinant methods known in the art. By screening libraries from cells or cells that contain the gene of interest By removing the gene from a vector known to be They can be obtained by direct isolation from cells and tissues using standard techniques. Alternatively, the ADR polynucleotide of interest can be isolated, rather than cloned, by It can also be produced synthetically.

[0103] In brief summary, expression of a synthetic polynucleotide encoding an ADR typically involves: A nucleic acid encoding an ADR polypeptide or a portion thereof is operably linked to a promoter. This is accomplished by incorporating the construct into an expression vector. A typical cloning vector is a transcription target. Initiator and translation terminator, initiation sequence, and regulating expression of a desired nucleic acid sequence The promoter serves to

[0104] The ADR polynucleotide can be cloned into several types of vectors. For example, but not limited to, plasmids, phagemids, phage derivatives, animal The nucleic acids can be cloned into vectors such as bacterial viruses and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and and sequencing vectors.

[0105] Additionally, the expression vector can be provided to the cell in the form of a viral vector. The chromatographic technique is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cl Oning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, and other Viruses useful as vectors are described in the virology and molecular biology manuals. The viruses include retroviruses, adenoviruses, adeno-associated viruses, and herpes viruses. In general, suitable vectors include, but are not limited to, vectors, vectors containing ... A suitable vector comprises an origin of replication, a promoter sequence, a Containing convenient restriction endonuclease sites, and one or more selectable markers ( See, for example, WO01 / 96584; WO01 / 29058; and U.S. Pat. No. 6,326,1 No. 93).

[0106] Several viral-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The gene is inserted into a vector using techniques known in the art and retroviral The recombinant virus can then be isolated and expressed as an infective virus. Delivery to cells of a subject can be either in vivo or ex vivo. Several retroviral systems are known. In some embodiments, an adenovirus vector is used. Several adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0107] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. In general, these are located in the region 30–110 bp upstream from the initiation site, but some promoter regions Motors have recently been shown to contain functional elements downstream of the initiation site. The spacing between promoter elements is often flexible, so it is possible to reverse the elements relative to each other. The promoter function is maintained even if the promoter is moved to another location. Thymidine kinase (tk) promoter In this study, increasing the spacing between promoter elements by up to 50 bp did not begin to reduce activity. Depending on the promoter, individual elements may function either in concert or independently to It appears to be able to activate transcription.

[0108] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. The promoter sequence may be any polynucleotide sequence operably linked thereto. A strong constitutive promoter sequence capable of driving high level expression of a suitable sequence. Another example of a promoter is the Elongation Growth Factor (EGF) promoter. ctor)-1 alpha (EF-1 alpha), but is not limited to , e.g., Simian Virus 40 (SV40) early promoter, Mouse Mammary Tumor Virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, M oMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus Rous immediate early promoter, Rous sarcoma virus promoter, and human gene promoter promoters, including but not limited to actin promoters, myosin promoters , the hemoglobin promoter and the creatine kinase promoter, etc. Constitutive promoter sequences may also be used. Furthermore, the present invention is not limited to the use of constitutive promoters. Inducible promoters are contemplated as part of the present invention. The use of a motor can regulate the expression of a polynucleotide sequence to which it is operably linked. If such expression is desired, turn it on, and if such expression is not desired, turn it off. An example of an inducible promoter is a molecular switch that can promoter, glucocorticoid promoter, progesterone promoter, and Examples of promoters include, but are not limited to, the tetracycline promoter and the ribosome promoter.

[0109] To assess the expression of the ADR polypeptide or a portion thereof, an expression vector is introduced into the cell. The vector is a cell that is to be transfected or infected with a viral vector. To facilitate identification and selection of expressing cells from a population of In another embodiment, the selectable gene may also contain a reporter gene or both. The marker is placed on a separate DNA fragment and used in the co-transfection procedure. Both selectable markers and reporter genes may be used to allow expression in the host cell. Useful selectable markers can be, for example, neo and other antibiotic resistance genes.

