Genetically Modified Anti-Third-Party Central Memory T Cells and Their Use in Immunotherapy

Abstract

The present invention relates to genetically modified anti-third-party central memory T cells and their use in immunotherapy. An isolated cell having a central memory T lymphocyte (Tcm) phenotype is disclosed, the cell being a tolerance-inducing cell and capable of homing to lymph nodes after transplantation, and the cell being transduced to express a cell surface receptor comprising a T cell receptor signaling module. Methods for generating and using the cells are also disclosed.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680053580.8, "Genetically Modified Anti-Third-Party Central Memory T Cells and Their Use in Immunotherapy", with a filing date of July 14, 2016. Technical Field

[0002] In some embodiments thereof, the present invention relates to genetically modified tolerance-inducing central memory T lymphocytes transduced to express cell surface receptors, and more particularly (but not exclusively) to their use in immunotherapy. Background Art

[0003] Adoptive cell therapy (ACT) is a treatment procedure in which a patient is given lymphocytes (such as T cells) to treat cancer or viral infections.

[0004] This method requires the ex vivo generation of tumor- or virus-specific T cells and their infusion into the patient. To support the acceptance of T cells, the patient is generally also treated with a conditioning regimen such as a conditioning regimen (e.g., irradiation or chemotherapy) and / or administration of lymphocyte growth factors (such as IL-2). Many methods have been described for generating tumor-specific lymphocytes, and two main methods are the use of genetically engineered antigen-specific T cell expansion or redirection of T cells.

[0005] According to one method, tumor-infiltrating lymphocytes (TILs) are isolated from the patient's own tumor mass (such as melanoma or renal carcinoma), expanded ex vivo, and re-infused into the patient. TILs are a promising cell source because they are a heterogeneous population of the patient's own cells that have T cell receptors (TCRs) specific for tumor-associated antigens (TAAs) present on the tumor. However, they are only applicable in cases where T cells can be isolated from the tumor mass.

[0006] This method has been promising in the treatment of metastatic melanoma.

[0007] According to another method, genetic modification is used to redirect lymphocytes against tumors by using transgenic TCR chains or chimeric receptors. Currently, target recognition depends on the single-chain variable domain (scFv) of monoclonal antibodies of chimeric antigen receptors (CARs) or T cell receptors (TCRs), and retroviral or lentiviral or electroporation transfer is generally used for the stable generation of therapeutic T cells (CAR-T cells or TCR-T cells, respectively) [Fujiwara, Pharmaceuticals (2014) 7:1049-1068].

[0008] TCR transgenic cells (TCR-T) require specific HLA molecules for the recognition of target antigens (i.e., HLA restriction) and have the ability to recognize intracellular proteins, which provides a wide range of target tumor-associated antigens or viral antigens. The therapeutic quality of TCR-T cells depends on their affinity. To generate higher affinity, several strategies have been implemented, including the use of TCRs selected from immunized human HLA transgenic mice with relevant epitopes and / or the insertion of targeted mutations in CDR regions 2 or 3 of the variable regions of the TCR α / β chains that interact with the HLA / epitope complex [Fujiwara, Pharmaceuticals (2014), see above].

[0009] Alternatively, CAR-T cells are not HLA restricted. Chimeric receptor (chimeric antigen receptor - CAR) constructs generally consist of an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain. Initial chimeric receptors (i.e., 'first generation') consisted of scFv fragments fused to the intracellular domain of the CD3ζ chain.

[0010] 'Second generation' chimeric receptors have also been generated, which add intracellular signaling domains from various co-stimulatory protein receptors (e.g., CD28, CD137, 4-1BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to the T cells. Preclinical studies have shown that'second generation' CARs improve the anti-tumor activity of T cells. Recently, 'third generation' CARs that combine multiple signaling domains, such as CD3ζ - CD28 - 4-1BB or CD3ζ - CD28 - OX40, have been generated to further enhance potency.

[0011] Tumor-specific CARs targeting multiple tumor antigens are in clinical trials for the treatment of a variety of different cancers. Examples of these cancers and their targeted antigens include follicular lymphoma (CD20 or GD2), neuroblastoma (CD171), non-Hodgkin lymphoma (CD20), lymphoma (CD19), glioblastoma (IL13Rα2), chronic lymphocytic leukemia or CLL and acute lymphocytic leukemia or ALL (both CD19). CARs that demonstrate activity against solid tumors including ovarian, prostate, breast, kidney, colon, neuroblastoma, etc. are being investigated. Virus-specific CARs have also been developed to attack cells carrying viruses such as HIV. For example, clinical trials have been initiated using a Gp100-specific CAR for the treatment of HIV (Chicaybam, ibid).

[0012] A major goal is to apply ACT, including genetically modified T cells, using fully or partially mismatched allogeneic cells rather than through bone marrow transplantation.

[0013] A variety of methods have been considered to modify T cells for adoptive cell therapy, some of which are described in Gilham et al., Human Gene Therapy (2015) 26:276-285; in Sharpe and Mount, Disease Models and Mechanisms (2015) 8:337-350 and Gouble et al. Blood (2014) 124(21) 4689.

[0014] A variety of methods have been considered for generating tolerance-inducing cells that lack graft-versus-host (GVH) activity and for their use in graft transplantation, some of which are outlined below.

[0015] Reisner and colleagues described a method for developing veto cytotoxic T lymphocytes (CTLs) that lack GVH activity, in which CTLs are stimulated against third-party stimulators in the absence of exogenous IL-2. This method is based on the observation that only activated cytotoxic T lymphocyte precursors (CTLps) can survive in primary cultures in IL-2 deprivation (IL-2 starvation leads to apoptosis of uninduced T cells). This method has been shown to deplete GVH reactivity from anti-third-party veto CTLs both in vitro and in vivo [PCT Publication No. WO 2001 / 049243, Bachar-Lustig et al., Blood. 2003; 102:1943-1950; Aviner et al., Hum Immunol. (2005) 66:644-652]. Introduction of these anti-third-party veto CTLs into recipients (along with the graft) prevents graft rejection without inducing graft-versus-host disease (GVHD) (PCT Publication No. WO 2001 / 049243).

[0016] PCT Publication No. WO 2010 / 049935 discloses an isolated cell population comprising non-GVHD-inducing anti-third-party cells having a central memory T lymphocyte (Tcm) phenotype, said cells being tolerance-inducing cells and capable of homing to lymph nodes after transplantation.

[0017] PCT Publication No. WO 2013 / 035099 discloses a new method for generating an isolated cell population comprising anti-third-party cells having a central memory T lymphocyte (Tcm) phenotype, said cells being tolerance-inducing cells and / or having disease-resistant activity and capable of homing to lymph nodes after transplantation. Summary of the Invention

[0018] One aspect of some embodiments of the present invention provides an isolated cell having a central memory T lymphocyte (Tcm) phenotype, the cell being a tolerance-inducing cell and capable of homing to lymph nodes after transplantation, and the cell being transduced to express a cell surface receptor comprising a T cell receptor signaling module.

[0019] One aspect of some embodiments of the present invention provides an isolated cell having a central memory T lymphocyte (Tcm) phenotype, the cell being a tolerance-inducing cell and capable of homing to lymph nodes after transplantation, and the cell being transduced to express a chimeric antigen receptor (CAR).

[0020] One aspect of some embodiments of the present invention provides an isolated cell having a central memory T lymphocyte (Tcm) phenotype, the cell being a tolerance-inducing cell and capable of homing to lymph nodes after transplantation, and the cell being transduced to express a chimeric antigen receptor (CAR), wherein the CAR comprises a co-stimulatory domain.

[0021] One aspect of some embodiments of the present invention provides an isolated cell having a central memory T lymphocyte (Tcm) phenotype, the cell being a tolerance-inducing cell and capable of homing to lymph nodes after transplantation, and the cell being transduced to express a chimeric antigen receptor (CAR), wherein the CAR comprises at least two co-stimulatory domains.

[0022] One aspect of some embodiments of the present invention provides a method for generating the isolated cells of some embodiments of the present invention, the method comprising transducing a cell having a central memory T lymphocyte (Tcm) phenotype, the cell being a tolerance-inducing cell and capable of homing to lymph nodes after transplantation, with a polynucleotide encoding a cell surface receptor comprising a T cell receptor signaling module or a chimeric antigen receptor (CAR).

[0023] One aspect of some embodiments of the present invention provides a cell population comprising the isolated cells of some embodiments of the present invention.

[0024] One aspect of some embodiments of the present invention provides a pharmaceutical composition comprising the cell population of some embodiments of the present invention and a pharmaceutically active carrier.

[0025] One aspect of some embodiments of the present invention provides a method for treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the cell population of some embodiments of the present invention, thereby treating the subject.

[0026] One aspect of some embodiments of the present invention provides a therapeutically effective amount of the cell population of some embodiments of the present invention for treating a disease in a subject in need thereof.

[0027] According to some embodiments of the present invention, the method is implemented ex vivo.

[0028] According to some embodiments of the present invention, cells are transduced with a vector comprising a polynucleotide.

[0029] According to some embodiments of the present invention, the polynucleotide encodes a transgenic T cell receptor (tg-TCR) or a chimeric antigen receptor (CAR).

[0030] According to some embodiments of the present invention, cells having a central memory T lymphocyte (Tcm) phenotype are against third-party cells.

[0031] According to some embodiments of the present invention, the cell surface receptor comprises a transgenic T cell receptor (tg-TCR) or a chimeric antigen receptor (CAR).

[0032] According to some embodiments of the present invention, the CAR comprises an antigen-binding domain that is an antibody or an antigen-binding fragment.

[0033] According to some embodiments of the present invention, the antigen-binding fragment is a Fab or an scFv.

[0034] According to some embodiments of the present invention, the CAR comprises CD3ζ.

[0035] According to some embodiments of the present invention, the CAR comprises at least one co-stimulatory domain selected from CD28, CD134 / OX40, CD137 / 4-1BB, Lck, ICOS, and DAP10.

[0036] According to some embodiments of the present invention, the CAR comprises at least two co-stimulatory domains selected from CD28, CD134 / OX40, CD137 / 4-1BB, Lck, ICOS, and DAP10.

[0037] According to some embodiments of the present invention, the cell surface receptor or CAR binds to an antigen selected from tumor antigens, viral antigens, bacterial antigens, fungal antigens, protozoal antigens, parasitic antigens, allergic antigens, and autoimmune antigens.

[0038] According to some embodiments of the present invention, the tumor antigen is associated with a solid tumor.

[0039] According to some embodiments of the present invention, the tumor antigen is associated with a hematological malignancy.

[0040] According to some embodiments of the present invention, the tumor antigen is selected from CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, Her2, GD2, gp100, p53, carcinoembryonic antigen (CEA), MART-1, telomerase reverse transcriptase (TERT), Caudin-6, extracellular domain of receptor tyrosine protein kinase (ErbB2-ECD), intracellular domain of receptor tyrosine protein kinase (ErbB2-ICD), histone H1.2, histone H4, tyrosinase, alpha-fetoprotein (AFP), MAGE A3, AIM-2a, AFP, ART-4, CLCA2, Cyp-B, EphA2, hTERT, iCE, FGF-5, G250, GnT-V, HST-2 (FGF-6), Livin (ML-IAP), MUC1, MUC2, PRAME, PSMA, P15, RAGE, RU1, RU2, SART-1, SART-3, SART-2, SOX10, survivin, survivin-2Bg, TRG, Neo-PAP, CAMEL and NY-ESO-1.

[0041] According to some embodiments of the present invention, the viral antigen belongs to a virus selected from the group consisting of: human immunodeficiency virus (HIV), influenza, cytomegalovirus (CMV), T-cell leukemia virus type 1 (TAX), hepatitis C virus (HCV), influenza virus, rabies virus, herpes virus, papillomavirus, hepatitis virus, varicella virus, encephalitis virus, cytomegalovirus, Ebola virus, human T-lymphotropic virus (HTLV), rubella virus, measles virus, rabies virus, lymphocytic choriomeningitis (LCM), rotavirus, mumps virus, adenovirus, adenovirus type 3 (HADV-3), adenovirus type 5 (HADV-5), adeno-associated virus 6 (AAV6), adeno-associated virus 8 (AAV8), BK polyomavirus (BKV), Candida, Epstein-Barr virus (EBV), human herpesvirus (HHV), varicella-zoster virus (VZV) and hepatitis B virus (HBV).

[0042] According to some embodiments of the present invention, the autoimmune antigen is associated with a disease selected from type 1 diabetes, multiple sclerosis, lupus, rheumatoid arthritis, Crohn's disease, celiac disease and stroke.

[0043] According to some embodiments of the present invention, the cell is further genetically modified to inhibit the expression of at least one endogenous immune checkpoint gene in the cell.

[0044] According to some embodiments of the present invention, the immune checkpoint gene is selected from the PD or CTLA genes.

[0045] In some embodiments of the present invention, cells having a central memory T lymphocyte (Tcm) phenotype (the cells being tolerance-inducing cells and capable of homing to lymph nodes after transplantation) are produced by a method comprising: (a) contacting peripheral blood mononuclear cells (PBMCs) with one or more third-party antigens in the presence of IL-21, so as to enrich antigen-reactive cells; and (b) culturing the cells generated from step (a) in the presence of IL-21, IL-15, and IL-7, so as to enable the proliferation of anti-third-party cells comprising the central memory T lymphocyte (Tcm) phenotype, thereby producing cells having the Tcm phenotype, the cells being tolerance-inducing cells and capable of homing to lymph nodes after transplantation.

[0046] In some embodiments of the present invention, the method further comprises: (c) separating the cells generated from step (b) into a single-cell suspension.

[0047] In some embodiments of the present invention, the method further comprises selecting activated cells after step (a) and before step (b).

[0048] In some embodiments of the present invention, the selection of activated cells is achieved by selecting CD137+ and / or CD25+ cells.

[0049] In some embodiments of the present invention, the Tcm phenotype comprises CD3 + , CD8 + , CD62L + , CD45RA - , CD45RO + characteristics.

[0050] In some embodiments of the present invention, at least 50% of the isolated cells are CD3+CD8+ cells, and at least 50% of the CD3+CD8+ cells have the said characteristics.

[0051] In some embodiments of the present invention, the diseases are selected from malignant diseases, viral diseases, bacterial diseases, fungal diseases, protozoal diseases, parasitic diseases, allergic diseases, and autoimmune diseases.

[0052] In some embodiments of the present invention, the malignant disease is a solid tumor or tumor metastasis.

[0053] In some embodiments of the present invention, the malignant disease is a hematological malignancy.

[0054] In some embodiments of the present invention, the hematological malignancy includes leukemia or lymphoma.

[0055] According to some embodiments of the present invention, the malignant disease is selected from leukemia, lymphoma, myeloma, melanoma, sarcoma, neuroblastoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, esophageal cancer, synovial cell carcinoma, and pancreatic cancer.

[0056] According to some embodiments of the present invention, the viral disease is selected from human immunodeficiency virus (HIV), influenza, cytomegalovirus (CMV), T-cell leukemia virus type 1 (TAX), hepatitis C virus (HCV), and hepatitis B virus (HBV).

[0057] According to some embodiments of the present invention, the autoimmune disease is selected from type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, celiac disease, and stroke.

[0058] According to some embodiments of the present invention, the cell population is non-homologous to the subject.

[0059] According to some embodiments of the present invention, the method further comprises pre-treating the subject under a sub-lethal, lethal, or supra-lethal pre-treatment regimen before administration.

[0060] According to some embodiments of the present invention, the therapeutically effective amount for use further comprises a sub-lethal, lethal, or supra-lethal pre-treatment regimen.

[0061] According to some embodiments of the present invention, the sub-lethal, lethal, or supra-lethal pre-treatment is selected from total body irradiation (TBI), regional irradiation, myeloablative pre-treatment, non-myeloablative pre-treatment, co-stimulatory blockade, chemotherapeutic agents, and antibody immunotherapy.

[0062] According to some embodiments of the present invention, the administration is achieved by a route selected from intratracheal, intrabronchial, intra-alveolar, intravenous, intraperitoneal, intranasal, subcutaneous, intramedullary, intrathecal, intraventricular, intracardiac, intramuscular, intra-serosal, intra-mucosal, transmucosal, transnasal, rectal, and enteral.

[0063] According to some embodiments of the present invention, the subject is a human subject.

[0064] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the following describes exemplary methods and / or materials. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] This invention will be described by way of example only and with reference to the accompanying drawings. Referring now specifically in detail to the drawings, it is to be emphasized that the details shown are by way of example only and for purposes of illustrative discussion of embodiments of the invention. In this regard, it will be apparent to those skilled in the art how the embodiments of the invention may be practiced in light of the description with reference to the drawings.

[0066] In the drawings:

[0067] Figures 1A - C are schematic diagrams of a model for studying the ability of Tcm cells to induce tolerance in the absence of the inductive properties of allogeneic BM. Tcm cell survival and proliferation were analyzed by FACS, and host CTL activity was tested by the 51 Cr assay for downregulation by Tcm cells.

[0068] Figures 2A - B are graphs illustrating the persistence of adoptively transferred F1 - Tcm cells in the context of syngeneic bone marrow graft (BMT). C57BL / 6 (H - 2b) mice were transplanted as outlined in Figure 1A. Using αH2D d (donor) and αH2K b , representative scatter plots of 1 mouse per group at 60 days post - transplantation (showing the percentage of Tcm cells in the peripheral whole blood of the mice) were analyzed by FACS to identify F1 (H2D d XH2K b ) - Tcm cells.

[0069] Figure 3 Is a graph to illustrate that Tcm cells specifically delete anti - donor T cells from the polyclonal host T cell (HTC) population while retaining other HTCs to exhibit cytotoxic activity. Mice were transplanted as outlined in Figure 1A. Mice were sacrificed 60 days post - transplantation, spleens and lymph nodes (LN) were harvested, and CD8 + cells (and H - 2D d was negatively selected to exclude Tcm) were selected. The C3H (H - 2 k ) or BALB / c (H - 2 d) Killing ability of the target. The bars present the following killing effects: HTC from mice receiving only BM (black bars, "only BM→C3H") or cells from mice also receiving Tcm (dark gray bars, "only BM + Tcm→C3H") kill C3H targets, or HTC from mice receiving only BM (white bars, "only BM→BALB") or T cells from mice also receiving Tcm (light gray bars, "BM + Tcm→BALB") kill BALB / c targets. Results are presented as the mean ± SD of the killing percentage in 12 wells per group. A representative experiment out of the 2 independent experiments conducted is presented. (**) represents a p-value less than 0.01, (***) represents a p-value less than 0.001.

[0070] Figure 4 Graph to illustrate the persistence of CB6 F1-derived Tcm cells in mice that received sublethal 5.5 Gy TBI and syngeneic T cell-depleted bone marrow (TDBMT). Balb / c (H2D d ) mice were irradiated sublethally (5.5 Gy) and transplanted as described in Figure 1B. Using αH2D d (host) and αH2K b , peripheral blood was analyzed by FACS at 40, 80, and 132 days post-transplantation to identify H2 db F1-Tcm cells. The scatter plot shows the percentage of CB6 Tcm cells in each mouse, with each point representing the Tcm cell population in 1 mouse belonging to the appropriate group, showing the mean and SD of each group.

[0071] Figure 5 Graph to illustrate the irradiation dose calibration that enables the survival of Tcm cells in the absence of TDBMT. Balb / c (H2D d ) mice were irradiated sublethally with 2 / 4 / 5.5 / 6.5 Gy on day -1. On day 0, the mice received adoptive transfer into the tail vein of 5 x 10 6 or 9 x 10 6 F1 CB6 (H-2 db ) Tcm cells. Using αH2D d (host) and αH2K b , a scatter plot depicting the percentage of CB6 Tcm cells in the peripheral whole blood of Balb / c host mice was generated by FACS analysis 42 days post-transplantation to identify H2 db F1-Tcm cells. The mean and SD of each group are shown.

[0072] Figures 6A-B are graphs showing the long-term persistence of fully allogeneic Tcm cells in 5.5 Gy Balb / c mice. As outlined in Figure 1C , Balb / c (H-2 d ) mice were transplanted. The Tcm cells were of CB6-F1 (H-2 db ) or C57BL / 6 (H-2 b ) origin. Mice were bled on the indicated days, and αH2D d (host) and αH2K b (donor) were used to analyze the Tcm cell population by FACS to identify H2 db F1-Tcm and H2 b Allo-Tcm cells. Figure 6A is a scatter plot depicting the percentage of Tcm cells in the Balb / c host. Each point represents the Tcm cell population in 1 mouse belonging to the appropriate group, showing the mean and SD for each group. Figure 6B is a time curve showing the long-term reduction of the Tcm cell population in peripheral blood.

[0073] Figure 7 are graphs showing the persistence of fully allogeneic Tcm cells in 5.5 Gy Balb / c mice and the promotion of additional donor T cell engraftment. Balb / c (H-2 b ) mice received 5.5 Gy TBI on day -1 and 5 x 10 6 Tcm cells of CB6 (H-2 db ) or C57BL / 6 (H-2 b ) origin on day 0. Eighty-nine days after Tcm cell injection, the mice were irradiated with 2 Gy TBI, and the next day they received 2 x 10 6 CD45.1 + , OT1 + , RAG + CD8 + cells. The scatter plot shows bleeding 120 days after Tcm cell transplantation and 30 days after OT-1 cell transplantation.

[0074] Figure 8 are graphs showing the analysis of OT-1 cells in the peripheral blood of sublethally irradiated Balb / c mice. Balb / c (H-2 d ) mice received 5.25 Gy TBI on day -1 and were then switched on day 0 to receive the CD8 + OT-1 + CD45.1 + RAG - cells of the first experiment, with or without the indicated number of CB6 (H-2db ) or C57BL / 6 (H-2 b )-derived Tcm cells. Sixty days after injection of the Tcm cells, the peripheral blood of the test mice was analyzed using FACS to detect the presence of OT-1 cells. The scatter plot shows the percentage of OT-1 cells in different groups among the total CD8 + H-2 b+ cells.

[0075] Figure 9 A graph showing the engraftment and survival of Tcm cells prepared from OT-1 mice transplanted in a reduced intensity conditioning (RIC) Balb / c mouse model. DETAILED DESCRIPTION

[0076] In some embodiments, the present invention relates to genetically modified tolerance-inducing central memory T lymphocytes transduced to express a cell surface receptor, and more particularly (but not exclusively) to their use in immunotherapy.

[0077] The principles and operations of the present invention can be better understood with reference to the accompanying drawings and the following description.

[0078] Before explaining in detail at least one embodiment of the present invention, it should be understood that the application of the present invention is not necessarily limited to the details set forth in the following description or illustrated by the examples. The present invention is capable of having other embodiments practiced or implemented in various ways. Moreover, it should be understood that the terminology and terms used herein are for the purpose of description and should not be regarded as restrictive.

[0079] Cell-based therapies using lymphocytes and antigen-presenting cells are promising methods for immunotherapy. Adoptive cell transfer (ACT), including the transfer of immunogenic cells from autologous or allogeneic sources, provides the goal of transferring immune functions and characteristics into the host. One method previously used for ACT includes genetically modified T cells (e.g., expressing a T cell receptor or a chimeric antigen receptor), where the specificity of the cells is redirected to a target antigen. However, the problems of graft rejection (due to the graft recipient) and / or graft-versus-host disease (due to the transplanted cells) are persistent problems that need to be overcome to pursue the potential of these cell therapies.

[0080] In practicing the present invention, the inventors have found that anti-third party central memory T (Tcm) cells without graft-versus-host reactivity have intrinsic veto tolerance-inducing activity and can induce tolerance on their own in the absence of hematopoietic progenitor cells. The inventors have further found that anti-third party Tcm cells can be genetically modified to express a T cell receptor (e.g., a transgenic T cell receptor or a chimeric antigen receptor) and can be used to combat diseases while inducing veto activity and having no graft-versus-host potential.

[0081] As shown below in the Examples section and subsequently herein, the inventors have shown that allogeneic donor-derived anti-third party Tcm cells can survive in a host for an extended period of time (e.g., greater than 120 days, see FIGS. 2A-B and 6A-B respectively) with or without bone marrow transplantation. In addition, anti-third party Tcm cells exert veto activity ( Figure 3 ). Thus, the application of anti-third party Tcm cells alone (i.e., in the absence of BM precursors) provides a useful tool for immunotherapy, particularly for targeting tumor antigens, pathogens (e.g., viral antigens), and autoantigens. Accordingly, these results further confirm that genetically modifying tolerogenic anti-third party Tcm cells from any cell donor to express heterologous T cell effector functions results in a universal product for immunotherapy for targeting disease antigens and avoiding graft rejection and graft-versus-host disease (GVHD).

[0082] Taken together, these cells provide a solution that has no graft-versus-host potential, graft rejection, and targets all specific antigens in a single cell. These cells do not require the use of autologous cells for treatment or the transplantation of hematopoietic cells. In addition, these cells overcome the need to manufacture cell-based therapies on a "per patient basis" and enable the manufacture of "off-the-shelf" products for therapy.

[0083] Accordingly, one aspect of the invention provides an isolated cell having a central memory T lymphocyte (Tcm) phenotype, the cell being a tolerance-inducing cell and capable of homing to lymph nodes after transplantation, the cell being transduced to express a cell surface receptor comprising a T cell receptor signaling module.

[0084] As used herein, the term "isolated cell" refers to a cell that has been isolated from its natural environment (e.g., from tissue such as from a human body).

[0085] As used herein, the phrase "central memory T lymphocyte (Tcm) phenotype" refers to a subset of T cytotoxic cells that home to lymph nodes. Cells in humans having a Tcm phenotype generally comprise the characteristics CD3+ / CD8+ / CD62L+ / CD45RO+ / CD45RA-. It should be appreciated that Tcm cells may express all of the characteristic markers on a single cell or may express only some of the characteristic markers on a single cell.

[0086] Tcm cells generally home to lymph nodes after transplantation.

[0087] According to some embodiments, the Tcm cells of the present invention can home to any of the lymph nodes after transplantation, such as peripheral lymph nodes and mesenteric lymph nodes. The homing properties of these cells enable them to exert their tolerogenic effects in a rapid and efficient manner.

[0088] As used herein, the phrase "tolerance-inducing cell" refers to a cell that, when contacted with recipient cells (e.g., recipient T cells), causes a decrease in the reactivity of the recipient cells as compared to the reactivity of the recipient cells in the absence of the administered tolerance-inducing cells. As previously described in PCT Publication Nos. WO 2001 / 049243 and WO 2002 / 102971, tolerance-inducing cells include veto cells (i.e., T cells that cause apoptosis of host T cells upon contact).

[0089] According to one embodiment, the Tcm cells of the present invention are also non-GVHD-inducing cells.

[0090] As used herein, the term "non-GVHD" refers to having a significantly reduced or no graft-versus-host-induced reactivity. Thus, the cells of the present invention are produced such that they do not significantly cause graft-versus-host disease (GVHD), as evidenced by the survival, body weight, and overall appearance of the transplanted subject 30 - 100 days after transplantation.

[0091] According to one embodiment, the reactivity of the cells of the present invention to the host is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or even 100% relative to T cell transplantation that is not anti-third party Tcm cells.

[0092] According to one embodiment, the cells of the present invention comprising a Tcm phenotype are genetically modified.

[0093] According to one embodiment, the cells of the present invention are transduced to express a cell surface receptor comprising a T cell receptor signaling module.

[0094] As used herein, the term "transduced" may be used interchangeably with the terms "transfected" or "transformed" and refers to a process of transferring or introducing exogenous nucleic acid (heterologous) into a cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. Cells include primary cells and their progeny or cell lines.

[0095] As used herein, the term "cell surface receptor" refers to a recombinant or synthetic molecule that is present on the cell membrane, binds to a ligand (e.g., an antigen), and mediates cell activation.

[0096] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. One of ordinary skill in the art will understand that any macromolecule (including substantially all proteins or peptides, as well as carbohydrates, lipids, and DNA) can be used as an antigen.

[0097] In some embodiments of the invention, the antigen is associated with a malignant disease, i.e., a tumor antigen (e.g., a tumor-specific antigen or a tumor-associated antigen), a viral protein antigen, a bacterial protein antigen, a fungal protein antigen, an antigen associated with allergy (i.e., an allergy antigen) or an antigen associated with autoimmunity (e.g., an "auto" antigen), as described in further detail below.

[0098] The cell surface receptor of the invention comprises a T cell receptor signaling module.

[0099] The term "T cell receptor signaling module" refers to the intracellular portion of a receptor that is responsible for activating at least one normal effector function of the T cell in which the receptor is placed. Normal effector functions of a T cell can include, for example, the secretion of immunostimulatory cytokines (e.g., IFN-γ, IL-2, TNF-α), antigen-specific cytotoxicity, and cell proliferation. Thus, the T cell receptor signaling module of the invention refers to the protein portion that transduces effector function signals and directs the cell to perform specialized functions.

[0100] According to one embodiment, the cell surface receptor comprises a transgenic T cell receptor (tg-TCR) or a chimeric antigen receptor (CAR).

[0101] As used herein, the term "transgenic T cell receptor" or "tg-TCR" refers to a recombinant or synthetic molecule that comprises the specificity of a T cell receptor (TCR), i.e., that recognizes an antigenic peptide (i.e., an antigen) presented by a major histocompatibility complex (MHC) protein.

[0102] The tg-TCR of the invention generally comprises two chains (i.e., polypeptide chains), such as the α chain of a T cell receptor (TCR), the β chain of a TCR, the γ chain of a TCR, the δ chain of a TCR, or a combination thereof (e.g., an αβ chain or a γδ chain). The polypeptides of the tg-TCR can comprise any amino acid sequence, provided that the tg-TCR has the antigen specificity and T cell effector functions as described above. It should be appreciated that the antigen specificity is determined by the TCR heterodimer (i.e., the αβ or γδ chain).

[0103] It should be appreciated that each of the two chains generally comprises two extracellular domains, i.e., a variable (V) region and a constant (C) region.

[0104] According to one embodiment, the tg-TCR comprises the variable regions of the TCR. According to a specific embodiment, the tg-TCR comprises the variable regions of the α and β chains of the TCR. According to another specific embodiment, the tg-TCR comprises the variable regions of the γ and δ chains of the TCR.

[0105] According to some embodiments of the invention, the variable regions of the tg-TCR comprise complementarity determining regions (CDRs) capable of specifically binding an antigen. The CDRs can be selected from any one of CDR1, CDR2, CDR3, and / or CDR4. According to a specific embodiment, the CDRs are present on a single chain, preferably the CDRs are present on both chains of the tg-TCR.

[0106] According to one embodiment, the tg-TCR comprises the constant regions of the TCR. According to a specific embodiment, the tg-TCR comprises the constant regions of the α and β chains of the TCR. According to another specific embodiment, the tg-TCR comprises the constant regions of the γ and δ chains of the TCR.

[0107] To avoid the formation of hybrid dimers between the endogenous TCR (i.e., the TCR derived from within the transduced cell) and the tg-TCR chains, the tg-TCR of the invention can comprise constant region murine (e.g., mouse) TCR. Another method that can be used to increase the specific pairing of the tg-TCR chains is to introduce additional cysteine residues within the constant regions of the tg-TCR chains (e.g., the α and β chains), which results in the formation of additional disulfide bonds. Alternatively, mutations can be introduced that reverse the key interacting amino acids within the constant regions of the tg-TCR chains (e.g., the α and β chains), which favors the pairing of the tg-TCR chains and also increases tg-TCR reactivity. Alternatively or additionally, the downregulation of the endogenous TCR can be effected using, for example, small interfering RNA (siRNA) for specifically downregulating the endogenous TCR. For further details see, for example, Zhang and Morgan, Adv Drug Deliv Rev. (2012) 64(8): 756-762, which is incorporated herein by reference.

[0108] As mentioned, the tg-TCR recognizes antigens in an MHC-dependent manner.

[0109] The phrase "major histocompatibility complex" or "MHC" as used herein refers to an antigen complex encoded by a group of linked loci, which are collectively referred to as H-2 in mice and as human leukocyte antigen (HLA) in humans. The two major classes of MHC antigens (class I and class II) each comprise a set of cell surface glycoproteins that play a role in determining tissue type and graft compatibility.

[0110] Major MHC class I molecules are contemplated herein.

[0111] Major histocompatibility complex (MHC) class I molecules are expressed on the surface of almost all cells. The function of these molecules is to present peptides mainly derived from endogenously synthesized proteins to CD8+ T cells via interaction with the αβ T cell receptor. In humans, there are several MHC haplotypes, such as HLA-A2, HLA-A1, HLA-A3, HLA-A24, HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45, and HLA-Cw8, the sequences of which can be found in the kabbat database at the website www.immuno.bme.nwu.edu. Further information on MHC haplotypes can be found in Paul, B. Fundamental Immunology Lippincott-Raven Press.

[0112] The selection of the tg-TCR depends on the type and number of antigens that define the target cell surface. For example, a tg-TCR can be selected to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state. Thus, for example, cell surface markers that can act as antigens for recognition by the tg-TCR can include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells. Examples are provided below.

[0113] To generate a successful tg-TCR, it is necessary to first identify a suitable target sequence. Thus, the TCR can be isolated from antigen-reactive T cells (e.g., tumor-reactive T cells), or if this is not possible, alternative techniques can be used. According to an exemplary embodiment, a transgenic animal (e.g., a rabbit or a mouse, preferably a human HLA transgenic mouse) is immunized with a human antigen peptide (e.g., a tumor or viral antigen) to generate T cells that express a TCR specific for the human antigen [as described, for example, in Stanislawski et al., Nat Immunol. (2001) 2(10):962-70]. According to another exemplary embodiment, antigen-specific T cells (e.g., tumor-specific T cells) are isolated from a patient who is experiencing remission of a disease (e.g., a tumor), and the reactive TCR sequences are isolated therefrom [as described, for example, in de Witte et al., Blood (2006) 108(3):870].

[0114] According to another exemplary embodiment, in vitro techniques are used to alter the sequence of an existing TCR to enhance the affinity of a weakly reactive antigen-specific TCR for the target antigen (this method is described below).

[0115] According to one embodiment, the tg-TCRs of the invention are selected to recognize antigenic peptide-HLA complexes with high avidity (i.e., the physical strength of the monomeric interaction between the TCR and the peptide-MHC complex).

[0116] Generation of cells with high functional avidity (i.e., cells that respond effectively to an antigen) can be achieved using any method known to those of ordinary skill in the art. Thus, according to one embodiment, the avidity of the tg-TCR is increased by increasing the affinity of the tg-TCR (i.e., the binding strength of the TCR to its ligand) or by increasing the expression of the tg-TCR on the cell surface. According to an exemplary embodiment, increasing the TCR affinity is effected by modifying the tg-TCR gene. For example, one possible modification of the tg-TCR gene includes modification of the complementarity-determining regions (CDRs) of the tg-TCR, such as the third CDR (CDR3). Thus, single or double amino acid substitutions in the CDR chains (such as the α or β chains) can be employed to increase the avidity of the tg-TCR and enhance antigen-specific reactivity in the transduced cells. According to another exemplary embodiment, increasing the functional avidity of the tg-TCR is effected by removing N-glycosylation motifs defined in the constant domains of the tg-TCR chains. According to another exemplary embodiment, increasing the avidity is effected by codon optimization.

[0117] Thus, rare codons of the tg-TCR are replaced with codons most frequently distributed in highly expressed human genes. During the optimization process, cis-acting AT- or GC-rich sequence segments, ambiguous splicing, and RNA instability motifs can also be removed. For further information see, e.g., Zhang and Morgan, Adv Drug Deliv Rev. (2012), supra, which is incorporated herein by reference.

[0118] According to one embodiment, the signaling module of the tg-TCR can comprise a single subunit or multiple signaling units. Thus, the tg-TCRs of the invention can use co-receptors that act in concert with the TCR (to initiate signal transduction after antigen receptor engagement) and any derivatives or variants thereof with the same functional capabilities.

[0119] According to one embodiment, the TCR signaling module comprises the CD3 complex (such as CD3 chains, such as CD3δ / ε, CD3γ / ε, and / or ζ chains, such as ζ / ζ or ζ / η).

[0120] Additionally or alternatively, the TCR signaling module can comprise co-stimulatory protein receptors to provide additional signals to the T cell. These are discussed in detail below.

[0121] According to one embodiment, the tg-TCR can comprise a transmembrane domain as described in detail below.

[0122] The method of transducing cells with TCR is described in detail below.

[0123] As used herein, the phrase "chimeric antigen receptor (CAR)" refers to a recombinant or synthetic molecule that combines specificity for a desired antigen with an intracellular domain that activates a T cell receptor (i.e., a T cell receptor signaling module) to generate cellular immune activity against a specific antigen-presenting cell.

[0124] Thus, the CARs of the invention generally comprise an extracellular domain (comprising an antigen-binding portion), a transmembrane domain, and an intracellular domain (i.e., a cytoplasmic domain) required for the T cell to respond effectively to the antigen.

[0125] Antigen-binding portion

[0126] In one embodiment, the CARs of the invention comprise a target-specific binding element, also referred to as an antigen-binding portion. The choice of portion depends on the type and number of ligands (i.e., antigens) that define the target cell surface. For example, an antigen-binding domain can be selected to recognize a ligand (i.e., an antigen) that functions as a cell surface marker on a target cell associated with a particular disease state. Thus, examples of cell surface markers that can function as ligands for the antigen portion domain in the CARs of the invention include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0127] According to some embodiments of the invention, the antibody-binding portion comprises complementarity determining regions (CDRs) capable of specifically binding an antigen. Such CDRs can be derived from an antibody.