[0110] Reporter genes are used to identify potentially transfected cells and to Reporter genes are generally used to evaluate the functionality of a reporter sequence. not present in the recipient organism or recipient tissue or It is not expressed by the tissue and is not expressed by any easily detectable property, e.g., enzymatic activity. A reporter gene is a gene that encodes a polypeptide whose expression can be elicited by a reporter gene. Expression of the gene is assayed at an appropriate time after introducing the DNA into the recipient cells. Suitable reporter genes include luciferase, beta-galactosidase, chloramphenicol, and tRNA. Muphenicol acetyltransferase or secretory alkaline phosphatase Examples of genes that encode the Ui-Tei gene include the green fluorescent protein gene. (Frank et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques. These are either prepared by hand or commercially available. Generally, they show the highest levels of reporter gene expression. The construct with the smallest 5' flanking region that satisfies the above criteria is identified as a promoter. The promoter region is linked to a reporter gene and regulates promoter-driven transcription. The assay can be used to evaluate agents for their ability to

[0111] Methods for introducing ADR polynucleotides into cells and expressing them therein are known in the art. In the case of an expression vector, the vector can be transformed into a vector by any method known in the art. and can be easily introduced into a host cell, such as a mammalian cell, a bacterial cell, a yeast cell or an insect cell. For example, an expression vector can be introduced into the host cell by physical, chemical, or biological means. The vector can be introduced into a host cell via the vector.

[0112] Physical methods for introducing ADR polynucleotides into host cells include calcium phosphate transferase. Cium precipitation, lipofection, particle bombardment, microinjection ion, electroporation, etc. Vectors and / or exogenous nucleic acids Methods for producing cells containing the . al.(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,Ne See, e.g., John Wiley & Sons, Inc., New York. One method for introducing polynucleotides into a host cell is by recombinant recombinant DNA technology. Calcium phosphate transfection.

[0113] As a biological method for introducing an ADR polynucleotide of interest into a host cell Vectors include DNA vectors and RNA vectors. Viral vectors, particularly retroviral vectors, Roviral vectors are the most popular vectors for inserting genes into mammalian cells, e.g., human cells. It has become a widely used method. Other viral vectors include lentiviruses, poxviruses, and Viruses, herpes simplex virus I, adenovirus and adeno-associated virus, etc. See, for example, U.S. Patents 5,350,674 and 5,585,3 See issue 62.

[0114] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems. , e.g., macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems used as delivery vehicles in vitro and in vivo are liposomes. and artificial membrane vesicles.

[0115] In instances where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. Lipid formulations for introducing nucleic acids into cells (in vitro, ex vivo or in vivo) In another embodiment, the nucleic acid may be associated with a lipid. The acid is either encapsulated in the aqueous interior of the liposome or dispersed within the lipid bilayer of the liposome. Alternatively, the liposome may be bound to the oligonucleotide by a linking molecule that is associated with both the liposome and the oligonucleotide. The antibodies were either attached to the membrane, entrapped in liposomes, or complexed with liposomes. They may be composed of lipids, dispersed in a solution containing lipids, mixed with lipids, or They may be combined, suspended in lipids, or contained with micelles. may be in a lipid-rich solution, complexed to a micelle, or otherwise associated with a lipid. The lipid, lipid / DNA or lipid / expression vector associated compositions may be in solution or in any particular They are not limited by structure either. For example, they may exist as micelles or as "collapsed ( They may exist in bilayer structures with a "collapsed" structure. They may simply be scattered in the solution. In some cases, the lipids may form aggregates that are not uniform in size or shape. is a fatty substance, which may be a natural lipid or a synthetic lipid. The lipid droplets naturally occurring in the cytoplasm, as well as long-chain aliphatic hydrocarbons and their derivatives, Bodies containing, for example, fatty acids, alcohols, amines, aminoalcohols and aldehydes Compound classes are included.