[0128] The term "antibody" as used in the present invention includes intact molecules and functional fragments thereof, such as Fab, Fab’, F(ab’)2, Fv, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments capable of binding to an antigen. These functional antibody fragments are defined as follows: (1) Fab, a fragment containing the monovalent antigen-binding fragment of an antibody molecule, which can be generated by digesting whole antibody with papain to obtain the intact light chain and a portion of one heavy chain; (2) Fab’, a fragment of an antibody molecule obtained by treating whole antibody with pepsin followed by reduction to obtain the intact light chain and a portion of the heavy chain; two Fab’ fragments are obtained per antibody molecule; (3) F(ab’)2, an antibody fragment obtained by treating whole antibody with pepsin without subsequent reduction; F(ab’)2 is a dimer of two Fab’ fragments linked together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; (5) single-chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, joined as a genetically fused single-chain molecule by a suitable polypeptide linker; (6) CDR peptide, a peptide encoding a single complementarity-determining region (CDR), and (7) single-domain antibody (also known as nanobody), a genetically engineered single monomer variable antibody domain that selectively binds to a specific antigen. The molecular weight of a nanobody is only 12 - 15 kDa, much smaller than that of a conventional antibody (150 - 160 kDa).

[0129] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation.

[0130] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation. κ- and λ-light chains refer to the two major antibody light chain isotypes.

[0131] The so-called term "synthetic antibody" as used herein means an antibody produced using recombinant DNA technology, such as an antibody expressed by bacterial phage as described herein. The term should also be construed to mean an antibody produced by synthesizing a DNA molecule encoding the antibody (and the DNA molecule expressing the antibody protein) or specifying the amino acid sequence of the antibody, wherein the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques available and well-known in the art.

[0132] Methods for generating polyclonal and monoclonal antibodies and their fragments are well known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, which is incorporated herein by reference).

[0133] Antibody fragments of the invention can be prepared by proteolytic cleavage of the antibody or by expressing DNA encoding the fragment in E. coli or mammalian cells (e.g., Chinese hamster ovary cell cultures or other protein expression systems). Antibody fragments can be obtained by digesting whole antibodies with pepsin or papain by conventional methods. For example, antibody fragments can be generated by enzymatic cleavage of the antibody with pepsin to provide a 5S fragment designated as F(ab’)2. This fragment can be further cleaved using a thiol reducing agent and optionally using a capping group for the sulfhydryl groups generated by disulfide bond cleavage to produce 3.5S Fab’ monovalent fragments. Alternatively, enzymatic cleavage with pepsin directly produces two monovalent Fab’ fragments and an Fc fragment. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 and the references contained therein, which patents are hereby incorporated by reference in their entirety. See also R.R. [Biochem. J. 73: 119-126 (1959)]. Other methods for cleaving antibodies can also be used, such as separating the heavy chain to form monovalent light-heavy chain fragments, further cleaving the fragments or other enzymatic, chemical or genetic techniques, so long as the fragment binds to the antigen recognized by the intact antibody.

[0134] The Fv fragment comprises the association of VH and VL chains. This association can be non-covalent, as described in Inbar et al. [Proc. Nat’l Acad. Sci. USA 69:2659-62 (1972)]. Alternatively, the variable chains can be linked by intermolecular disulfide bonds or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragment comprises VH and VL chains linked by a peptide linker. These single-chain antigen-binding proteins (sFv) are prepared by constructing a structural gene comprising a DNA sequence encoding VH and VL domains linked by an oligonucleotide. The structural gene is inserted into an expression vector, which is then introduced into a host cell such as Escherichia coli. The recombinant host cell synthesizes a single polypeptide chain using a linker peptide that bridges the two V domains. Methods for generating sFv are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio / Technology 11:1271-77 (1993); and U.S. Patent No. 4,946,778, which is hereby incorporated by reference in its entirety].

[0135] CDR peptides (“minimal recognition units”) can be obtained by constructing genes encoding the CDRs of a target antibody. Such genes are prepared, for example, by synthesizing the variable region from the RNA of antibody-producing cells using polymerase chain reaction. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].

[0136] Once the CDRs of an antibody have been identified, using conventional genetic engineering techniques, an expressible polynucleotide encoding any form or fragment of the antibody described herein can be synthesized and modified in one of a variety of ways to produce a family of related products.

[0137] In some embodiments of the invention, the CDRs are derived from an αβ T cell receptor (TCR) that specifically binds to an antigen.

[0138] In some embodiments of the invention, the CDRs are derived from a γδ T cell receptor (TCR) that specifically binds to an antigen.

[0139] In some embodiments of the invention, the CDRs are derived from an engineered affinity-enhanced αβ T cell receptor or γδ T cell receptor (TCR) that specifically binds to an antigen (as discussed in detail above).

[0140] According to some embodiments of the present invention, the CDR is derived from an engineered αβ T cell receptor or γδ T cell receptor (TCR) having improved stability or any other biophysical property.

[0141] According to some embodiments of the present invention, the CDR is derived from a T cell receptor-like (TCRL) antibody that specifically binds to an antigen. Examples of TCRLs and methods for their production are described in WO03 / 068201, WO2008 / 120203, WO2012 / 007950, WO2009125395, WO2009 / 125394, each of which is incorporated herein by reference in its entirety.

[0142] According to some embodiments of the present invention, the antigen-binding domain comprises a single-chain Fv (scFv) molecule.

[0143] Cytoplasmic domain

[0144] The cytoplasmic domain of the CAR molecule of the present invention (also referred to as the "intracellular signaling domain" or "T cell receptor signaling module") is responsible for activating at least one normal effector function of the cell in which the CAR has been placed.

[0145] Although the entire intracellular signaling domain can generally be used, it is not necessary to use the entire chain in many cases. In terms of using truncated portions of the intracellular signaling domain, such truncated portions can be used in place of the full chain as long as they transduce effector function signals. The term intracellular signaling domain is thus intended to include any truncated portion of the intracellular signaling domain that is sufficient to transduce effector function signals.

[0146] Preferred examples of the intracellular signaling domain for the CAR molecule of the present invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act together to initiate signal transduction after antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional ability.

[0147] It is known that the signals generated solely through the TCR are not sufficient to fully activate T cells, and co-stimulatory signals are also required. Thus, T cell activation can be mediated by two different classes of cytoplasmic signaling sequences: those cytoplasmic signaling sequences that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those cytoplasmic signaling sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0148] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs called immunoreceptor tyrosine-based activation motifs (ITAMs).

[0149] Examples of ITAMs containing primary cytoplasmic signaling sequences that are particularly useful in the present invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Particularly preferably, the cytoplasmic signaling molecule in the CAR of the present invention comprises a cytoplasmic signaling sequence derived from CD3ζ.

[0150] In a preferred embodiment, the cytoplasmic domain of the CAR can be designed to itself comprise the CD3ζ signaling domain or in combination with any other desired cytoplasmic domain useful in the context of the CAR of the present invention. For example, the cytoplasmic domain of the CAR can comprise a CD3ζ chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to a portion of the CAR that comprises the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules required for lymphocytes to respond effectively to an antigen in addition to the antigen receptor or its ligand. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, etc. Thus, although the present invention is mainly illustrated by 4-1BB as the co-stimulatory signaling element, other co-stimulatory elements are within the scope of the present invention.

[0151] According to some embodiments of the present invention, the intracellular domain comprises a co-stimulatory signaling region and a ζ chain portion. The co-stimulatory signaling region refers to a portion of the CAR molecule that comprises the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules required for lymphocytes to respond effectively to an antigen in addition to the antigen receptor or its ligand.

[0152] As used herein, the term "costimulatory ligand" includes molecules on antigen-presenting cells (such as aAPC (artificial antigen-presenting cells), dendritic cells, B cells, etc.) that specifically bind to cognate costimulatory molecules on T cells, thereby providing signals that mediate T cell responses (including but not limited to proliferation, activation, differentiation, etc.) in addition to the primary signal provided by, for example, the binding of the TCR / CD3 complex to peptide-loaded MHC molecules. Costimulatory ligands can include but are not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also particularly include antibodies that specifically bind to costimulatory molecules present on T cells, such as but not limited to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0153] "Costimulatory molecule" refers to the cognate binding partner on T cells that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response in T cells such as but not limited to proliferation. Costimulatory molecules include but are not limited to MHC class I molecules, BTLA, and Toll ligand receptors.

[0154] As used herein, the term "costimulatory signal" refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, results in T cell proliferation and / or upregulation or downregulation of key molecules.

[0155] The so-called term "stimulation" means the binding of a stimulatory molecule (such as the TCR / CD3 complex) to its cognate ligand, thereby mediating signal transduction events such as but not limited to the primary response induced by signal transduction through the TCR / CD3 complex. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-β and / or reorganization of the cytoskeletal structure, etc.

[0156] As used herein, the term "stimulatory molecule" means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0157] As used herein, "stimulatory ligand" means a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner (referred to herein as "stimulatory molecule") on a T cell, thereby mediating a primary response of the T cell (including but not limited to activation, initiation of an immune response, proliferation, etc.). Stimulatory ligands are well known in the art and include, in particular, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0158] With respect to the cytoplasmic domain, the CAR molecules of some embodiments of the invention can be designed to inherently include a CD28 and / or 4-1BB signaling domain, or in combination with any other desired cytoplasmic domain useful in the context of the CAR molecules of some embodiments of the invention. In some embodiments, the cytoplasmic domain of the CAR can be designed to further include the signaling domain of CD3ζ. For example, the cytoplasmic domain of the CAR can include, but is not limited to, CD3ζ, 4-1BB, and CD28 signaling modules and combinations thereof.

[0159] According to some embodiments of the invention, the intracellular domain comprises at least one, such as at least two, at least three, at least four, at least five, such as at least six, of the polypeptides selected from: CD3ζ (CD247, CD3z), CD27, CD28, 4-1BB / CD137, ICOS, OX40 / CD134, DAP10, tumor necrosis factor receptor (TNFr), and Lsk.

[0160] According to some embodiments of the invention, the intracellular domain comprises the CD3ζ chain [CD247 molecule, also known as "CD3-ZETA (ζ)" and "CD3z"; GenBank accession numbers NP_000725.1 and NP_932170.1], which is the primary transmitter of signals from the endogenous TCR.

[0161] In some embodiments of the present invention, the intracellular domain comprises various co-stimulatory protein receptors of the CAR cytoplasmic tail to provide additional signals to the T cells ("second-generation" CAR). Examples include, but are not limited to, CD28 [e.g., GenBank accession numbers NP_001230006.1, NP_001230007.1, NP_006130.1], 4-1BB [tumor necrosis factor receptor superfamily member 9 (TNFRSF9), also known as "CD137", e.g., GenBank accession number NP_001552.2], ICOS [inducible T cell co-stimulator, e.g., GenBank accession number NP_036224.1], DAP10 [hematopoietic cell signal transduction molecule, e.g., GenBank accession numbers NP_001007470, NP_055081.1], and Lsk [LCK proto-oncogene, Src family tyrosine kinase, e.g., GenBank accession numbers NP_001036236.1, NP_005347.3]. Preclinical studies have shown that the "second-generation CAR design improves the anti-tumor activity of T cells.

[0162] In some embodiments of the present invention, the intracellular domain comprises multiple signaling domains, such as CD3z-CD28-4-1BB or CD3z-CD28-OX40, to further enhance potency. The term "OX40" refers to tumor necrosis factor receptor superfamily member 4 (TNFRSF4), e.g., GenBank accession number NP_003318.1 ("third-generation" CAR).

[0163] In some embodiments of the present invention, the intracellular domain comprises CD28-CD3z, CD3z, CD28-CD137-CD3z. The term "CD137" refers to tumor necrosis factor receptor superfamily member 9 (TNFRSF9), e.g., GenBank accession number NP_001552.2.

[0164] In some embodiments of the present invention, the intracellular domain comprises CD3z, CD28, and tumor necrosis factor receptor (TNFr).

[0165] In some embodiments of the present invention, the CAR comprises a CD3ζ chain.

[0166] In some embodiments of the present invention, the CAR comprises at least one co-stimulatory domain selected from CD28, CD134 / OX40, CD137 / 4-1BB, Lck, ICOS, and DAP10.

[0167] According to some embodiments of the present invention, the CAR comprises at least two co-stimulatory domains selected from CD28, CD134 / OX40, CD137 / 4-1BB, Lck, ICOS, and DAP10.

[0168] Transmembrane domain

[0169] The transmembrane domain of the CAR can be derived from a natural source or from a synthetic source. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Particularly useful transmembrane regions in the present invention can be derived from (i.e., at least comprise their transmembrane regions) the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain can be synthetic, in which case it will mainly comprise hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0170] Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the connection between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0171] According to some embodiments of the present invention, the transmembrane domain included in the CAR molecule in some embodiments of the present invention is a transmembrane domain naturally associated with a domain in the CAR. According to some embodiments of the present invention, the transmembrane domain can be selected or modified by amino acid substitution to avoid this domain binding to the transmembrane domains of the same or different surface membrane proteins, so as to minimize the interaction with other members of the receptor complex.

[0172] According to some embodiments of the present invention, the transmembrane domain is the CD8α hinge domain.

[0173] According to some embodiments, a spacer domain can be incorporated between the extracellular domain and the transmembrane domain of the CAR molecule or between the cytoplasmic domain and the transmembrane domain of the CAR molecule. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide that serves to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of the polypeptide chain. The spacer domain can contain up to 300 amino acids, preferably 10-100 amino acids and most preferably 25-50 amino acids.

[0174] As mentioned, the cell surface receptors (e.g., tg-TCR and / or CAR) of the cells of the present invention bind to an antigen (e.g., on a target cell).

[0175] According to one embodiment, the antigen may comprise a tumor-associated antigen, a viral antigen, a bacterial antigen, a fungal antigen, a protozoal antigen, a parasitic antigen, an allergy-related antigen, and / or an autoimmune antigen.

[0176] As used herein, the phrase "tumor antigen" refers to an antigen that is common to a particular hyperproliferative disorder such as cancer. A tumor antigen is a protein produced by tumor cells that elicits an immune response, particularly a T cell-mediated immune response. The selection of the antigen-binding portion of the present invention depends on the specific type of cancer to be treated.

[0177] According to one embodiment, the tumor antigen is associated with a solid tumor.

[0178] According to one embodiment, the tumor antigen is associated with a hematological malignancy.

[0179] The types of tumor antigens mentioned in the present invention include tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). "TSA" refers to a protein or polypeptide antigen that is unique to tumor cells and does not occur on other cells of the body. "TAA" refers to a protein or polypeptide antigen expressed by tumor cells. For example, a TAA may be one or more surface proteins or polypeptides, nuclear proteins, or glycoproteins of tumor cells or fragments thereof.

[0180] The antigens discussed herein are included only as examples. The list is not intended to be exclusive, and further examples will be readily apparent to those skilled in the art.

[0181] Tumor antigens are well known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mutant hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin, and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, (IGF)-II, IGF-1 receptor, and mesothelin.

[0182] These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphoma, the tumor-specific idiotype immunoglobulins constitute the true tumor-specific immunoglobulin antigens unique to an individual tumor. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidates for target antigens in B-cell lymphoma. Some of these antigens (e.g., CEA, HER-2, CD19, CD20, idiotype) have been used as targets for monoclonal antibody passive immunotherapy, but with limited success.

[0183] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-1 / MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2; and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens generated by chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-901\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, YLP, and TPS.

[0184] Further examples of tumor antigens include, but are not limited to, A33, BAGE, Bcl-2, β-catenin, CAl25, CA19-9, CD5, CD19, CD20, CD21, CD22, CD33, CD37, CD45, CD123, CEA, c-Met, CS-1, Cyclin B1, DAGE, EBNA, EGFR, Ephrin B2, Estrogen receptor, FAP, Ferritin, Folate binding protein, GAGE, G250, GD-2, GM2, gp75, gp100 (Pmel 17), HER-2 / neu, HPV E6, HPV E7, Ki-67, LRP, Mesothelin, p53 and PRAME. Further tumor antigens are provided in van der Bruggen P, Stroobant V, Vigneron N, Van den Eynde B. Peptide database: Tcell-defined tumor antigens. Cancer Immun (2013), www.cancerimmunity.org / peptide / , which is incorporated herein by reference.

[0185] According to a specific embodiment, the tumor antigens include, but are not limited to, CD19, CD20, CD22, ROR1, Mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, Her2, GD-2, gp100, p53, carcinoembryonic antigen (CEA), MY-ESO-1, MART-1, MAGE A3, etc.

[0186] According to one embodiment, the target antigen is CD19.

[0187] According to some embodiments of the present invention, the antigen is a viral antigen. The viral antigen can be derived from any virus, such as, but not limited to, human immunodeficiency virus (HIV), influenza, cytomegalovirus (CMV), T-cell leukemia virus type 1 (TAX), hepatitis C virus (HCV), (HBV), Epstein-Barr virus (EBV), adenovirus (Adv), cold virus, influenza virus, hepatitis A, B and C viruses, herpes simplex, Japanese encephalitis, measles, polio, rabies, respiratory syncytial, rubella, smallpox, varicella zoster, rotavirus, West Nile virus, polyomavirus (e.g., BK virus) and / or Zika virus.

[0188] According to some embodiments of the present invention, the viral antigen includes, but is not limited to, viral epitopes from polypeptides selected from the group consisting of: human T-lymphotropic virus type 1 (HTLV-1) transcription factor (TAX), influenza matrix protein epitope, Epstein-Barr virus (EBV)-derived epitope, HIV-1 RT, HIV Gag, HIV Pol, influenza membrane protein M1, influenza hemagglutinin, influenza neuraminidase, influenza nucleoprotein, influenza nucleoprotein, influenza matrix protein (M1), influenza virus ion channel (M2), influenza non-structural protein NS-1, influenza non-structural protein NS-2, influenza PA, influenza PB1, influenza PB2, influenza BM2 protein, influenza NB protein, influenza nucleocapsid protein, cytomegalovirus (CMV) phosphorylated matrix protein (pp65), TAX, hepatitis C virus (HCV), HBV pre-S protein 85-66, HTLV-1 tax 11-19, HBV surface antigen 185-194.

[0189] According to some embodiments of the present invention, the antigen is a bacterial antigen. The bacterial antigen can be derived from any bacterium, such as, but not limited to, anthrax, Gram-negative bacilli, chlamydia, diphtheria, Haemophilus influenzae, Helicobacter pylori, malaria, Mycobacterium tuberculosis, pertussis toxin, Streptococcus pneumoniae, Rickettsia, Staphylococcus, Streptococcus, and tetanus.

[0190] According to some embodiments of the present invention, the bacterial antigen includes, but is not limited to, anthrax antigen (including, but not limited to, anthrax protective antigen), Gram-negative bacilli antigen (including, but not limited to, lipopolysaccharide), Haemophilus influenzae antigen (including, but not limited to, capsular polysaccharide), diphtheria antigen (including, but not limited to, diphtheria toxin), Mycobacterium tuberculosis antigen (including, but not limited to, mycolic acid, heat shock protein 65 (HSP65), 30 kDa major secreted protein, and antigen 85A), pertussis toxin antigen (including, but not limited to, hemagglutinin, Bordetella pertussis adhesin, FIM2, FIM3, and adenylate cyclase), Streptococcus pneumoniae antigen (including, but not limited to, pneumolysin and pneumococcal capsular polysaccharide), Rickettsia antigen (including, but not limited to, rompA), Streptococcus antigen (including, but not limited to, M protein), and tetanus antigen (including, but not limited to, tetanus toxin).

[0191] According to some embodiments of the present invention, the antigen is a superbug antigen (e.g., a multi-drug resistant bacterium). Examples of superbugs include, but are not limited to, Enterococcus faecium, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae (including Escherichia coli), Klebsiella pneumoniae, Enterobacter spp..

[0192] According to some embodiments of the present invention, the antigen is a fungal antigen. Examples of fungi include, but are not limited to, candida, coccidiodes, cryptococcus, histoplasma, leishmania, plasmodium, protozoa, parasites, schistosomae, tinea, toxoplasma, and trypanosoma cruzi.

[0193] According to some embodiments of the present invention, fungal antigens include, but are not limited to, coccidiodes antigens (including, but not limited to, spherule antigens), cryptococcus antigens (including, but not limited to, capsular polysaccharides), histoplasma antigens (including, but not limited to, heat shock protein 60 (HSP60)), leishmania antigens (including, but not limited to, gp63 and lipophosphoglycan), plasmodium falciparum antigens (including, but not limited to, merozoite surface antigen, sporozoite surface antigen, circumsporozoite antigen, gametocyte / gamete surface antigen), protozoa and other parasite antigens (including blood stage antigen pf 155 / RESA), schistosomae antigens (including, but not limited to, glutathione-S-transferase and paramyosin), tinea fungi antigens (including, but not limited to, trichophytin), toxoplasma antigens (including, but not limited to, SAG-1 and p30), and trypanosoma cruzi antigens (including, but not limited to, 75-77 kDa antigen and 56 kDa antigen).

[0194] According to some embodiments of the present invention, the antigen is an antigen expressed by cells associated with allergic disorders. Examples of allergic antigens include but are not limited to pollen antigens such as Cryptomeria japonica pollen antigen, ragweed pollen antigen, ryegrass pollen antigen, animal-derived antigens (such as dust mite antigen and cat antigen), histocompatibility antigens, and penicillin and other therapeutic drugs.

[0195] According to some embodiments of the present invention, the antigen is an autoantigen associated with an autoimmune disease.

[0196] As used herein, the term "autoimmune disease" is defined as a disorder caused by an autoimmune reaction. Autoimmune diseases are the result of an inappropriate overreaction to autoantigens.

[0197] Examples of autoimmune diseases include but are not limited to Addison's disease, alopecia greata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, inflammatory bowel disease (IBD), celiac disease, dermatitis (including atopic dermatitis and eczematous dermatitis), type I diabetes, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus (SLE), multiple sclerosis (MS), myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), sarcoidosis, scleroderma, Sjögren's syndrome, Stevens-Johnson syndrome, Wegener's granulomatosis, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, anemia, asthma, pernicious anemia, ulcerative colitis, and stroke, etc.

[0198] As used herein, the phrase "autoantigenic peptide" refers to an antigen derived from an endogenous (i.e., self-protein) or consumed protein (e.g., through food) that elicits an inflammatory response as part of an autoimmune inflammatory reaction.

[0199] It should be noted that the phrases "endogenous" and "self" are relative expressions referring to the individual in whom the autoimmune reaction is triggered.

[0200] Autoantigens include but are not limited to cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

[0201] According to some embodiments of the present invention, the autoantigenic peptide is associated with a disease selected from diabetes, multiple sclerosis, rheumatoid arthritis, celiac disease, and stroke.

[0202] Autoantigens in multiple sclerosis include, but are not limited to, myelin proteins, such as myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG).

[0203] Autoantigens associated with rheumatoid arthritis include, but are not limited to, autoantigenic peptides derived from collagen II (COL2A1), matrix metalloproteinase-1 (MMP1), aggrecan core protein precursor (ACAN), matrix metalloproteinase-16 (MMP16), tenascin (TNXB), and heterogeneous nuclear ribonucleoprotein A2 (HNRNPA2B1).

[0204] Autoantigens in type 1 diabetes (T1D) include, but are not limited to, antigens expressed in pancreatic islets, including glutamate decarboxylase (GAD65) and β-cell autoantigenic peptides.

[0205] Autoantigens in celiac disease (Celiac or Coeliac) include, but are not limited to, gliadin (e.g., α-gliadin, γ-gliadin) and heat shock protein 20.

[0206] Autoantigens in Crohn's disease, ulcerative colitis, or inflammatory bowel disease (IBD) include, but are not limited to, FAM84A, glycoprotein 2 (GP2), CUB and zona pellucida-like domain-containing protein 1 (CUZD1), complement C3, catalase, and α-enolase.

[0207] According to some embodiments of the present invention, stroke-related autoantigens include, but are not limited to, autoantigenic peptides derived from brain antigens, such as myelin basic protein, neurofilament, and the NR2A / 2B subtype of the N-methyl-D-aspartic acid receptor (MOG-35-55).

[0208] One aspect of some embodiments of the present invention provides a method for generating isolated cells of some embodiments of the present invention, the method comprising transducing cells having a central memory T lymphocyte (Tcm) phenotype with a polynucleotide encoding a cell surface receptor comprising a T cell receptor signaling module, the cells being tolerance-inducing cells and capable of homing to lymph nodes after transplantation.

[0209] According to one embodiment, cells having a central memory T lymphocyte (Tcm) phenotype, being tolerance-inducing cells and capable of homing to lymph nodes after transplantation are anti-third-party cells.

[0210] As used herein, the phrase "anti-third-party cells" refers to lymphocytes (i.e., T lymphocytes) that are specific for (i.e., recognize through T cells) one or more third-party antigens.

[0211] As used herein, the phrase "one or more third-party antigens" refers to one or more soluble or insoluble (such as membrane-associated) antigens that are not present in the donor or recipient, as described in detail below.

[0212] For example, the third-party antigen can be a third-party cell, a cell antigen (such as a cell surface antigen), an antigen of a virus (i.e., a viral antigen) such as Epstein-Barr virus (EBV) or cytomegalovirus (CMV), or an antigen of a bacterium (i.e., a bacterial antigen) such as flagellin. The viral or bacterial antigen can be presented by a cell (such as a cell line) that is infected with it or otherwise causes the expression of viral / bacterial proteins.

[0213] Autologous or allogeneic antigen-presenting cells or artificial carriers or artificial antigen-presenting cells can be used to present short synthetic peptides that are fused to or loaded onto them or to present protein extracts or purified proteins. Such short peptides, protein extracts, or purified proteins can be peptides of viral or bacterial origin or peptides representing any other antigen.

[0214] Specialized software can be used to analyze viral or other sequences to identify immunogenic short peptides, i.e., peptides that can be presented in the context of class I MHC or class II MHC.

[0215] The third-party cells can be allogeneic or xenogeneic with respect to the recipient (explained in further detail below). In the case of allogeneic third-party cells, such cells have HLA antigens that are different from those of the donor, but they do not cross-react with the recipient HLA antigens such that anti-third-party cell antibodies generated against such cells do not react against the graft or recipient antigens.

[0216] According to one embodiment of the invention, the allogeneic or xenogeneic third-party cells are stimulatory cells selected from the group consisting of cells purified from peripheral blood lymphocytes (PBL), spleen or lymph nodes, cytokine-mobilized PBL, in vitro-expanded antigen-presenting cells (APC), in vitro-expanded dendritic cells (DC), and artificial antigen-presenting cells.

[0217] The artificial APCs of the invention can be engineered to present autologous MHC with a third-party peptide or third-party MHC not pulsed with exogenous peptide. Thus, according to one embodiment, the artificial APCs comprise K562 tumor cells transfected with third-party MHC determinants and costimulatory molecules [such as previously described, e.g., Suhoski MM et al., Mol Ther. (2007) 15(5): 981-8] or fibroblasts transfected with the same substances.

[0218] A third-party antigen can be presented on the surface of a cell, virus or bacterium, or derived and / or purified therefrom. Additionally, a viral or bacterial antigen can be presented on an infected cell, and a cellular antigen can be presented on an artificial vector such as a liposome or an artificial antigen-presenting cell (e.g., a leukemia or fibroblast cell line transfected with one or more third-party antigens).

[0219] The third-party antigen can further comprise a synthetic peptide presented by an autologous antigen-presenting cell, a non-autologous antigen-presenting cell or on an artificial vector or on an artificial antigen-presenting cell.

[0220] Additionally, the third-party antigen can be, for example, a protein extracted or purified from a variety of sources. An example of a purified protein that can be used as the third-party antigen of the present invention is ovalbumin. Other examples are conceivable.

[0221] It is particularly advantageous to use a cell, a virus-infected cell, a bacterium-infected cell, a viral peptide-presenting cell or a bacterial peptide-presenting cell as the third-party antigen because such a third-party antigen includes a wide variety of antigenic determinants and thus leads to the formation of a diverse population of anti-third-party cells, which can be further used to more rapidly reconstitute T cells in situations where such reconstitution is needed, for example, after a lethal or sub-lethal irradiation or chemotherapy procedure.

[0222] Furthermore, when the anti-third-party cells are directed against the third-party antigen, the cells have anti-disease activity. The term "anti-disease activity" refers to the activity (e.g., killing ability) of Tcm cells against diseased cells (e.g., cancer cells, such as graft-versus-leukemia (GVL) activity). This activity is generally due to TCR-independent killing mediated by LFA1-I / CAM1 binding [Arditti et al., Blood (2005) 105(8):3365-71. Epub 2004 Jul 6].

[0223] According to one embodiment, the third-party cell comprises a dendritic cell.

[0224] According to one embodiment, the third-party cell comprises a mature dendritic cell.

[0225] Methods for generating third - party dendritic cells, which can be used as stimulatory cells for inducing Tcm cells, are well - known in the art. Thus, as a non - limiting example, peripheral blood mononuclear cells (PBMCs) can be obtained from a third - party non - homologous cell donor [e.g., in the case where the Tcm cells are homologous such as autologous, the dendritic cells (DCs) can be non - homologous such as allogeneic for the subject; and if the Tcm cells are non - homologous such as allogeneic, the DCs are selected from donors that are non - homologous such as allogeneic and HLA - mismatched with both the subject and the Tcm cells]. The monocytes can then be separated by plastic adherence and cultured (e.g., in a cell culture plate) in DC cell medium (e.g., Cellgro DC medium) supplemented with human serum (e.g., 1% human serum), penicillin / streptomycin, and GM - CSF (e.g., 800 IU / ml) and IL - 4 (e.g., 20 ng / ml) (available from, for example, Peprotech, Hamburg, Germany). After culturing for about 24 - 72 hours (e.g., 48 hours), DC medium containing GM - CSF (e.g., 1600 IU / ml) and IL - 4 (e.g., 20 ng / ml) can be added. After about 12 - 36 hours (e.g., 24 hours), the non - adherent cells can be harvested, and the large cells (mostly immature DCs) can be resuspended in fresh medium containing GM - CSF (e.g., 800 IU / ml), IL - 4 (e.g., 20 ng / ml), LPS (e.g., from Escherichia coli O55:B5, at, for example, 10 ng / ml), and IFNγ (e.g., 100 IU / ml) (available from, for example, Peprotech, Hamburg, Germany), plated, and incubated overnight. The next day, the non - adherent cells can be discarded, and the adherent DCs can be gently removed using, for example, cold PBS / 1% HS after incubating on ice for about 15 - 30 minutes (e.g., 20 minutes), thereby obtaining large cells composed of mature DCs.

[0226] According to one embodiment, the third - party cells comprise irradiated dendritic cells.

[0227] Thus, according to one embodiment, the DCs are irradiated with about 5 - 10 Gy, about 10 - 20 Gy, about 20 - 30 Gy, about 20 - 40 Gy, about 20 - 50 Gy, about 10 - 50 Gy. According to a specific embodiment, the DCs are irradiated with about 10 - 50 Gy (e.g., 30 Gy).

[0228] Any method for generating anti - third - party Tcm cells can be used in accordance with the present invention, as previously described in PCT Publication Nos. WO2010 / 049935, WO 2012 / 032526, and WO 2013 / 035099, which are hereby incorporated by reference.

[0229] According to one embodiment, generating anti-third-party cells with a Tcm phenotype can be implemented by a method comprising: (a) contacting peripheral blood mononuclear cells (PBMCs) with one or more third-party antigens in the presence or absence of IL-21 so as to enable enrichment of antigen-reactive cells; and (b) culturing the cells generated from step (a) in an antigen-free environment in the presence of IL-21, IL-15, and IL-7 so as to enable proliferation of cells comprising a central memory T lymphocyte (Tcm) phenotype.

[0230] According to one embodiment, the PBMCs in step (a) are contacted with one or more third-party antigens in the absence of IL-21.

[0231] According to one embodiment, the PBMCs in step (a) are contacted with one or more third-party antigens in the presence of IL-21.

[0232] According to one embodiment, the cells generated from step (a) are cultured in an antigen-free environment (such as not adding antigens to the cell culture) in the presence of only IL-15. IL-21 and / or IL-7 can be optionally added.

[0233] The anti-third-party Tcm cells of the present invention are generally generated by first contacting homologous (such as autologous) or non-homologous (such as non-autologous, such as allogeneic or xenogeneic, as described in further detail below) peripheral blood mononuclear cells (PBMCs) with one or more third-party antigens (such as those described above) in a culture supplemented with IL-21 (such as in a cytokine-free culture, i.e., without adding any additional cytokines). This step is generally carried out for about 12 - 24 hours, about 12 - 36 hours, about 12 - 72 hours, 24 - 48 hours, 24 - 36 hours, about 24 - 72 hours, about 48 - 72 hours, 1 - 2 days, 2 - 3 days, 1 - 3 days, 2 - 4 days, 1 - 5 days, 2 - 5 days, 2 - 6 days, 1 - 7 days, 5 - 7 days, 2 - 8 days, 8 - 10 days, or 1 - 10 days, and enables enrichment of antigen-reactive cells.

[0234] According to a specific embodiment, contacting homologous or non-homologous PBMCs with one or more third-party antigens (such as those described above) for 1 - 5 days (such as 3 days) is achieved in a culture supplemented with IL-21 (a cytokine-free culture originally).

[0235] Contacting autologous or non-autologous PBMCs with one or more third-party antigens (such as those described above) in a culture supplemented with IL-21 is generally carried out in the presence of about 0.001 - 3000 IU / ml, 0.01 - 3000 IU / ml, 0.1 - 3000 IU / ml, 1 - 3000 IU / ml, 10 - 3000 IU / ml, 100 - 3000 IU / ml, 1000 - 3000 IU / ml, 0.001 - 1000 IU / ml, 0.01 - 1000 IU / ml, 0.1 - 1000 IU / ml, 1 - 1000 IU / ml, 10 - 1000 IU / ml, 100 - 1000 IU / ml, 250 - 1000 IU / ml, 500 - 1000 IU / ml, 750 - 1000 IU / ml, 10 - 500 IU / ml, 50 - 500 IU / ml, 100 - 500 IU / ml, 250 - 500 IU / ml, 100 - 250 IU / ml, 0.1 - 100 IU / ml, 1 - 100 IU / ml, 10 - 100 IU / ml, 30 - 100 IU / ml, 50 - 100 IU / ml, 1 - 50 IU / ml, 10 - 50 IU / ml, 20 - 50 IU / ml, 30 - 50 IU / ml, 1 - 30 IU / ml, 10 - 30 IU / ml, 20 - 30 IU / ml, 10 - 20 IU / ml, 0.1 - 10 IU / ml or 1 - 10 IU / ml of IL-21.

[0236] According to a specific embodiment, the concentration of IL-21 is 50 - 150 IU / ml (e.g., 100 IU / ml).

[0237] According to a specific embodiment, contacting autologous or non-autologous PBMCs with one or more third-party antigens is achieved in a cytokine-free culture (e.g., supplemented only with IL-21), and such culture conditions only allow those cells that have been stimulated and activated with one or more third-party antigens (i.e., the antigens of antigen-reactive cells) to survive and be enriched because these cells secrete cytokines (such as IL-2) that enable their survival (all the remaining cells die under these culture conditions).

[0238] The ratio of one or more third-party antigens (such as dendritic cells) to PBMCs is generally about 1:2 - about 1:10, such as about 1:4, about 1:6, about 1:8 or about 1:10.

[0239] According to a specific embodiment, the ratio of one or more third-party antigens (such as dendritic cells) to PBMCs is about 1:2 - about 1:8 (e.g., 1:5).

[0240] Next, the anti-third-party cells are cultured in an antigen-free environment in the presence of IL-21, IL-15, and / or IL-7 so that cells with a Tcm phenotype can proliferate. This step is generally carried out for about 12 - 24 hours, about 12 - 36 hours, about 12 - 72 hours, 24 - 48 hours, 24 - 36 hours, about 24 - 72 hours, about 48 - 72 hours, 1 - 20 days, 1 - 15 days, 1 - 10 days, 1 - 5 days, 5 - 20 days, 5 - 15 days, 5 - 10 days, 1 - 2 days, 2 - 3 days, 1 - 3 days, 2 - 4 days, 2 - 5 days, 2 - 8 days, 2 - 10 days, 4 - 10 days, 4 - 8 days, 6 - 8 days, 8 - 10 days, 7 - 9 days, 7 - 11 days, 7 - 13 days, 7 - 15 days, 10 - 12 days, 10 - 14 days, 12 - 14 days, 14 - 16 days, 14 - 18 days, 16 - 18 days, or 18 - 20 days. According to a specific embodiment, the anti-third-party cells are cultured in an antigen-free environment in the presence of IL-21, IL-15, and IL-7 for about 7 - 11 days (e.g., 8 days).

[0241] This step is generally carried out in the presence of IL-21 at a concentration of about 0.001 - 3000 IU / ml, 0.01 - 3000 IU / ml, 0.1 - 3000 IU / ml, 1 - 3000 IU / ml, 10 - 3000 IU / ml, 100 - 3000 IU / ml, 1000 - 3000 IU / ml, 0.001 - 1000 IU / ml, 0.01 - 1000 IU / ml, 0.1 - 1000 IU / ml, 1 - 1000 IU / ml, 10 - 1000 IU / ml, 100 - 1000 IU / ml, 250 - 1000 IU / ml, 500 - 1000 IU / ml, 750 - 1000 IU / ml, 10 - 500 IU / ml, 50 - 500 IU / ml, 100 - 500 IU / ml, 250 - 500 IU / ml, 100 - 250 IU / ml, 0.1 - 100 IU / ml, 1 - 100 IU / ml, 10 - 100 IU / ml, 30 - 100 IU / ml, 50 - 100 IU / ml, 1 - 50 IU / ml, 10 - 50 IU / ml, 20 - 50 IU / ml, 30 - 50 IU / ml, 1 - 30 IU / ml, 10 - 30 IU / ml, 20 - 30 IU / ml, 10 - 20 IU / ml, 0.1 - 10 IU / ml, or 1 - 10 IU / ml IL-21.