[0116] Suitable lipids for use are available from commercial sources. For example, dimyristyl Phosphatidylcholine ("DMPC") was purchased from Sigma (St. Louis, MO). Dicetyl phosphate ("DCP") is available from K&K Laboratories. ries (Plainview, NY), and cholesterol ( "Choi" is available from Calbiochem-Behring, and Jimmy Lysylphosphatidylglycerol ("DMPG") and other lipids are available from Avanti Available from Polar Lipids, Inc. (Birmingham, Alabama). Stock solutions of lipids in chloroform or chloroform / methanol are stored at approximately -20°C. Since chloroform evaporates more easily than methanol, it is possible to Liposomes are the formation of closed lipid bilayers or lipid aggregates. Total lipid vesicles including a variety of single and multilamellar lipid vehicles formed by Liposomes are small vesicular structures consisting of a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes are composed of multiple vesicles 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, resulting in the formation of water molecules between the lipid bilayers. and dissolved solutes (Ghosh et al., 1991 Glycobiology 5:505-10). Compositions having a dissolved structure that differs from the normal vesicular structure are also included. For example, lipids may be They can exist in micellar structures or simply as non-uniform aggregates of lipid molecules. Min-nucleic acid complexes are also envisaged.

[0117] In some instances, the ADR molecule may be incorporated into the endogenous nucleic acid of a cell. If integration at a specific locus is desired, a target site for homologous recombination is used. For example, materials and methods known in the art for homologous recombination may be used. Use it to knock out the endogenous gene and then insert it (at the same locus or elsewhere) (if applicable), can be replaced by the gene that the construct encodes. Either the OMEGA vector or the O-vector may be used. CRISPR / Cas9, zinc finger nuclease, TALE nuclease, meganuclease, and other site-specific nucleases may be used.

[0118] Exemplary T cells that have been engineered to contain an ADR expression construct are selected The cells are then grown in culture under physiological conditions and selected for carrying the construct. and then performing a polymerase chain reaction to determine the presence of the construct in the host cell, for example. The engineered host cell may be identified and further analyzed using techniques such as enzyme chain reaction. They can then be used as planned, e.g., grown in culture and incubated in a host It can be introduced into an organism.

[0119] Depending on the nature of the cells, the cells can be introduced into a host organism, e.g., a mammal, in a variety of ways. In a specific aspect, the cells may be introduced into a tumor site, but in an alternative embodiment, the cells may be The cells are modified to home to the host or to home to the infected tissue. The number of cells transferred will vary depending on several circumstances, the purpose of the transfer, the lifespan of the cells, the protocol used, and This will depend, for example, on the number of doses, the doubling ability of the cells, and the stability of the recombinant construct. The cells can be applied as a dispersion and typically injected at or near the site of interest. The cells may be present in a physiologically acceptable medium.

[0120] It is not necessary in all cases for the introduction of DNA to result in integration. In some cases, transient maintenance of the introduced DNA may be sufficient. and after a predetermined time after the cells are introduced into the host, e.g., the cells are After being able to home to a fixed site, the cells could be turned on. EXAMPLES

[0121] The following examples are presented to more particularly illustrate certain embodiments of the present disclosure, where: However, they should in no way be construed as limiting the scope of the broad present disclosure.

[0122] Example 1: Self / Alloimmune Defense Receptors for Selective Targeting of Pathogenic T Cells Herein, we present a method to detect autologous / alloimmune defense receptors (ADRs) expressed on normal T cells. A novel approach is disclosed for specifically targeting pathogenic T cells through ADR expression. T cells find and eliminate only activated T cells, and not the resting T cells that make up the majority of circulating lymphocytes. Nonpathogenic naive and memory T cells are spared.

[0123] The concept of ADR-mediated targeting is that T cells target their Transiently express the costimulatory genes 4-1BB, OX40, and / or CD40L on the cell surface Based on the findings that 4-1BB, OX40 and / or Expression of CD40L or CD40L is maintained only if the T cell is actively cytotoxic, and TCR Once signaling is stopped, it is gradually downregulated within 4-5 days. In particular, activated CD8 +T cells showed more intense 4-1BB expression, whereas CD4 + T cells preferentially expressed OX40 and / or CD40L. In addition to activated T cells, ADR ligands activate NK cells and several other non-essential recruitable cell subsets. This expression pattern of 4-1BB, OX40 and CD40L is consistent with These genes can be expressed in a variety of ways, while avoiding permanent damage to important immune and non-immune tissues. This makes activated cells attractive targets for targeting with high specificity.