[0242] According to a specific embodiment, the concentration of IL-21 is 50-150 IU / ml (e.g., 100 IU / ml).

[0243] This step is further carried out in the presence of IL-15 at a concentration of about 0.001-3000 IU / ml, 0.01-3000 IU / ml, 0.1-3000 IU / ml, 1-3000 IU / ml, 10-3000 IU / ml, 100-3000 IU / ml, 125-3000 IU / ml, 1000-3000 IU / ml, 0.001-1000 IU / ml, 0.01-1000 IU / ml, 0.1-1000 IU / ml, 1-1000 IU / ml, 10-1000 IU / ml, 100-1000 IU / ml, 125-1000 IU / ml, 250-1000 IU / ml, 500-1000 IU / ml, 750-1000 IU / ml, 10-500 IU / ml, 50-500 IU / ml, 100-500 IU / ml, 125-500 IU / ml, 250-500 IU / ml, 250-500 IU / ml, 125-250 IU / ml, 100-250 IU / ml, 0.1-100 IU / ml, 1-100 IU / ml, 10-100 IU / ml, 30-100 IU / ml, 50-100 IU / ml, 1-50 IU / ml, 10-50 IU / ml, 20-50 IU / ml, 30-50 IU / ml, 1-30 IU / ml, 10-30 IU / ml, 20-30 IU / ml, 10-20 IU / ml, 0.1-10 IU / ml or 1-10 IU / ml. According to a specific embodiment, the concentration of IL-15 is 100-150 IU / ml (e.g., 125 IU / ml).

[0244] This step is further carried out in the presence of IL-7 at a concentration of about 0.001 - 3000 IU / ml, 0.01 - 3000 IU / ml, 0.1 - 3000 IU / ml, 1 - 3000 IU / ml, 10 - 3000 IU / ml, 30 - 3000 IU / ml, 100 - 3000 IU / ml, 1000 - 3000 IU / ml, 0.001 - 1000 IU / ml, 0.01 - 1000 IU / ml, 0.1 - 1000 IU / ml, 1 - 1000 IU / ml, 10 - 1000 IU / ml, 30 - 1000 IU / ml, 100 - 1000 IU / ml, 250 - 1000 IU / ml, 500 - 1000 IU / ml, 750 - 1000 IU / ml, 10 - 500 IU / ml, 30 - 500 IU / ml, 50 - 500 IU / ml, 100 - 500 IU / ml, 250 - 500 IU / ml, 100 - 250 IU / ml, 0.1 - 100 IU / ml, 1 - 100 IU / ml, 10 - 100 IU / ml, 30 - 100 IU / ml, 50 - 100 IU / ml, 1 - 50 IU / ml, 10 - 50 IU / ml, 20 - 50 IU / ml, 30 - 50 IU / ml, 1 - 30 IU / ml, 10 - 30 IU / ml, 20 - 30 IU / ml, 10 - 20 IU / ml, 0.1 - 10 IU / ml or 1 - 10 IU / ml. According to a specific embodiment, the concentration of IL-7 is 10 - 50 IU / ml (e.g., 30 IU / ml).

[0245] The present inventors have collected through painstaking experiments and screening many criteria that can be used to improve the anti-third-party cell proliferation including the central memory T lymphocyte (Tcm) phenotype, and the anti-third-party cells have no graft-versus-host (GVH) reactive cells and / or are enhanced disease-resistant (e.g., GVL) reactive cells.

[0246] According to one embodiment, before contacting with one or more third-party antigens in the presence of IL-21, PBMCs are depleted of adherent cells.

[0247] According to one embodiment, before contacting with one or more third-party antigens in the presence of IL-21, PBMCs are depleted of CD4+ and / or CD56+ cells.

[0248] According to one embodiment, before contacting with one or more third-party antigens in the presence of IL-21, CD45RA+ cells of PBMCs are selected.

[0249] Deplete CD4 + and / or CD56 + Depletion of CD4 and / or CD56 cells can be carried out using any method known in the art, such as affinity-based purification (e.g., by using MACS beads, FACS sorters, and / or capture ELISA tags). To increase the purity of CD8 + cells in the culture (i.e., to remove other lymphocytes from the cell culture, such as T CD4 + cells or NK cells) or to increase the number of CD8 + T cells, such steps can be beneficial.

[0250] According to one embodiment, the PBMCs comprise non-adherent cells.

[0251] According to one embodiment, the PBMCs comprise CD8+ T cells.

[0252] According to one embodiment, the PBMCs comprise CD8+ T cells from a first experiment.

[0253] Selection of CD8+ T cells from a first experiment can be achieved by selecting cells that express CD45RA+ and / or cells that express CD45RO-, and can be carried out using any method known in the art, such as affinity-based purification (e.g., by using MACS beads, FACS sorters, and / or capture ELISA tags).

[0254] According to one embodiment, the PBMCs comprise CD45RA+ cells.

[0255] Additional steps that can be carried out in accordance with this teaching include culturing the PBMC cells with one or more third-party antigens in the presence of IL-21, IL-15, and IL-7 before removing one or more third-party antigens from the cell culture (i.e., before creating an antigen-free environment).

[0256] This step is generally carried out for about 12 - 24 hours, about 12 - 36 hours, about 12 - 72 hours, 24 - 48 hours, 24 - 36 hours, about 24 - 72 hours, about 48 - 72 hours, 1 - 2 days, 2 - 3 days, 1 - 3 days, 2 - 4 days, 1 - 5 days, or 2 - 5 days, and with the same doses of IL-21, IL-15, and IL-7 as specified above. According to a specific embodiment, culturing the PBMC cells with one or more third-party antigens in the presence of IL-21, IL-15, and IL-7 is carried out for 12 hours - 4 days (e.g., 1 - 2 days).

[0257] Additionally or alternatively, an additional two-step process enabling the selection and isolation of activated cells can be implemented. This selection step helps to remove potential host-reactive T cells (such as alloreactive cells) in cases where the PBMCs are non-homologous to the subject (as described in further detail below).

[0258] Thus, the isolation of activated cells can be implemented in a two-stage method. In the first stage, the activated cells are selected prior to culturing the cells in the presence of IL-15 and IL-7. This first stage is generally implemented after the initial contact of PBMCs with one or more third-party antigens in the presence of IL-21. This selection process only picks those cells activated by the third-party antigens (such as expressing activation markers as described below), and generally occurs about 12 - 24 hours, about 24 - 36 hours, about 12 - 36 hours, about 36 - 48 hours, about 12 - 48 hours, about 48 - 60 hours, about 12 - 60 hours, about 60 - 72 hours, about 12 - 72 hours, about 72 - 84 hours, about 12 - 84 hours, about 84 - 96 hours, about 12 - 96 hours after the initial contact of PBMCs with one or more third-party antigens.

[0259] According to a specific embodiment, the selection process occurs about 12 - 24 hours (such as 14 hours) after the initial contact of PBMCs with one or more third-party antigens.

[0260] The isolation of activated cells can be achieved by affinity-based purification (such as by using MACS beads, FACS sorters, and / or capture ELISA labeling), and can be achieved for any activation marker, including cell surface markers such as but not limited to CD69, CD44, CD25, CFSE, CD137 or non-cell surface markers such as but not limited to IFN-γ and IL-2. The isolation of activated cells can also be achieved by morphology-based purification (such as isolating large cells) using any method known in the art (such as by FACS). Generally, activated cells are also selected for CD8+ cell expression. Additionally, any combination of the above methods can be used to effectively isolate activated cells.

[0261] According to an embodiment of the present invention, the selection of activated cells is achieved by selecting CD137+ and / or CD25+ cells.

[0262] Generally, a second stage of isolating activated cells is performed at the end of culturing (i.e., after culturing with IL-21, IL-15, and IL-7 in an antigen-free environment). This stage depletes alloreactive cells by depleting those cells that are activated after central memory T lymphocytes (Tcm) contact irradiated host antigen-presenting cells (APCs, such as dendritic cells). As mentioned above, isolating activated cells can be achieved by affinity-based purification (e.g., by using MACS beads, FACS sorters, and / or capture ELISA labeling), and can be achieved for any activation marker, including cell surface markers such as but not limited to CD69, CD44, CD25, CFSE, CD137, or non-cell surface markers such as but not limited to IFN-γ and IL-2.

[0263] According to one embodiment of the invention, depleting alloreactive cells is achieved by depleting CD137+ and / or CD25+ cells and / or IFN-γ capture.

[0264] The following are exemplary protocols that can be used according to some embodiments of the invention.

[0265] One embodiment of the invention provides a method of generating isolated cells with a central memory phenotype, the cells being tolerance-inducing cells and capable of homing to lymph nodes after transplantation, the method comprising: (a) contacting peripheral blood mononuclear cells (PBMCs) with one or more third-party antigens in the presence of IL-21 (e.g., for 12 hours to 5 days), so as to enrich antigen-reactive cells; and (b) culturing the cells generated from step (a) in an antigen-free environment in the presence of IL-21, IL-15, and IL-7 (e.g., for 5 to 20 days), so that anti-third-party cells comprising a central memory T lymphocyte (Tcm) phenotype can proliferate.

[0266] According to one embodiment, the method further comprises: (c) separating the cells generated from step (b) into a single cell suspension.

[0267] According to one embodiment, the method further comprises depleting adherent cells from PBMCs before step (a).

[0268] According to one embodiment, the method further comprises depleting CD4+ and / or CD56+ cells from PBMCs before step (a).

[0269] According to one embodiment, the method further comprises selecting activated cells after step (a) and before step (b).

[0270] According to one embodiment, the method further comprises selecting activated cells by selecting CD137+ and / or CD25+ cells.

[0271] One embodiment of the invention provides a method for generating isolated cells having a central memory phenotype, the cells being tolerance-inducing cells and capable of homing to lymph nodes after transplantation, the method comprising: (a) treating non-adherent peripheral blood mononuclear cells (PBMCs) with a substance capable of depleting CD4+ and / or CD56+ cells so as to obtain CD8+ T cells; (b) contacting the CD8+ T cells with third-party dendritic cells in the presence of IL-21 (e.g., for 12 hours to 5 days) so as to enrich antigen-reactive cells; (c) culturing the cells generated from step (b) with third-party dendritic cells in the presence of IL-21, IL-15 and IL-7 (e.g., for 12 hours to 3 days); and (d) culturing the cells generated from step (c) in an antigen-free environment in the presence of IL-21, IL-15 and IL-7 (e.g., for 5 to 20 days) so as to allow the proliferation of cells comprising a central memory T lymphocyte (Tcm) phenotype.

[0272] According to one embodiment, the method further comprises separating the cells generated from step (d) into a single cell suspension.

[0273] According to one embodiment, the anti-third-party cells comprising the Tcm phenotype comprise CD3 + , CD8 + , CD62L + , CD45RA - , CD45RO + characteristics.

[0274] It should be appreciated that at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or even 100% of the anti-third-party cells are CD3+CD8+ cells. According to a specific embodiment, the anti-third-party cells comprise about 70-90% CD3+CD8+ cells.

[0275] It should be appreciated that at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or even 100% of the CD3+CD8+ cells have Tcm cell characteristics. According to a specific embodiment, about 30-80% of the CD3+CD8+ cells have Tcm cell characteristics (e.g., 40-50%).

[0276] According to one embodiment, at least 50% of the cells are CD3+CD8+ cells, and at least 50% of the CD3+CD8+ cells have the characteristics.

[0277] Thus, the cells of the invention having a central memory T lymphocyte (Tcm) phenotype are not naturally occurring and are not a product of nature. These cells are generally generated by ex vivo manipulation (i.e., exposure to one or more third-party antigens in the presence of specific cytokines).

[0278] As mentioned, the Tcm cells of the invention are transduced with a polynucleotide encoding a cell surface receptor comprising a T cell receptor signaling module.

[0279] As used herein, the term "polynucleotide" refers to a single-stranded or double-stranded nucleic acid sequence provided in isolated form as an RNA sequence, complementary polynucleotide sequence (cDNA), genomic polynucleotide sequence, and / or composite polynucleotide sequence (e.g., a combination of the above).

[0280] The term "isolated" refers to being at least partially separated from its natural environment, such as being separated from a cell or separated from a tissue, such as being separated from a human body.

[0281] Isolated polynucleotides can be obtained using recombinant methods known in the art, such as using standard techniques, screening libraries from cells expressing the gene, deriving the gene from a vector known to contain the gene, or directly isolating from cells and tissues containing the gene. Alternatively, the target gene can be synthesized rather than cloned.

[0282] The polynucleotides of some embodiments of the invention can comprise a single polynucleotide comprising a nucleic acid sequence encoding an extracellular domain, transmembrane domain, and / or signaling module of a cell surface receptor (e.g., tg-TCR and / or CAR). Alternatively, two or more polynucleotides can be used, where one polynucleotide can comprise a nucleic acid sequence encoding, for example, an extracellular domain and a transmembrane domain, and another polynucleotide can comprise a nucleic acid sequence encoding a signaling module.

[0283] One aspect of some embodiments of the invention provides a nucleic acid construct comprising an isolated polynucleotide comprising a nucleic acid sequence encoding a molecule of some embodiments of the invention and cis-acting regulatory elements for directing transcription of the isolated polynucleotide in a host cell.

[0284] Thus, expression of a native or synthetic nucleic acid encoding a cell surface receptor of the invention (e.g., a tg-TCR or CAR molecule) is generally achieved by operably linking a nucleic acid encoding a cell surface receptor (e.g., a tg-TCR or CAR) polypeptide or a portion thereof to a cis-acting regulatory element (e.g., a promoter sequence) and incorporating the construct into an expression vector.

[0285] The nucleic acid constructs of the invention may also include enhancers, transcription and translation initiation sequences, transcription and translation terminators and polyadenylation signals, 5' LTR, tRNA binding sites, packaging signals, origins of second strand DNA synthesis, and 3' LTR or portions thereof; additional polynucleotide sequences that enable, for example, the translation of several proteins from a single-stranded mRNA (such as an internal ribosome entry site (IRES)), and sequences for genomic integration of promoter-chimeric polypeptides; sequences engineered to enhance the stability, production, purification, yield, or toxicity of the expressed peptide.

[0286] Enhancers regulate the frequency of transcriptional initiation. Generally, promoter elements are located in the region from 30 to 110 bp upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible such that promoter function is maintained when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased up to 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can act cooperatively or independently to activate transcription.

[0287] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of inducible promoters provides a molecular switch that can turn on the expression of an operably linked polynucleotide sequence when such expression is desired, or turn off the expression when expression is not desired. Examples of inducible promoters include but are not limited to metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0288] The isolated polynucleotides of the present invention can be cloned into many types of vectors. For example, the isolated polynucleotides can be cloned into vectors including but not limited to: plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particularly interesting vectors include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.

[0289] Examples of mammalian expression vectors include but are not limited to pcDNA3, pcDNA3.1 (+ / -), pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 (available from Invitrogen), pCI (available from Promega), pMbac, pPbac, pBK-RSV, and pBK-CMV (available from Strategene), pTRES (available from Clontech), and their derivatives.

[0290] Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can also be used. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papillomavirus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A +, pMTO10 / A + , pMAMneo-5, baculovirus pDSVE, and any other vector that enables the expression of a protein under the direction of an SV-40 early promoter, SV-40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoter that has been shown to be effective for expression in eukaryotic cells.

[0291] Currently preferred in vivo or in vitro nucleic acid transfer techniques include transfection with viral or non-viral constructs such as adenovirus, lentivirus, herpes simplex virus type I, or adeno-associated virus (AAV). Recombinant viral vectors offer advantages such as lateral infection and target specificity. Introduction of nucleic acids by viral infection offers several advantages over other methods such as liposome transfection and electroporation, as higher transfection efficiencies can be achieved due to the infectious nature of the virus.

[0292] Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain an origin of replication that is functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584, WO 01 / 29058, and U.S. Patent No. 6326193).

[0293] According to some embodiments of the present invention, the nucleic acid construct of the present invention is a viral vector.

[0294] Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they enable long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus because they can transduce non-proliferating cells, such as hepatocytes.

[0295] In addition, lentiviral vectors offer greater gene insertion capacity and also have the additional advantage of low immunogenicity. Alternatively, γ-retroviral vectors can be used. γ-retroviral vectors have good transduction efficiency and no vector-related toxicity [see, for example, Zhang and Morgan, Adv Drug Deliv Rev. (2012), supra].

[0296] For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered in vivo or ex vivo to the cells of a subject.

[0297] To assess the expression of a cell surface receptor (e.g., tg-TCR or CAR) polypeptide or a portion thereof, the nucleic acid construct to be introduced into the cells can also contain a selectable marker gene or a reporter gene or both to facilitate the identification and selection of expressing cells from a population of cells that have been transfected or infected with a viral vector. In other aspects, the selectable marker can be carried on a separate DNA fragment and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

[0298] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and encodes a polypeptide whose expression is manifested by some readily detectable property such as enzyme activity. After introducing the DNA into the recipient cells, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes can include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, constructs having a minimal 5' flanking region that shows the highest level of reporter gene expression are identified as promoters. Such promoter regions can be ligated to the reporter gene and used to assess the ability of a substance to modulate promoter-driven transcription.

[0299] The nucleic acid constructs of the present invention can be introduced into host cells, such as mammalian, bacterial, yeast or insect cells, using a variety of methods. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995); Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995); Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. [Biotechniques 4 (6): 504-512, 1986], and include physical, chemical or biological means (such as stable or transient transfection, liposome transfection, electroporation and infection with recombinant viral vectors). Additionally, for positive-negative selection methods see U.S. Patent Nos. 5,464,764 and 5,487,992.

[0300] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0301] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0302] Biological methods for introducing a target polynucleotide into a host cell include the use of DNA and RNA vectors (as described above). Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses, adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0303] In cases where a non-viral delivery system is employed, an exemplary delivery vehicle is a liposome.

[0304] "Liposome" is an umbrella term that includes a variety of single and multi-layer lipid carriers formed by the generation of closed lipid bilayers or aggregates. Liposomes can be characterized as vesicular structures having a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous media. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.