[0124] To explore the feasibility of targeting these activated T cells, The 4-terminally linked CD3 ζ chain was directly linked via a spacer to the CD3 ζ chain encoded in the vector SFG. -1BB-specific ligand, OX40-specific ligand or CD40L-specific receptor The autologous / alloimmune defense receptor (ADR) was designed to be capable of binding to the endothelial cell. ) Incorporating type II proteins 4-1BBL and OX40L into the type I backbone of ADR and b) to detect ADR on the cell surface by FACS staining. To facilitate this, we incorporated the construct into T cells. These ADR T cells efficiently suppressed ADR expression on the surface of the tumour cells. 0 and CD40L expressing cells, and has potent and robust cytotoxicity against 9 of the target cells. 0-99% of the 4-1BB and OX40 antibodies were eliminated within 48 hours. and demonstrate the feasibility of generating CD40L-specific ADR T cells.

[0125] ADR signaling in T cells results in the upregulation of 4-1BB, OX40 and CD40 Upregulation of L promotes sibling killing and effector cell proliferation CRISPR / Cas9 of ADR target genes in effector T cells prevents We investigated the effect of genome disruption. We previously demonstrated that primary human T cells expressing a D7-specific CAR can prevent sibling killing. In this regard, the present inventors have used CRISPR / Cas9 to 1BB expression can be knocked out in approximately 70% of ADR T cells, which Consistently, 4-1BB was inhibited without affecting cytotoxicity. + 48 After co-culture with target cells At that time point, ADR T cell proliferation increased more than two-fold.

[0126] Next, we tested the ability of ADR T cells to selectively eliminate activated T cells. , OX40, and CD40L ADR T cells were cultured using fluorescently labeled resting T cells or CD 3 / CD28 activated T cells were co-cultured with remaining live CD4 + and CD8 + T cells, cells Quantification was performed by flow cytometry using counting beads. 4-1BB, OX40 or CD After 72 h of co-culture with T cells expressing 40L-specific ADR, the effect of autologous T cells on resting T cells was In contrast, 4-1BB ADR T cells and CD3 / CD 28 In co-culture with activated T cells, most CD8 + T cells and some CD4 + T cells and The cells were eliminated within 48 hours. Incubation with OX40 ADR T cells inhibited the activation of CD4 + Reciprocally high levels of T cell depletion and activated CD8 + Modest depletion of T cells and CD40L ADR T cells were activated by CD4 + Moderate for T cells Although it produced a cytotoxic effect, CD8 + No effect was observed on activated CD4+ T cells. 4 + T cells and CD8 + OX40, CD40L and 4-1BB AD on T cells Differential targeting profiles of RT cells are mediated by OX40, CD40, and OX40 expression on each T cell subset. This correlates with the observed differences in the intensity and kinetics of L and 4-1BB expression. The antibody preferentially targets either alloreactive or autoreactive T cells as appropriate. Therefore, ADR expression can be used to determine whether T cells are activated (pathogenic). This allows the specific targeting of active T cells while sparing resting cells, and their clinical This suggests that the use of

[0127] Virus-specific T cells (VSTs) expressing ADR were in vitro In a mixed lymphocyte reaction (MLR) assay, We evaluated whether CMV-specific T cells could resist rejection by HLA-A2 negative control. The control non-transduced VSTs or ADR-transduced VSTs were generated from a single donor and alloreactive HLA-A2 + The cells were mixed with PBMCs at a cell-to-cell ratio of 1:2. The cells were cultured for 12 days. At the end of the coculture, control VSTs were + PBMC was almost completely eliminated by 4-1BB ADR or OX40 VSTs expressing either ADR were resistant to rejection. Taken together, these results By targeting activated T cells using a newly developed ADR platform embodiment, The feasibility and selectivity of

[0128] Example 2: Self / Alloimmune Defense Receptors for Selective Targeting of NK Cells ADR is HLA low Key cells mediating rapid rejection of erythrocytes or HLA-mismatched cells It exhibits specific cytotoxic activity against the NK cell population.

[0129] NK cells act as a part of antitumor and antiviral immune surveillance to protect against HLA-mismatched or Therefore, immune sufficiency (IMS) is a condition in which the immune system is unable to recognize cells with low or no HLA expression. Adoptive transfer of allogeneic cells into patients with plete NK cells activates NK cells and rejects HLA-incompatible cells. Here, we investigated the role of 4-1BB and OX40-specific auto / alloimmune defense receptors (A Co-culture of T cells expressing ADR leads to the elimination of NK cells and thus to the induction of ADR. These results suggest that the NK cell-mediated host rejection of allogeneic T cells may be counteracted. , ADR not only inhibits alloreactive T cell responses but also suppresses NK cell-mediated rejection, Further support for the application of ADRs to enhance the survival and activity of 'off-the-shelf' therapeutic T cells can be done.

[0130] [Example 3] ADR-expressing T cells eliminate target cells. ADRs are expressed on the cell surface of immune cells and mediate cytotoxicity against their respective targets. FIG. 1A illustrates an example of a schematic diagram of ADR (labels such as GFP can be used as needed). The expression of ADR on the surface of T cells was confirmed (Figure 1B). The cells proliferated in a manner comparable to that of the control. (Fig. 1C). (Fig. 1D) ADR-expressing T cells express the corresponding ADR ligand. The 4-1BB ADR-expressing mAb was cytotoxic to target cells (Figure 1D). Proliferation of wild-type and 4-1BB KO T cells and their expression against 4-1BB+ targets The cytotoxicity of these is shown in comparison. By combining ADR and its ligases, proliferation and cytotoxicity can be further enhanced. Coexpression of the agonist and agonist on T cells is not required for ADR-T cell proliferation or function. (Figure 1E).

[0131] Selective expression of ADR ligands on activated T cells may mediate their selection by ADR T cells. Expression of ADR ligands in resting and activated T cells after TCR stimulation The expression of resting CD4+ T cells and resting CD8+ There was no cytotoxicity of ADR T cells against T cells, but ADR after 48 h of co-culture There was clearance of activated CD4+ T cells and activated CD8+ T cells by T cells (Figure 2 E).

[0132] As an example, expression of the 4-1BB ADR inhibits T cells from immune rejection in the MLR model. After co-culture with allogeneic PBMCs at an ADR T:PBMC ratio of 1:10, Representative dot plots showing that TCRKO T cells co-expressing β-actin are protected from rejection. (Figure 3A). The absolute numbers of donor T cells and allogeneic T cells in PBMCs during coculture (Figure 3 B–C) is also seen in virus-specific ADR T cells (Figures 3D–F).

[0133] Expression of ADR was determined by in vitro mixed lymphocyte reaction in which virus-specific allogeneic T cells ADR VST protects cells from immune rejection by recipient allogeneic PBMCs. Representative dot plots (Figure 4A) showing protection from IL-1 at various time points during the MLR. The absolute numbers of recipient T cells and donor VSTs in the 10-well plate are shown in Figures 4B and 4C. do.

[0134] In Figure 5, ADR VST retains antiviral function. The ADR VST was co-cultured with monocytes pulsed with Iruspepmix. Monocyte counts showed that the virus-infected cells were effectively eliminated.

[0135] Activated NK cells upregulate ADR ligands and are selected by ADR T cells. 4-1BB expression has been confirmed in resting and activated NK cells. Resting N cells after 24 h of co-culture with 4-1BB ADR T cells The remaining numbers of K cells and activated NK cells are determined (Figure 6C). ADR T cells lacking HC suppress the proliferation of NK cells and thus inhibit the proliferation of allogeneic PBMCs. Figure 6E shows that the donor T cells and allogeneic NK cells in co-culture are protected from immune rejection by the donor T cells. The logarithm is determined. Upon 48 h co-culture at an E:T ratio of 1:1, ADR lacking MHC T cells resist immune rejection by NK cells (Figure 6F). ADR T cells are expressed in PBMCs. During MLR with IL-1, it controls the proliferation of alloreactive NK cells (Figure 6G). The absolute numbers are plotted.

[0136] ADR expression protects allogeneic T cells from immune rejection in vivo. T cells from 2+ donors were given to mice after sublethal irradiation, followed 4 days later by allogeneic H An example of a mouse model of immune rejection in which LA-A2-T cells were administered is shown. HL Control T cells from A-A2- donors were rejected by day 18, whereas ADR-expressing cells were preserved. The results are shown in Fig. 7B. The absolute number of T cells from the donor was determined (Figure 7C). Dell uses whole PBMCs from donor 1 (both T and NK cells) instead of allogeneic T cells. A representative flow plot in Figure 7E shows the ADR T cell The cells protect mice from immune rejection and from the rapid development of lethal GvHD. This shows that.