[0305] The lipid components undergo self-rearrangement before the formation of the closed structure and trap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions having structures different from normal vesicular structures in solution are also included. For example, the lipids can assume a micellar structure or exist only as non-uniform aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0306] The use of lipid formulations for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo) is contemplated. On the other hand, nucleic acids can be associated with lipids. Nucleic acids associated with lipids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, linked to liposomes via linking molecules that associate with both liposomes and oligonucleotides, trapped within liposomes, complexed with liposomes, dispersed in solutions containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contain micelles or be complexed with micelles, or otherwise associated with lipids. Compositions associated with lipids, lipid / DNA, or lipid / expression vectors are not limited to any particular structure in solution. For example, it can exist in a bilayer structure, as micelles or with a "collapsed" structure. It can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids can be naturally occurring fatty substances or synthetic lipids. For example, lipids include the fat droplets naturally present in the cytoplasm and the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0307] Suitable lipids for use are available from commercial sources. For example, dimyristoyl phosphatidylcholine (“DMPC”) is available from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) is available from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) is available from Calbiochem-Behring; dimyristoyl phosphatidylglycerol (“DMPG”); and other lipids are available from Avanti Polar Lipids, Inc, (Birmingham, Ala.). Additionally or alternatively, DOTMA, DOPE, and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)] lipids can be used. Lipid stock solutions in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol.

[0308] Another exemplary non-viral delivery system that can be used in accordance with the present invention is a transposon-based non-viral gene delivery system, such as, for example, Sleeping Beauty or PiggyBac.

[0309] Regardless of the method used to introduce exogenous nucleic acid into a host cell, in order to confirm the presence of a recombinant DNA sequence in the host cell, a variety of assays can be performed. Such assays include, for example, “molecular biology” assays well known to those skilled in the art, such as Southern and Northern blots, RT-PCR, and PCR; “biochemical” assays, such as, for example, detecting the presence or absence of a specific peptide by immunological means (ELISA and Western blot) or by the assays described herein to identify substances that fall within the scope of the present invention.

[0310] It should be appreciated that cells transduced with cell surface receptors (e.g., tg-TCR and / or CAR) can be further genetically modified to inhibit the expression of at least one endogenous immune checkpoint gene in the cell.

[0311] The immune checkpoint gene can comprise a PD or CTLA gene.

[0312] As used herein, the term “immune checkpoint gene” refers to any gene that participates in an inhibitory process (e.g., a feedback loop) used to regulate the amplitude of an immune response, such as an immunosuppressive feedback loop that reduces the uncontrolled spread of a harmful immune response.

[0313] Non-limiting examples of immune checkpoint genes include members of the extended CD28 family of receptors and their ligands, as well as genes that participate in co-inhibitory pathways (e.g., CTLA-4 and PD-1).

[0314] Thus, according to one embodiment, PD1 and / or CTLA-4 targeted nucleases or transcription repressors can be employed, as discussed in U.S. Patent Application No. 20140120622, which is incorporated herein by reference.

[0315] Additionally or alternatively, immune checkpoint proteins that regulate T cell activation or function include, for example, PD1, PDL-1, B7H2, B7H4, CTLA-4, CD80, CD86, LAG-3, TIM-3, KIR, IDO, CD19, OX40, 4-1BB (CD137), CD27, CD70, CD40, GITR, CD28, and / or ICOS (CD278), and can be regulated (e.g., upregulated or downregulated as needed) in transduced cells by using immune checkpoint regulators.

[0316] According to specific embodiments, the immune checkpoint regulators are selected from anti-CTLA4, anti-PD-1, anti-PDL-1, CD40 agonists, 4-1BB agonists, GITR agonists, and OX40 agonists.

[0317] One aspect of some embodiments of the present invention provides a cell population comprising the isolated cells of some embodiments of the present invention.

[0318] The isolated cells or cell populations of some embodiments of the present invention can be administered to an organism, either alone or in a pharmaceutical composition mixed with a suitable carrier or excipient.

[0319] As used herein, "pharmaceutical composition" refers to a formulation of one or more active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to an organism.

[0320] As used herein, the term "active ingredient" refers to the cells of some embodiments of the present invention that can elicit a biological effect.

[0321] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier", which can be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to an organism and does not eliminate the biological activity and properties of the administered compound. Adjuvants are included in these phrases.

[0322] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of the active ingredient. Examples of excipients (non-limitingly) include calcium carbonate, calcium phosphate, various sugars, various types of starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0323] Techniques for formulating and administering drugs can be found in “Remington’s Pharmaceutical Sciences”, Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0324] For example, suitable routes of administration can include oral, rectal, transmucosal (especially nasal), enteral or parenteral delivery, including intramuscular, subcutaneous and intramedullary injection as well as intrathecal, direct intraventricular, intracardiac (e.g., into the right or left ventricular cavity, into the common coronary artery), intravenous, intraperitoneal, intranasal or intraocular injection.

[0325] Conventional methods for delivering drugs to the central nervous system (CNS) include: neurosurgery (e.g., intracerebral injection or lateral ventricular infusion), molecular manipulation of substances that attempt to utilize one of the endogenous transport pathways of the BBB (e.g., generating chimeric fusion proteins that contain a transport peptide with affinity for endothelial cell surface molecules in combination with a substance that cannot cross the BBB itself); pharmacological strategies designed to increase the lipophilicity of the substance (e.g., conjugation of a water-soluble substance with a lipid or cholesterol carrier); and transient disruption of the integrity of the BBB due to hyperosmotic disruption (caused by infusion of mannitol solution into the carotid artery or use of bioactive substances such as angiotensin peptides). However, each of these strategies has limitations, such as the inherent risks associated with invasive surgery, size limitations imposed by the limitations inherent in the endogenous transport system, potential unwanted biological side effects associated with systemic administration of chimeric molecules containing carrier motifs that may be active outside the CNS, and the risk of brain injury that may exist in the brain regions where the BBB is disrupted, which makes them not the most ideal delivery methods.

[0326] Alternatively, the pharmaceutical composition can be administered in a local rather than a systemic manner, for example by directly injecting the pharmaceutical composition into a tissue area of the patient.

[0327] According to one embodiment, the route of administration includes, for example, injection, ingestion, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullarily, intramuscularly, by intravenous (i.v.) injection or intraperitoneally. In one embodiment, the pharmaceutical composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the pharmaceutical composition of the present invention is preferably administered by i.v. injection. The pharmaceutical composition can be directly injected into a tumor, lymph node or site of infection.

[0328] The pharmaceutical compositions of some embodiments of the present invention can be prepared by methods well known in the art, such as by conventional mixing, dissolving, granulating, sugar coating, levitating, emulsifying, encapsulating, entrapping or lyophilizing methods.

[0329] Thus, the pharmaceutical compositions for use in some embodiments of the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers, which carriers include excipients and auxiliaries that facilitate the processing of the active ingredient into a pharmaceutically acceptable preparation. The appropriate formulation depends on the chosen route of administration.

[0330] For injection, the active ingredient of the pharmaceutical composition can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution or physiological saline buffer. For transmucosal administration, penetrants suitable for the permeation barrier are used in the formulation. Such penetrants are generally known in the art.

[0331] For oral administration, the pharmaceutical composition can be readily formulated by combining the active compound with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by a patient. Solid excipients can be used to prepare the pharmaceutical preparations for oral use, and the resulting mixture is optionally ground, and if necessary, the particulate mixture is processed after adding suitable auxiliaries to obtain tablets or dragee cores. Suitable excipients are specifically fillers such as saccharides, including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If necessary, disintegrants can be added, such as cross-linked polyvinylpyrrolidone, agar or alginic acid or its salts such as sodium alginate.

[0332] The dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions can be used which optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solution and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to the tablet or dragee coatings for identifying or characterizing different combinations of the active compound dosage.

[0333] Pharmaceutical compositions for oral use include push-fit capsules made of gelatin and soft-sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules may contain an active ingredient mixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In the soft capsules, the active ingredient may be dissolved or suspended in a suitable liquid such as a fatty oil, liquid paraffin, or liquid polyethylene glycol. Additionally, a stabilizer may be added. All formulations for oral administration should be present in a dosage suitable for the chosen route of administration.

[0334] For buccal administration, the composition may take the form of tablets or lozenges formulated in a conventional manner.

[0335] For administration by nasal inhalation, the active ingredient for use in some embodiments of the present invention is conveniently delivered in the form of an aerosol spray from a pressurized pack or a nebulizer, wherein a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide is used. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Gelatin capsules and cartridges for use in a dispenser may be formulated to contain a powder mixture of the compound and a suitable powder matrix such as lactose or starch.

[0336] The pharmaceutical compositions described herein may be formulated for parenteral administration, for example, by bolus injection or continuous infusion.

[0337] Preparations for injection may be presented in unit dosage form, for example, in ampoules or multi-dose containers, optionally with the addition of a preservative. The composition may be a suspension, solution, or emulsion in an oily or aqueous vehicle and may contain formulating agents such as suspending agents, stabilizers, and / or dispersing agents.

[0338] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active agent in water-soluble form. Additionally, suspensions of the active ingredient may be prepared as suitable oily or aqueous-based injectable suspensions. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes.

[0339] Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or substances that increase the solubility of the active ingredient to enable the preparation of highly concentrated solutions.

[0340] Alternatively, the active ingredient may be present in powder form for constitution with a suitable vehicle such as a sterile pyrogen-free aqueous solution before use.

[0341] The pharmaceutical compositions of some embodiments of the present invention may also be formulated as rectal compositions such as suppositories or retention enemas using, for example, conventional suppository bases such as cocoa butter or other glycerides.

[0342] Pharmaceutical compositions suitable for use in the context of the present invention include those in which the active ingredient is included in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of the active ingredient effective to prevent, alleviate or ameliorate pathological symptoms or to prolong the survival of the subject being treated.

[0343] Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, particularly in light of the detailed disclosure provided herein.

[0344] When referring to a "therapeutic amount", the exact amount of the composition of the present invention to be administered can be determined by a physician taking into account the age, weight, disease state of the patient (subject), such as the size of the tumor, the degree of infection or metastasis, and individual variations in condition. Generally speaking, a pharmaceutical composition comprising the cells described herein can be administered at a dose of 10 4 -10 9 cells / kg body weight, including all integer values within those ranges.

[0345] For example, the number of cells infused into the recipient should be more than 1 x 10 4 / Kg body weight. The number of cells infused into the recipient should generally be in the range of 1 x 10 3 / Kg body weight - 1 x 10 4 / Kg body weight, in the range of 1 x 10 4 / Kg body weight - 1 x 10 5 / Kg body weight, in the range of 1 x 10 4 / Kg body weight - 1 x 10 6 / Kg body weight, in the range of 1 x 10 4 / Kg body weight - 1 x 10 7 / Kg body weight, in the range of 1 x 10 4 / Kg body weight - 1 x 10 8 / Kg body weight, in the range of 1 x 10 3 / Kg body weight - 1 x 10 5 / Kg body weight, in the range of 1 x 10 4 / Kg body weight - 1 x 10 6 / Kg body weight, in the range of 1 x 10 6 / Kg body weight - 1 x 10 7 / Kg body weight, in the range of 1 x 10 5 / Kg body weight - 1 x 10 7in the range of 1 x 10 6 / Kg body weight - 1 x 10 8 / Kg body weight, or in the range of 1 x 10 6 / Kg body weight - 1 x 10 9 / Kg body weight. According to a specific embodiment, the number of cells infused to the recipient should be in the range of 1 x 10 6 / Kg body weight - 1 x 10 8 / Kg body weight.

[0346] The cell compositions of some embodiments of the present invention can also be administered multiple times at these doses. The cells can be administered by using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a particular patient can be readily determined by those skilled in the medical art by monitoring the patient's signs of disease and adjusting the treatment accordingly.

[0347] For example, the effect of the active ingredient (such as the cells of some embodiments of the present invention) on the pathology can be evaluated by monitoring the levels of markers (such as hormones, glucose, peptides, carbohydrates, etc.) in biological samples using well-known methods (such as ELISA, FACS, etc.) or by monitoring the tumor size using well-known methods (such as ultrasound, CT, MRI, etc.).

[0348] For any formulation used in the methods of the present invention, the therapeutically effective amount or dose can initially be estimated from in vitro and cell culture assays. For example, the dose can be formulated in an animal model to obtain the desired concentration or titer. This information can be used to more accurately determine the useful dose for humans.

[0349] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined in vitro by standard pharmaceutical procedures, using cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used to formulate a dose range for humans.

[0350] The dose can vary depending on the dosage form used and the route of administration employed. The exact dosage form, route of administration, and dose can be selected by the individual physician in view of the patient's condition (see, for example, Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics” Ch. 1 p.1).

[0351] The dosage and interval can be adjusted individually to provide a level of active ingredient sufficient to induce or inhibit a biological effect (minimum effective concentration, MEC). The MEC varies for each formulation but can be estimated from in vitro data. The dosage necessary to obtain the MEC depends on individual characteristics and the route of administration. Assay analysis can be used to determine plasma concentration.

[0352] Depending on the severity and responsiveness of the condition to be treated, administration can be single or multiple, and the course of treatment can last from several days to several weeks or until cure is achieved or a reduction in the disease state is realized.

[0353] Of course, the amount of the composition to be administered will depend on the subject being treated, the severity of the affliction, the mode of administration, the judgment of the prescribing physician, etc.

[0354] According to some embodiments of the present invention, the therapeutic agent of the present invention can be provided to a subject together with other drugs designed to treat a pathology [combination therapy (e.g., before, simultaneously, or after)].

[0355] In certain embodiments of the present invention, the cells of some embodiments of the present invention are administered to a patient together with any number of the following related treatment modalities: including but not limited to treatment with the following drugs, such as antiviral drugs (e.g., ganciclovir, valacyclovir, acyclovir, valganciclovir, foscarnet, cidofovir, maribavir, leflunomide), chemotherapeutic drugs (e.g., anti-tumor drugs, such as but not limited to alkylating agents, including for example cyclophosphamide, busulfan, nitrogen mustard or mechlorethamine (HN2), uramustine or uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, nitrosoureas, carmustine, lomustine, streptozocin, thiotepa, platinum, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, procarbazine, hexamethylmelamine, triazenes (dacarbazine, mitozolomide, temozolomide), dacarbazine, temozolomide, busulfan, busulphan, fludarabine, dimethylbusulfan or cytarabine), drugs for treating MS (e.g., natalizumab), or drugs for treating psoriasis (e.g., efalizumab).

[0356] In a further embodiment, the cells of some embodiments of the present invention can be used in combination with chemotherapy, radiation, immunosuppressive drugs (e.g., cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506), antibodies, or other immune-depleting drugs (discussed in further detail below).

[0357] In a further embodiment, the cell composition of some embodiments of the present invention is administered to a patient together with a bone marrow transplant (e.g., before, simultaneously, or after).

[0358] In a further embodiment, the cell compositions of some embodiments of the invention are administered to a patient following T cell depletion therapy using, for example, chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide or antibodies such as OKT3 or CAMPATH.

[0359] In another embodiment, the cell compositions of the invention are administered following B cell depletion therapy such as a drug reactive with CD20, for example Rituxan.

[0360] Combination therapy can increase the therapeutic effect of the drugs of the invention in the subject being treated.

[0361] If desired, the compositions of some embodiments of the invention can be presented in a package or dispenser device, such as an FDA-approved kit, which can contain one or more unit dosage forms containing the active ingredient. The package can, for example, comprise a metal or plastic foil, such as a blister pack. The package or dispenser device can be accompanied by instructions for administration. The package or dispenser can also bear a notice associated with the container in a form prescribed by a government agency regulating the manufacture, use or sale of pharmaceuticals, which notice reflects the approval of the composition for use in humans or veterinarians. For example, such notice can be a label approved by the U.S. Food and Drug Administration for prescription drugs or an approved product insert. Compositions containing the formulations of the invention formulated with a compatible pharmaceutical carrier can also be prepared, placed in an appropriate container, and labeled for the treatment of the indicated conditions as further detailed above.

[0362] The kit can, for example, comprise a metal or plastic foil, such as a blister pack. The package or dispenser device can be accompanied by instructions for administration. The package or dispenser can also bear a notice associated with the container in a form prescribed by a government agency regulating the manufacture, use or sale of pharmaceuticals, which notice reflects the approval of the composition for use in humans or veterinarians. For example, such notice can be a label approved by the U.S. Food and Drug Administration for prescription drugs or an approved product insert. Compositions containing the formulations of the invention formulated with a compatible pharmaceutical carrier can also be prepared, placed in an appropriate container, and labeled for the treatment of the indicated conditions as further detailed above.

[0363] According to one embodiment, the kit further comprises a chemotherapeutic agent (e.g., an anti-tumor agent as discussed in detail above).

[0364] According to one embodiment, the kit further comprises an antiviral agent (as discussed in detail above).

[0365] One aspect of some embodiments of the present invention provides a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a cell population of some embodiments of the present invention, thereby treating the subject.

[0366] One aspect of some embodiments of the present invention provides a therapeutically effective amount of a cell population of some embodiments of the present invention for treating a disease in a subject in need thereof.

[0367] The term "treatment" refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and / or causing a reduction, remission or regression of the pathology. Those skilled in the art will understand that various methodologies and assays can be used to evaluate the development of a pathology, and similarly, various methodologies and assays can be used to evaluate the reduction, remission or regression of a pathology.

[0368] As used herein, the term "subject" includes mammals, preferably humans of any age or sex suffering from a pathology.

[0369] The pathology can be, but is not limited to, a malignant disease (cancer), an infectious disease (e.g., viral infection, bacterial infection, fungal infection, protozoal infection or parasitic infection), an allergy and / or an autoimmune disease.

[0370] Cancerous diseases

[0371] The malignant diseases (also referred to as cancers) that can be treated by the methods of some embodiments of the present invention can be any solid or non-solid tumor and / or tumor metastasis.

[0372] Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia. More specifically, examples of such cancers include squamous cell carcinoma, soft tissue sarcoma, Kaposi's sarcoma, melanoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, rectal cancer, endometrial cancer or uterine cancer, carcinoid carcinoma, salivary gland cancer, renal cancer or kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, mesothelioma, multiple myeloma, post-transplant lymphoproliferative disorder (PTLD) and various types of head and neck cancers (e.g., brain tumors). Cancerous conditions suitable for treatment by the present invention include metastatic cancers.

[0373] According to one embodiment, the malignant disease is a hematological malignancy. Exemplary hematological malignancies include, but are not limited to, leukemia [such as acute lymphoblastic leukemia, acute lymphocytic leukemia, acute lymphoblastic precursor B-cell leukemia, acute lymphoblastic T-cell leukemia, acute megakaryocytic leukemia, monocytic leukemia, acute myeloid leukemia, acute myelogenous leukemia, acute myeloid leukemia with eosinophilia, B-cell leukemia, basophilic leukemia, chronic myelogenous leukemia, chronic leukemia, B-cell leukemia, eosinophilic leukemia, Friend leukemia, granulocytic leukemia or myelocytic leukemia, hairy cell leukemia, lymphocytic leukemia, megakaryocytic leukemia, monocytic leukemia, monocyte-macrophage leukemia, myeloblastic leukemia, myelogenous leukemia, myelomonocytic leukemia, plasma cell leukemia, precursor B-cell leukemia, promyelocytic leukemia, subacute leukemia, T-cell leukemia, lymphoid neoplasms, predisposition to myeloid malignancies, acute non-lymphocytic leukemia, T-cell acute lymphoblastic leukemia (T-ALL), and B-cell chronic lymphocytic leukemia (B-CLL)] and lymphoma [such as Hodgkin's disease, non-Hodgkin lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, histiocytic lymphoma, lymphoblastic lymphoma, T-cell lymphoma, thymic lymphoma, B-cell lymphoma (including low-grade / follicular lymphoma), small lymphocyte (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia].

[0374] According to a specific embodiment, the malignant disease is leukemia, lymphoma, myeloma, melanoma, sarcoma, neuroblastoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, esophageal cancer, synovial cell carcinoma, or pancreatic cancer.

[0375] According to some embodiments of the present invention, the pathology is a solid tumor.

[0376] According to some embodiments of the present invention, the pathology is tumor metastasis.

[0377] According to some embodiments of the present invention, the pathology is a hematological malignancy.

[0378] According to some embodiments of the present invention, the pathology is leukemia or lymphoma.

[0379] Exemplary malignant diseases that can be treated by the methods of some embodiments of the present invention are listed in Tables 1 and 2 below.

[0380] Table 1: Clinical applications of tg-TCR-transduced cells and optional pretreatment regimens

[0381] (Adapted from Fujiwara, Pharmaceuticals (2014), 7: 1049-1068)

[0382] Table 2: Clinical applications of CAR-transduced cells and optional pretreatment regimens

[0383] (Adapted from Fujiwara, Pharmaceuticals (2014), 7: 1049-1068)

[0384] According to a specific embodiment, the malignant disease is leukemia, lymphoma, myeloma, melanoma, sarcoma, neuroblastoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, esophageal cancer, synovial cell carcinoma, and pancreatic cancer.

[0385] Infectious diseases

[0386] Examples of infectious diseases include, but are not limited to, chronic infectious diseases, subacute infectious diseases, acute infectious diseases, viral diseases, bacterial diseases, protozoal diseases, parasitic diseases, fungal diseases, mycoplasmal diseases, and prion diseases.

[0387] Specific types of viral pathogens that cause infectious diseases treatable according to the teachings of the present invention include, but are not limited to, retroviruses, circoviruses, parvoviruses, papovaviruses, adenoviruses, herpesviruses, iridoviruses, poxviruses, hepadnaviruses, picornaviruses, caliciviruses, togaviruses, flaviviruses, reoviruses, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, coronaviruses, arenaviruses, and filoviruses.

[0388] Specific examples of viral infections treatable according to the teachings of the present invention include, but are not limited to, acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV), influenza, rhinovirus infection, viral meningitis, Epstein-Barr virus (EBV) infection, hepatitis A, B, or C virus infection, measles, papillomavirus infection / warts, cytomegalovirus (CMV) infection, herpes simplex virus infection, yellow fever, Ebola virus infection, and rabies.

[0389] According to a specific embodiment, the viral disease is selected from immunodeficiency virus (HIV), influenza, cytomegalovirus (CMV), T-cell leukemia virus type 1 (TAX), hepatitis C virus (HCV), and hepatitis B virus (HBV).

[0390] Allergic diseases

[0391] Examples of allergic diseases include, but are not limited to, asthma, urticaria, rubella, pollen allergy, dust mite allergy, venom allergy, cosmetic allergy, latex allergy, chemical allergy, drug allergy, insect bite allergy, animal fur allergy, stinging plant allergy, poison ivy allergy, and food allergy.

[0392] Autoimmune diseases

[0393] include, but are not limited to, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, skin diseases, liver diseases, nerve diseases, muscle diseases, kidney diseases, reproductive-related diseases, connective tissue diseases, and systemic diseases.

[0394] Examples of autoimmune cardiovascular diseases include, but are not limited to, atherosclerosis (Matsuura E. et al. , Lupus. 1998; 7 Suppl 2:S135); myocardial infarction (Vaarala O. Lupus. 1998; 7 Suppl 2:S132); thrombosis (Tincani A. et al. , Lupus 1998; 7 Suppl 2:S107-9); Wegener's granulomatosis, Takayasu arteritis, Kawasaki syndrome (Praprotnik S. et al. , Wien Klin Wochenschr 2000 Aug 25; 112(15-16):660); autoimmune diseases against factor VIII (Lacroix-Desmazes S. et al. , Semin ThrombHemost. 2000; 26 (2):157); necrotizing small-vessel vasculitis, microscopic polyangiitis, Churg-Strauss syndrome, pauci-immune focal necrotizing and crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris).2000 May; 151 (3):178); antiphospholipid syndrome (Flamholz R. et al. , J Clin Apheresis 1999;14 (4):171); antibody-induced heart failure (Wallukat G. et al. , Am J Cardiol. 1999 Jun 17; 83(12A):75H); thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 Apr-Jun; 14 (2):114; Semple JW. et al. ,Blood 1996 May 15; 87 (10):4245); Autoimmune hemolytic anemia (Efremov DG, et al. , Leuk Lymphoma 1998 Jan; 28 (3-4):285; Sallah S, et al. , Ann Hematol 1997 Mar; 74 (3):139); Cardiac autoimmunity in Chagas disease (Cunha-Neto E, et al. , J Clin Invest 1996 Oct 15; 98 (8):1709) and anti-helper T lymphocyte autoimmunity (Caporossi AP, et al. , Viral Immunol 1998; 11 (1):9).

[0395] Examples of autoimmune rheumatic diseases include, but are not limited to, rheumatoid arthritis [Krenn V, et al. , Histol Histopathol (2000) 15 (3):791; Tisch R and McDevitt HO. Proc Natl Acad Sci USA (1994) 18; 91(2): 437-438] and ankylosing spondylitis [Jan Voswinkel, et al. , Arthritis Res (2001) 3 (3): 189].

[0396] Examples of autoimmune glandular diseases include, but are not limited to, pancreatic diseases, type I diabetes, thyroid diseases, Graves' disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune antisperm infertility, autoimmune prostatitis, and type I autoimmune polyglandular syndrome. Diseases include, but are not limited to, pancreatic autoimmune diseases, type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 Oct; 34 Suppl:S125); autoimmune thyroid diseases, Graves' disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 Jun; 29 (2):339; Sakata S, et al. ,Mol Cell Endocrinol 1993 Mar;92 (1):77); Spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec 15; 165 (12):7262); Hashimoto's thyroiditis (Toyoda N. et al. , Nippon Rinsho 1999 Aug;57 (8):1810); Idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug; 57 (8):1759); Ovarian autoimmunity (Garza KM. et al. , J Reprod Immunol 1998 Feb; 37 (2):87); Autoimmune antisperm infertility (Diekman AB. et al. , Am J Reprod Immunol. 2000 Mar; 43 (3):134); Autoimmune prostatitis (Alexander RB. et al. , Urology 1997 Dec; 50 (6):893) and type I autoimmune polyglandular syndrome (Hara T. et al. , Blood. 1991 Mar 1; 77 (5):1127).

[0397] Examples of autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory bowel disease (Garcia Herola A. et al. , Gastroenterol Hepatol. 2000 Jan; 23 (1):16); Celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16; 138 (2):122); Colitis, ileitis, and Crohn's disease.

[0398] Examples of autoimmune skin diseases include, but are not limited to, autoimmune bullous skin diseases, such as, but not limited to, pemphigus vulgaris, bullous pemphigoid, and pemphigus foliaceus.

[0399] Examples of autoimmune liver diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol 1990 Mar; 54 (3):382); primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov; 91 (5):551; Strassburg CP. et al. , Eur J Gastroenterol Hepatol. 1999 Jun; 11 (6):595) and autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug; 33 (2):326).

[0400] Examples of autoimmune neurological diseases include, but are not limited to, multiple sclerosis (Cross AH. et al., JNeuroimmunol 2001 Jan 1; 112 (1-2):1); Alzheimer's disease (Oron L. et al. , J NeuralTransm Suppl. 1997; 49:77); myasthenia gravis (Infante AJ. And Kraig E, Int RevImmunol 1999; 18 (1-2):83; Oshima M. et al. , Eur J Immunol 1990 Dec; 20 (12):2563); neuropathy, motor neuropathy (Kornberg AJ. J Clin Neurosci. 2000 May; 7 (3):191); Guillain-Barré syndrome and autoimmune neuropathy (Kusunoki S. Am J Med Sci. 2000 Apr; 319 (4):234); myasthenia, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr; 319(4):204); paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy and stiff-person syndrome (Hiemstra HS. et al. ,Proc Natl Acad Sci U S A 2001 Mar 27; 98 (7):3988); non-paraneoplastic stiff-person syndrome, progressive cerebellar atrophy, encephalitis, Rasmussen encephalitis, amyotrophic lateral sclerosis, Sydenham chorea, Gilles de la Tourette syndrome, and autoimmune polyendocrine diseases (Antoine JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan; 156 (1):23); immunodeficiency neuropathy (Nobile-Orazio E. et al. , Electroencephalogr Clin Neurophysiol Suppl 1999; 50:419); acquired neuromyotonia, congenital multiple arthrogryposis (Vincent A. et al. , Ann N Y Acad Sci. 1998 May 13; 841:482); neuritis, optic neuritis (Soderstrom M. et al. , J Neurol Neurosurg Psychiatry 1994 May; 57 (5):544) and neurodegenerative diseases.

[0401] Examples of autoimmune muscle diseases include, but are not limited to, myositis, autoimmune myositis, and primary Sjogren's syndrome (Feist E. et al. , Int Arch Allergy Immunol 2000 Sep; 123 (1):92) and autoimmune diseases of smooth muscle (Zauli D. et al. , Biomed Pharmacother 1999 Jun; 53 (5-6):234).

[0402] Examples of autoimmune kidney diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug; 1 (2):140).

[0403] Examples of autoimmune diseases related to reproduction include, but are not limited to, recurrent fetal loss (Tincani A. et al. , Lupus 1998; 7 Suppl 2:S107-9).

[0404] Examples of autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo TJ. et al., Cell Immunol 1994 Aug; 157 (1):249), and autoimmune inner ear diseases (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec 29; 830:266).

[0405] Examples of autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et al. , Immunol Res 1998; 17 (1-2):49) and systemic sclerosis (Renaudineau Y. et al. , Clin DiagnLab Immunol. 1999 Mar; 6 (2):156); Chan OT. et al., Immunol Rev 1999 Jun; 169:107).

[0406] According to one specific embodiment, the autoimmune disease is selected from type 1 diabetes, multiple sclerosis, rheumatoid arthritis, celiac disease, and stroke.

[0407] As mentioned, the cells of the present invention can be obtained from any cell donor. Thus, the subject to be treated can be a human subject, and the cells can be obtained from a homologous (e.g., autologous) or non-homologous donor (e.g., allogeneic or xenogeneic with respect to the subject).

[0408] As used herein, the term "homologous" cells refers to cells that are substantially genetically identical to the subject or substantially all of the lymphocytes of the subject. Examples of homologous cells include cells derived from the subject (also referred to in the art as "autologous"), clones derived from the subject, or cells derived from an identical twin of the subject.

[0409] As used herein, the term "non-homologous" cells refers to cells that are substantially genetically different from the subject or substantially all of the lymphocytes of the subject, such as allogeneic cells or xenogeneic cells.

[0410] As used herein, the term "allogeneic" refers to cells derived from a donor of the same species as the subject but that are clearly non-clonal with respect to the subject. Generally, outbred, non-zygotic twin mammals of the same species are allogeneic to each other. It should be appreciated that, with respect to the subject, allogeneic cells can be HLA-identical, partially HLA-identical, or HLA-different (i.e., presenting one or more different HLA determinants).

[0411] As used herein, the term "allogeneic" refers to cells that express significantly different classes of antigens relative to a substantial proportion of the lymphocytes of a subject. Generally, outbred mammals of different species are allogeneic to each other.

[0412] The present invention contemplates that allogeneic cells are derived from a variety of species. Thus, according to one embodiment, the cells can be derived from any mammal. Suitable species sources of the cells include primary domesticated or livestock animals and primates. Such animals include, but are not limited to, porcine (e.g., pigs), bovine (e.g., cows), equine (e.g., horses), ovine (e.g., goats, sheep), feline (e.g., domestic cats (Felis domestica)), canine (e.g., domestic dogs (Canis domestica)), rodents (e.g., mice, rats, rabbits, guinea pigs, gerbils, hamsters), and primates (e.g., chimpanzees, rhesus monkeys, macaques, marmosets).

[0413] Cells from an allogeneic source (e.g., porcine source) are preferably obtained from a source known to be free of zoonoses such as porcine endogenous retrovirus. Similarly, human cells or tissues are preferably obtained from a source that is substantially pathogen-free.

[0414] According to one embodiment, the cells are non-homologous to the subject.

[0415] According to one embodiment, the cells are allogeneic to the subject.

[0416] According to one embodiment, the cells are homologous to the subject (e.g., autologous).

[0417] According to one embodiment of the present invention, the subject is a human, and the cells are from a human source (i.e., non-autologous).

[0418] According to one embodiment, the subject is a human, and the cells are from an allogeneic source (e.g., porcine source).

[0419] Any method known in the art can be used to obtain cells for transplantation. Thus, for example, immune cells (e.g., T cells, B cells, NK cells, DCs) can be obtained by collecting peripheral blood from a donor. Methods for collecting peripheral blood are well known in the art and include, but are not limited to, taking up to 500 - 1000 ml of whole blood from a donor and collecting it in a container containing an anticoagulant (e.g., heparin or citrate), or by apheresis, which is a procedure in which a subject's peripheral blood is passed through a device that yields the major components (e.g., monocytes such as lymphocytes, monocytes, or dendritic cells) and returns the other components to the subject's circulation. Alternatively, the cells can be obtained by in vitro or ex vivo culture of the cells. It should be appreciated that the cells of the present invention can be part of a fresh or frozen (e.g., cryopreserved) preparation.

[0420] Depending on the transplantation context, to promote engraftment of the implanted cells, the method may further advantageously include pre-treating the subject under sub-lethal, lethal or supra-lethal conditions prior to transplantation.

[0421] When referring to pre-treating the subject of the present invention, the terms "sub-lethal", "lethal" and "supra-lethal" as used herein refer to myeloablative and / or lymphoablative treatments which, when applied to a representative population of subjects, are generally, respectively: non-lethal to substantially all members of the population; lethal to some but not all members of the population; or lethal to substantially all members of the population under normal non-fertile conditions.

[0422] According to some embodiments of the present invention, the sub-lethal, lethal or supra-lethal pre-treatment includes total body irradiation (TBI), total lymphoid irradiation (TLI, i.e., exposure of all lymph nodes, thymus and spleen), regional irradiation (e.g., specific exposure of colon, breast, etc.), myeloablative pre-treatment and / or non-myeloablative pre-treatment, e.g., with different combinations including but not limited to co-stimulatory blockade, chemotherapeutic agents and / or antibody immunotherapy. According to some embodiments of the present invention, the pre-treatment includes a combination of any of the pre-treatment regimens described above (e.g., chemotherapeutic agent with TBI, co-stimulatory blockade with chemotherapeutic agent, antibody immunotherapy with chemotherapeutic agent, etc.).

[0423] According to one embodiment, the TBI comprises a single or fractionated irradiation dose in the range of: 0.5 - 1 Gy, 0.5 - 1.5 Gy, 0.5 - 2.5 Gy, 0.5 - 5 Gy, 0.5 - 7.5 Gy, 0.5 - 10 Gy, 0.5 - 15 Gy, 1 - 1.5 Gy, 1 - 2 Gy, 1 - 2.5 Gy, 1 - 3 Gy, 1 - 3.5 Gy, 1 - 4 Gy, 1 - 4.5 Gy, 1 - 5.5 Gy, 1 - 7.5 Gy, 1 - 10 Gy, 2 - 3 Gy, 2 - 4 Gy, 2 - 5 Gy, 2 - 6 Gy, 2 - 7 Gy, 2 - 8 Gy, 2 - 9 Gy, 2 - 10 Gy, 3 - 4 Gy, 3 - 5 Gy, 3 - 6 Gy, 3 - 7 Gy, 3 - 8 Gy, 3 - 9 Gy, 3 - 10 Gy, 4 - 5 Gy, 4 - 6 Gy, 4 - 7 Gy, 4 - 8 Gy, 4 - 9 Gy, 4 - 10 Gy, 5 - 6 Gy, 5 - 7 Gy, 5 - 8 Gy, 5 - 9 Gy, 5 - 10 Gy, 6 - 7 Gy, 6 - 8 Gy, 6 - 9 Gy, 6 - 10 Gy, 7 - 8 Gy, 7 - 9 Gy, 7 - 10 Gy, 8 - 9 Gy, 8 - 10 Gy, 10 - 12 Gy or 10 - 15 Gy.

[0424] According to one specific embodiment, the TBI comprises a single or fractionated irradiation dose in the range of 1 - 7.5 Gy.

[0425] According to one embodiment, the preconditioning step is achieved by preconditioning a subject under supra - lethal conditions, such as under myeloablative conditions.

[0426] Alternatively, the preconditioning step can be achieved by preconditioning the subject under lethal or sub - lethal conditions, such as by preconditioning the subject under myeloreductive conditions or non - myeloablative conditions.

[0427] According to one embodiment, the preconditioning step is achieved by preconditioning the subject with a myeloablative agent (such as busulfan or melphalan) or a non - myeloablative agent (such as cyclophosphamide and / or fludarabine).

[0428] Examples of preconditioning substances that can be used to precondition a subject include (without limitation) irradiation, drugs, and tolerance - inducing cells (as described herein).

[0429] Examples of drugs include myelotoxic drugs, lymphocytotoxic drugs, and immunosuppressive drugs (discussed in detail below).

[0430] Examples of myelotoxic drugs include (without limitation) busulfan, dimethylmyleran, melphalan, and thiotepa.

[0431] Additionally or alternatively, the method can further comprise preconditioning the subject with an immunosuppressive regimen before, simultaneously with, or after the transplanted cells.

[0432] Examples of suitable types of immunosuppressive regimens include administration of immunosuppressive drugs and / or immunosuppressive irradiation.

[0433] Adequate guidance for selecting and administering an appropriate immunosuppressive regimen for transplantation is provided in the literature of the art (e.g., see: Kirkpatrick CH. and Rowlands DT Jr., 1992. JAMA. 268, 2952; Higgins RM. et al., 1996. Lancet 348, 1208; Suthanthiran M. and Strom TB., 1996. New Engl. J. Med. 331, 365; Midthun DE. et al., 1997. Mayo Clin Proc. 72, 175; Morrison VA. et al., 1994. Am J Med. 97, 14; Hanto DW., 1995. Annu Rev Med. 46, 381; Senderowicz AM. et al., 1997. Ann Intern Med. 126, 882; Vincenti F. et al., 1998. New Engl. J. Med. 338, 161; Dantal J. et al 1998. Lancet 351, 623).