[0137] Coexpression of CAR and ADR maintains the function of both receptors. Figure 1. Illustrated example of immune cells co-expressing CAR and ADR. Co-expression of CAR and ADR on the cell surface was confirmed. Figure 8C shows the results of the analysis of NALM-6 (CD19+CAR) as an example of a target. The cytotoxicity of CAR-ADR T cells against activated CAR-ADR T cells is shown in Figure 8D. The cytotoxicity of CAR-ADR T cells against T cells (ADR targets) was also determined. In E, CAR-ADR T cells against both targets when simultaneously co-cultured with both cellular targets. Cytotoxic activity of has been demonstrated.

[0138] CAR-ADR T cells are protected from immune rejection and exert potent antitumor activity. An example of a transplant model and regimen is illustrated in Figure 9A. Mice were then given allogeneic T cells from these mice and b2mKO NALM6 with a 24-hour interval, followed by dosing. A single dose of CAR-ADR T cells from donor 2 was administered. T cells from donor 2 in peripheral blood The kinetics of the cells is shown in Figure 9B, and the kinetics of T cells from donor 1 in the experimental group is shown in Figure 9C. Figure 9D shows the leukemia burden in the mice, and the overall survival rate of the mice was also determined (Figure 9C). 9E).

[0139] Figure 13A-C. CAR-ADR T cells prevent immune rejection in solid tumor models. The mouse model and treatment are outlined in Fig. 10A. Here, allogeneic T cells from donor 1 and the b2mKO neuroblastoma cell line, CHLA Mice were then given 255 mg of CAR-ADR T cells from donor 2, separated by 24 hours. Donor 2's GD2 CAR T cells were rejected by D18, but C AR-ADR T cells resisted allogeneic rejection and persisted in the peripheral blood (Fig. 10B). The tumor burden in is shown in FIG. * in the ATC+GD2 CAR T group Shown is heterologous GvHD-associated death.

[0140] TCR knockout CAR-ADR T cells are protected from immune rejection and have potent antitumor activity Allogeneic T cells from donor 1 and b2mKO NALM6 were incubated for 24 hours. Mice were then given a single dose of TCR-edited CAR-ADR T cells from donor 2. A schematic of the mouse model is shown in Fig. 11A. T cells from donor 2 in peripheral blood The dynamics of donor 1 T cells in the experimental group are shown (Figure 11C). The leukemia burden in mice (Figure 11D) and overall survival rate of mice (Figure 11E) are shown. do.

[0141] ADR T cells protect mice from lethal xenogeneic GvHD. A schematic of the model is shown in Figure 12A. and demonstrate proliferation of FFLuc-labeled ADR T cells in vivo (FIG. 12B). The kinetics of weight gain and loss in mice was determined (FIG. 12C). Overall survival rate of mice is illustrated. (Figure 12D).

[0142] As an example, a second-generation ADR ("AD An example of the structure of ADR.28zeta is shown in Figure 1. 3A). The in vitro cytotoxicity of ADR.28zeta against target-expressing cells was determined. (Figure 13B and 13C). ADR.28zeta inhibited mice from heterologous GvHD lines. The FFLuc targeting in vivo was performed by protecting the target from the GFP (Figure 13B). The proliferation of ADR.28 zeta T cells was confirmed (Figure 13E). (FIG. 13F) and overall survival of the mice (FIG. 13G) were determined.

[0143] The present disclosure and its advantages have been described in detail, but are defined by the appended claims. Various changes, substitutions and alterations are permitted herein without departing from the spirit and scope of the design. It should be understood that the scope of the present application is limited to the It is to be understood that the present invention is limited to particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described herein. It is not intended to be a substitute for the above-mentioned. It will be readily understood by those of ordinary skill in the art. As such, they perform substantially the same functions as the corresponding embodiments described herein or substantially the same functions as the corresponding embodiments described herein. Any existing or hereafter developed process, machine, manufacture, composition of matter or means which accomplishes the same result; Any method or process may be utilized in accordance with the present disclosure. includes within its scope such processes, machines, manufacture, compositions of matter, means, methods, or steps. This includes:

Claims

1. An isolated polynucleotide comprising a sequence encoding a polypeptide, Petite is (1) OX40-specific ligand, 4-1BB-specific ligand, CD40L-specific ligand or one or more of their functional derivatives (2) Signaling domain that promotes T cell activation An isolated polynucleotide comprising the nucleic acid sequence operably linked to

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

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

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

5. The OX40-specific ligand is OX40L, an antibody targeting OX40, OX40L -Fc fusion, or a combination thereof. Do.

6. The 4-1BB specific ligand is 4-1BBL, an antibody targeting 4-1BB, 4- 1BBL-Fc fusion, or a combination thereof. A polynucleotide according to any one of claims 1 to 4.

7. The CD40L-specific ligand is an antibody that targets CD40, CD40L, or CD40 -Fc fusion, or any other engineered antibody capable of specific binding to CD40L. The polynucleotide according to any one of claims 1 and 4, which is a protein. 。

8. The polypeptide further comprises one, two or more costimulatory domains. A polynucleotide according to any one of 1 to 7.

9. The polynucleotide further comprises a sequence encoding a spacer between (1) and (2). The polynucleotide according to any one of claims 1 to 8.

10. The polynucleotide of claim 9, wherein the spacer is 10 to 220 amino acids in length. 。

11. The polypeptide of claim 10, wherein the spacer has a sequence that facilitates surface detection by an antibody. Ligands.

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

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

14. The polynucleotide further encodes a chimeric antigen receptor, a T cell receptor, or both. The polynucleotide according to any one of claims 1 to 13.

15. A polynucleotide encoding the polypeptide of claim 1 and a chimeric antigen receptor. , a 2A element or an IRE on the polynucleotide encoding the T cell receptor, or both The polynucleotide of claim 14, comprising an S element.

16. 1 , wherein the chimeric antigen receptor comprises one, two or more costimulatory domains.

6. The polynucleotide according to claim 5.

17. The method according to any one of claims 1 to 16, wherein the polynucleotide is present on a vector. A polynucleotide of.

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

19. The viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, The polynucleotide of claim 18, which is a viral vector or an adeno-associated viral vector. Cleotide.

20. The polynucleotide according to any one of claims 1 to 19, which is present in a cell.

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

22. 22. The polynucleotide of claim 20 or claim 21, wherein the cell is an immune cell.

23. The polynucleotide according to any one of claims 20 to 22, wherein the cell is engineered. Punch.

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

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

26. 2. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises one, two or more costimulatory domains.

6. The polynucleotide according to claim 5.

27. 2. The method of claim 1, wherein the T cells comprise one or more engineered T cell receptors (TCRs).

5. The polynucleotide according to claim 4.

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

29. (1) one or more of OX40-specific ligand, 4-1BB-specific ligand, and CD40 Above, (2) Signaling domain that promotes T cell activation A polypeptide comprising:

30. The signaling domain that promotes T cell activation is a CD3 zeta subunit, D 30. The polypeptide of claim 29, derived from AP12, an Fc receptor, or a combination thereof. Chid.

31. The polypeptide further comprises one, two or more costimulatory domains. 29 or 30. A polypeptide according to claim 30.

32. A polynucleotide according to any one of claims 1 to 27 or any one of claims 29 to 31. A chimeric receptor expressing cell comprising the polypeptide described in any one of claims 1 to 4.

33. The cell of claim 32, which is an immune cell.

34. 34. The cell of claim 32 or claim 33, which is engineered.

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

36. 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. 38. The method of claim 32, which is engineered to lack endogenous expression of one or more genes. A cell described in any one of claims 1 to 4.

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

40. CRISPR / Cas9, zinc finger nucleases, TALE nucleases, etc. or meganuclease engineered according to claim 38 or 39. cells.

41. The cell according to any one of claims 32 to 40, contained in a cell repository.

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

43. 43. The method of claim 42, further comprising depositing the cells in a cell repository.

44. Expressing one or more chimeric antigen receptors and / or one or more recombinant T cell receptors 44. The method of claim 42 or claim 43, further comprising modifying the cell so that 。

45. 43. The method of claim 42, further comprising providing an effective amount of said cells to an individual in need thereof.

45. The method according to any one of claims 1 to 44.

46. 46. ​​The method of claim 45, wherein the cells are allogeneic with respect to the individual.

47. A method of treating an individual with allogeneic therapeutic cells for a medical condition, comprising administering to said individual a therapeutically effective amount of allogeneic therapeutic cells. providing cells to the individual, the cells comprising (1) an OX40-specific ligand, 4-1 BB-specific ligand, CD40L-specific ligand, or functional derivatives thereof (2) one or more of the T cell receptors are operably linked to a signaling domain that promotes T cell activation. A method for expressing a polypeptide comprising:

48. 48. The method of claim 47, wherein the cells are obtained from a cell repository.

49. The cells include one or more chimeric antigen receptors and / or one or more recombinant T cell receptors The method of claim 47 or claim 48, wherein the gene expresses

50. 1. A method for avoiding rejection of allogeneic cells, tissues or organs in an individual, comprising: An extracellular domain that targets a compound selectively present on activated T cells, in an effective amount for an individual. and expressing an engineered chimeric receptor comprising CD3 zeta. wherein said delivering step results in said individual: (1) inhibition of endogenous alloreactive T cells in the individual; and / or (2) suppression of NK cell activation in said individual.

51. 51. The method of claim 50, wherein the allogeneic cell is an allogeneic immune cell expressing a chimeric receptor. 。

52. the allogeneic cells express a chimeric antigen receptor or an engineered T cell receptor; 52. The method of claim 50 or claim 51.

53. The allogeneic immune cells are administered prior to, during and after tissue and / or organ transplantation in the individual.

51. The method of claim 50, wherein the method is delivered to the individual and / or at a later time.

54. The method according to any one of claims 50 to 53, wherein the activated T cells are pathogenic T cells. Law.

55. 1. A method of selectively targeting activated T cells in an individual, comprising administering to the individual an effective amount of The method includes providing an immune cell that expresses an engineered chimeric receptor, Condition (1) an extracellular domain that targets a compound that is selectively present on activated T cells; and (2) Signaling domain that promotes T cell activation The method includes:

56. The signaling domain that promotes T cell activation is a CD3 zeta subunit, D 56. The method of claim 55, which is derived from AP12, an Fc receptor, or a combination thereof.

57. 57. The method of claim 55 or claim 56, wherein the activated T cells are pathogenic T cells.

58. 1. A method for preventing or treating a medical condition associated with activated T cells in an individual, comprising: an effective amount of an engineered chimera that selectively targets the activated T cells to the individual; 2. A method comprising the step of delivering an immune cell expressing a 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.

59. The chimeric receptor further comprises one, two or more costimulatory domains.

58. The method according to claim 58.

60. 60. The method of claim 58 or claim 59, wherein the medical condition is an autoimmune disorder.

61. The medical condition is selected from the group consisting of graft rejection, graft-versus-host disease, type I diabetes, multiple sclerosis, autoimmune diseases, and the like. The method according to any one of claims 58 to 60, comprising administering to the patient a therapeutically effective amount of any of the following: Law.

62. To avoid NK cell-mediated host rejection of allogeneic T cells, allogeneic tissue or allogeneic organ in an individual administering to said individual an effective amount of a compound that is selectively present on activated T cells. The method further comprises the step of: providing a method for treating a T cell comprising administering to said patient an amount of at least one of said antibodies a T cell activation inhibitor, said method ... The method comprises the step of providing an immune cell that expresses an engineered chimeric receptor.

63. 2. The method of claim 1, wherein the immune cells expressing the engineered chimeric receptor are allogeneic T cells.

62. The method according to claim 62.

64. the immune cells express a chimeric antigen receptor or an engineered T cell receptor; 64. The method of claim 62 or claim 63.

65. The amount of immune cells expressing the engineered chimeric receptor provided to the individual is 1 ml ２ 10 per ２ ~10 １２ The method according to any one of claims 62 to 64, Law.

66. The cells expressing the chimeric receptor are provided to the individual systemically or locally. The method according to any one of claims 47 to 65.

67. The method of any one of claims 47 to 66, wherein the immune cell is a T cell.

68. 67. The immune cells are delivered to the individual once or more than once.

2. The method according to claim 1 .

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