[0434] Examples of immunosuppressive drugs include, but are not limited to, tacrolimus (also known as FK-506 or fujimycin, trade names: Prograf, Advagraf, Protopic), mycophenolate mofetil, sodium mycophenolate, prednisone, methotrexate, cyclophosphamide, cyclosporine, cyclosporine A, chloroquine, hydroxychloroquine, sulfasalazine (sulfasalazine or sulphasalazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, TNF-α blockers (biological drugs targeting inflammatory cytokines), and non-steroidal anti-inflammatory drugs (NSAIDs). Examples of NSAIDs include, but are not limited to, acetylsalicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salicylsalicylic acid, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, tramadol, rapamycin (sirolimus), and rapamycin analogs (such as CCI-779, RAD001, AP23573). These drugs can be administered singly or in combination.

[0435] As used herein, the term "about" means ±10%.

[0436] The terms "comprising", "including", "containing", "having", and their variations mean "including but not limited to".

[0437] The term "consisting of" means "including and limited to".

[0438] The term "consisting essentially of" means that a composition, method, or structure may include additional ingredients, steps, and / or parts, but only if the additional ingredients, steps, and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0439] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0440] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as the individual numerical values within that range. For example, a range description such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as the individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0441] Whenever a numerical range is indicated herein, it is meant to include any recited numerical value (fractional or integral) within the indicated range. The phrases "range between a first recited number and a second recited number" and "range from a first recited number to a second recited number" are used interchangeably herein and are meant to include the first and second recited numbers and all the fractional and integral numerical values therebetween.

[0442] As used herein, the term "method" refers to a way, means, technique, and procedure for accomplishing a given task, including but not limited to those ways, means, techniques, and procedures that are known to, or are readily developed from known ways, means, techniques, and procedures by practitioners in the fields of chemistry, pharmacology, biology, biochemistry, and medicine.

[0443] It should be appreciated that certain features of the invention that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination or, as appropriate, in any other described embodiment of the invention. Certain features that are described in the context of various embodiments are not considered essential features of those embodiments unless the embodiment does not function without those elements.

[0444] The various embodiments and aspects of the invention as depicted above and as claimed in the following claims section find experimental support in the following examples.

[0445] An isolated cell having a central memory T lymphocyte (Tcm) phenotype, the cell being a tolerance-inducing cell and capable of homing to lymph nodes after transplantation, the cell being transduced to express a cell surface receptor comprising a T cell receptor signaling module.

[0446] 2. An isolated cell having a central memory T lymphocyte (Tcm) phenotype, the cell being a tolerance-inducing cell and capable of homing to lymph nodes after transplantation, the cell being transduced to express a chimeric antigen receptor (CAR).

[0447] 3. An isolated cell having a central memory T lymphocyte (Tcm) phenotype, the cell being a tolerance-inducing cell and capable of homing to lymph nodes after transplantation, the cell being transduced to express a chimeric antigen receptor (CAR), wherein the CAR comprises a co-stimulatory domain.

[0448] 4. An isolated cell having a central memory T lymphocyte (Tcm) phenotype, the cell being a tolerance-inducing cell and capable of homing to lymph nodes after transplantation, the cell being transduced to express a chimeric antigen receptor (CAR), wherein the CAR comprises at least two co-stimulatory domains.

[0449] 5. A method of generating the isolated cell of any one of embodiments 1-4, the method comprising transducing a cell having a central memory T lymphocyte (Tcm) phenotype, the cell being a tolerance-inducing cell and capable of homing to lymph nodes after transplantation, with a polynucleotide encoding the cell surface receptor comprising a T cell receptor signaling module or the chimeric antigen receptor (CAR).

[0450] 6. The method of embodiment 5, wherein the method is carried out ex vivo.

[0451] 7. The method of embodiment 5, wherein the cell is transduced with a vector comprising the polynucleotide.

[0452] 8. The method of embodiment 5 or 7, wherein the polynucleotide encodes a transgenic T cell receptor (tg-TCR) or a chimeric antigen receptor (CAR).

[0453] 9. The isolated cell of any one of embodiments 1-4 or the method of any one of embodiments 5-8, wherein the cell having a central memory T lymphocyte (Tcm) phenotype is an anti-third party cell.

[0454] 10. The isolated cell of embodiment 1 or the method of embodiment 5, wherein the cell surface receptor comprises a transgenic T cell receptor (tg-TCR) or a chimeric antigen receptor (CAR).

[0455] 11. The isolated cell of any one of embodiments 2-4 or 10 or the method of embodiment 5 or 10, wherein the CAR comprises an antigen-binding domain that is an antibody or an antigen-binding fragment.

[0456] 12. The isolated cell or method of embodiment 11, wherein the antigen-binding fragment is a Fab or an scFv.

[0457] 13. The isolated cell of any one of embodiments 2-4 or 10 or the method of embodiment 5 or 10, wherein the CAR comprises CD3ζ.

[0458] 14. The isolated cell of any one of embodiments 2-4 or 10 or the method of embodiment 5 or 10, wherein the CAR comprises at least one co-stimulatory domain selected from CD28, CD134 / OX40, CD137 / 4-1BB, Lck, ICOS, and DAP10.

[0459] 15. The isolated cell of any one of embodiments 2-4 or 10 or the method of embodiment 5 or 10, wherein the CAR comprises at least two co-stimulatory domains selected from CD28, CD134 / OX40, CD137 / 4-1BB, Lck, ICOS, and DAP10.

[0460] 16. The isolated cell of any one of embodiments 1-4 or 9-15 or the method of any one of embodiments 5-15, wherein the cell surface receptor or the CAR binds to an antigen selected from tumor antigens, viral antigens, bacterial antigens, fungal antigens, protozoan antigens, parasite antigens, allergic antigens, and autoimmune antigens.

[0461] 17. The isolated cell or method of embodiment 16, wherein the tumor antigen is associated with a solid tumor.

[0462] 18. The isolated cell or method of embodiment 16, wherein the tumor antigen is associated with a hematological malignancy.

[0463] 19. The isolated cell or method of any one of embodiments 16-18, wherein the tumor antigen is selected from CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, Her2, GD2, gp100, p53, carcinoembryonic antigen (CEA), MART-1, telomerase reverse transcriptase (TERT), Caudin-6, extracellular domain of receptor tyrosine protein kinase (ErbB2-ECD), intracellular domain of receptor tyrosine protein kinase (ErbB2-ICD), histone H1.2, histone H4, tyrosinase, alpha-fetoprotein (AFP), MAGE A3, AIM-2a, AFP, ART-4, CLCA2, Cyp-B, EphA2, hTERT, iCE, FGF-5, G250, GnT-V, HST-2 (FGF-6), Livin (ML-IAP), MUC1, MUC2, PRAME, PSMA, P15, RAGE, RU1, RU2, SART-1, SART-3, SART-2, SOX10, survivin, survivin-2Bg, TRG, Neo-PAP, CAMEL, and NY-ESO-1.

[0464] 20. The isolated cell or method of embodiment 16, wherein the viral antigen belongs to a virus selected from the group consisting of human immunodeficiency virus (HIV), influenza, cytomegalovirus (CMV), T-cell leukemia virus type 1 (TAX), hepatitis C virus (HCV), influenza virus, rabies virus, herpes virus, papillomavirus, hepatitis virus, varicella virus, encephalitis virus, cytomegalovirus, Ebola virus, human T-lymphotropic virus (HTLV), rubella virus, measles virus, rabies virus, lymphocytic choriomeningitis (LCM), rotavirus, mumps virus, adenovirus, adenovirus type 3 (HADV-3), adenovirus type 5 (HADV-5), adeno-associated virus 6 (AAV6), adeno-associated virus 8 (AAV8), BK polyomavirus (BKV), Candida, Epstein-Barr virus (EBV), human herpesvirus (HHV), varicella-zoster virus (VZV), and hepatitis B virus (HBV).

[0465] 21. The isolated cell or method of embodiment 16, wherein the autoimmune antigen is associated with a disease selected from type 1 diabetes, multiple sclerosis, lupus, rheumatoid arthritis, Crohn's disease, celiac disease, and stroke.

[0466] 22. The isolated cells of any one of embodiments 1-4 or 9-15, or the method of any one of embodiments 5-15, wherein the cells are further genetically modified to inhibit the expression of at least one endogenous immune checkpoint gene in the cells.

[0467] 23. The isolated cells or method of embodiment 22, wherein the immune checkpoint gene is selected from the PD or CTLA genes.

[0468] 24. The method of any one of embodiments 5-15, wherein the cells having a central memory T lymphocyte (Tcm) phenotype are tolerance-inducing cells and are capable of homing to lymph nodes after transplantation, and the cells are produced by a method comprising:

[0469] (a) contacting peripheral blood mononuclear cells (PBMCs) with one or more third-party antigens in the presence of IL-21 so as to enable enrichment of antigen-reactive cells; and

[0470] (b) culturing the cells generated from step (a) in the presence of IL-21, IL-15, and IL-7 so as to enable proliferation of anti-third-party cells comprising the central memory T lymphocyte (Tcm) phenotype, thereby producing cells having a Tcm phenotype, which are tolerance-inducing cells and are capable of homing to lymph nodes after transplantation.

[0471] 25. The method of embodiment 24, further comprising:

[0472] (c) separating the cells generated from step (b) into a single cell suspension.

[0473] 26. The method of embodiment 24, further comprising selecting activated cells after step (a) and before step (b).

[0474] 27. The method of embodiment 26, wherein the selection of activated cells is achieved by selecting CD137+ and / or CD25+ cells.

[0475] 28. The isolated cells of any one of embodiments 1-4 or the method of embodiment 24, wherein the Tcm phenotype comprises CD3 + , CD8 + , CD62L + , CD45RA - , CD45RO + characteristics.

[0476] 29. The isolated cell or method of embodiment 28, wherein at least 50% of the isolated cells are CD3+CD8+ cells, and at least 50% of the CD3+CD8+ cells have the characteristics.

[0477] 30. A cell population comprising the isolated cells of any one of embodiments 1-4, 9-23 or 28-29.

[0478] 31. A pharmaceutical composition comprising the cell population of embodiment 30 and a pharmaceutically active carrier.

[0479] 32. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the cell population of embodiment 30, thereby treating the subject.

[0480] 33. A therapeutically effective amount of the cell population of embodiment 30 for use in treating a disease in a subject in need thereof.

[0481] 34. The method of embodiment 32 or the therapeutically effective amount for use of embodiment 33, wherein the disease is selected from the group consisting of malignant diseases, viral diseases, bacterial diseases, fungal diseases, protozoal diseases, parasitic diseases, allergic diseases and autoimmune diseases.

[0482] 35. The method of embodiment 34 or the therapeutically effective amount for use, wherein the malignant disease is a solid tumor or tumor metastasis.

[0483] 36. The method of embodiment 34 or the therapeutically effective amount for use, wherein the malignant disease is a hematological malignancy.

[0484] 37. The method of embodiment 36 or the therapeutically effective amount for use, wherein the hematological malignancy comprises leukemia or lymphoma.

[0485] 38. The method of any one of embodiments 34-36 or the therapeutically effective amount for use, wherein the malignant disease is selected from the group consisting of leukemia, lymphoma, myeloma, melanoma, sarcoma, neuroblastoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, esophageal cancer, synovial cell carcinoma and pancreatic cancer.

[0486] 39. The method of embodiment 34 or the therapeutically effective amount for use, wherein the viral disease is selected from the group consisting of human immunodeficiency virus (HIV), influenza, cytomegalovirus (CMV), human T-cell leukemia virus type 1 (TAX), hepatitis C virus (HCV) and hepatitis B virus (HBV).

[0487] 40. The method of embodiment 34 or a therapeutically effective amount for use, wherein the autoimmune disease is selected from type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, celiac disease, and stroke.

[0488] 41. The method of any one of embodiments 32 or 34 - 40 or a therapeutically effective amount for use of any one of embodiments 33 - 40, wherein the cell population is non - homologous to the subject.

[0489] 42. The method of any one of embodiments 32 or 34 - 40, further comprising pre - treating the subject under a sub - lethal, lethal, or supra - lethal pre - treatment regimen prior to said administration.

[0490] 43. A therapeutically effective amount for use of any one of embodiments 33 - 40, further comprising a sub - lethal, lethal, or supra - lethal pre - treatment regimen.

[0491] 44. The method of embodiment 42 or a therapeutically effective amount for use of embodiment 43, wherein the sub - lethal, lethal, or supra - lethal pre - treatment is selected from total body irradiation (TBI), partial body irradiation, myeloablative pre - treatment, non - myeloablative pre - treatment, co - stimulatory blockade, chemotherapeutic agents, and antibody immunotherapy.

[0492] 45. The method of embodiment 32, wherein the administration is effected by a route selected from intratracheal, intrabronchial, intra - alveolar, intravenous, intraperitoneal, intranasal, subcutaneous, intramedullary, intrathecal, intraventricular, intracardiac, intramuscular, intra - serosal, intra - mucosal, transmucosal, transnasal, rectal, and intestinal.

[0493] 46. The method of any one of embodiments 32 or 34 - 45 or a therapeutically effective amount for use of any one of embodiments 33 - 45, wherein the subject is a human subject. Examples

[0494] Reference is now made to the following examples, which, together with the above description, illustrate the invention in a non - limiting manner.

[0495] Generally, the nomenclature used herein and the laboratory procedures employed in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are well explained in the literature. See, for example, "Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); the methodologies described in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J.E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J.E., ed. (1994); Stites et al (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W.H. Freeman and Co., New York (1980); Available immunoassays are widely described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M.J., ed. (1984); “Nucleic Acid Hybridization” Hames, B.D., and Higgins S.J., eds. (1985); “Transcription and Translation” Hames, B.D., and Higgins S.J., Eds. (1984); “Animal Cell Culture” Freshney, R.I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, CA (1990); Marshak et al., “Strategies for Protein Purification and Characterization - A Laboratory Course Manual” CSHL Press (1996); all of these references are incorporated by reference as if fully set forth herein. Additional general references are provided throughout this document. It is believed that the procedures therein are well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

[0496] General Materials and Experimental Procedures

[0497] Animals

[0498] Female 6 - 12 - week - old BALB / c, CB6 (F1), and C57BL / 6 mice were obtained from Harlan Laboratories or were grown in the animal facilities of the Weizmann Institute of Science. All mice were kept in small cages (5 animals per cage) and were fed sterile food and acidified water. All studies were approved by the Weizmann Institute of Science Institutional Animal Care and Use Committee.

[0499] Preparation of host - non - reactive mouse anti - third - party Tcm

[0500] Anti - third - party Tcm was prepared as previously described [Ophir E et al., Blood (2010) 115: 2095 - 2104]. Briefly, splenocytes from donor mice were cultured against irradiated third - party splenocytes for 60 h under cytokine deprivation. Subsequently, CD8 + cells were positively selected using magnetic beads (BD Pharmingen) and cultured in an Ag - free environment. rhIL - 15 (20 ng / mL; R&D Systems) was added every other day. To obtain a purified population at the end of the culture (day 16), Tcm cells were positively selected for CD62L expression by magnetic - activated cell sorting [MACS, Miltenyi, Bergisch Gladbach, Germany].

[0501] Bone marrow transplantation

[0502] 1. Long bones were harvested from Balb / c or C57BL / 6 mice [Nude or wild - type (WT)]. Bone marrow was extracted by flushing or grinding the bones to obtain a single - cell suspension. Some preparations harvested from WT mice were subjected to T - cell depletion by magnetic - activated cell sorting. The bone marrow was counted and brought to the correct concentration and then injected intravenously into the mouse tail vein or intraorbitally.

[0503] 2. Before transplantation, mice were subjected to a pre - conditioning regimen. Reduced - intensity conditioning (RIC) consisted of subjecting the mice to a sub - lethal irradiation dose (i.e., a dose of irradiation from which the recipient mice could spontaneously recover) or subjecting the mice to a low dose of myeloablative (e.g., busulfan) or non - myeloablative (e.g., cyclophosphamide) drugs. Total - body irradiation (TBI) was given using a gamma - ray machine or X - rays (e.g., XRAD - 320). The drugs were given i.v., s.c., i.p., or orally.

[0504] OT1+ cell transplantation

[0505] Lymph nodes and / or spleens were harvested from OT-1 transgenic mice. The mice were OT-1 mice carrying the CD45.1 gene and / or on a RAG- / - mutant background. Alternatively, the OT-1 mice were F1-OT1 mice, the offspring of host XOT-1 mice, which could be used to eliminate allogeneic phenomena. Single cell suspensions were created and then subjected to T cell purification by magnetic activated cell sorting [MACS, Miltenyi, Bergisch Gladbach, Germany]. The purity of the OT-1 T cell population generated was tested by FACS. The cells were then injected as "fresh" cells or cultured ex vivo to generate Tcm cells as described above, i.e., by activating irradiated splenocytes from ovalbumin-expressing mice by a third party. Then the OT-1 Tcm cells were injected as described herein.

[0506] Flow cytometry analysis

[0507] Fluorescence-activated cell sorting (FACS) analysis was performed using a modified Becton Dickinson FACScan. Cells were stained with labeled antibodies specific for Vα2, Vβ5, H2Dd, H2Kb, CD45.1, CD45.2, CD8a, CD4, CD25, CD69, CD19 (Biolegend; BD; Miltenyi).

[0508] CTL activity assay ( 51 Cr assay)

[0509] Mice were sacrificed, spleens and LNs were harvested and CD8 + cells were selected (and H-2D was negatively selected d to exclude 'Tcm'). The killing ability of HTC from these first experiments against C3H (H-2 k ) or BALB / c (H-2 d ) targets was tested by the chromium release assay. BALB / c and C3H splenocytes to be used as target cells were pretreated with 2 μg / ml concanavalin A (Sigma, St. Louis, MO) for 48 hours and exposed to 70 μCi 51 Cr (Perkin Elmer, Wellesley, MA) for 1 hour. Effector cells were prepared from CD8+-selected cells of C57BL / 6 mice and incubated with BALB / c or C3H splenocytes at different dilutions in 96-well plates containing IL-2 (20 U / ml) 12 times for each dilution for 6 days. On day 6, titrated numbers of effector cells and 5×10 3 cells 51 Cr-labeled targets were mixed in V-bottom plates at various effector / target (E:T) ratios. For 4 hours51 Cr release assays measure cytotoxic activity. The percentage of specific lysis is calculated as (experimental release - spontaneous release) / (maximum release - spontaneous release) × 100. The 51 Cr release of target cells cultured alone in medium or lysed with 1% SDS was defined as spontaneous release or total release, respectively.

[0510] Peripheral blood mononuclear cells (PBMCs)

[0511] PBMCs were isolated from the whole blood of patients and healthy volunteers by Ficoll density gradient centrifugation. When indicated, cells were typed for class I HLA by serological methods as previously described [Manual of Tissue Typing Techniques. Washington DC, National Institute of Allergy and Infectious Diseases, NIHDHEW Publication 76 - 545, 1976, p. 22].

[0512] Dendritic cells were generated

[0513] Monocytes were isolated by plastic adherence and cultured in 6 - well plates using 3 ml of Cellgro DC medium (Peprotech, Hamburg, Germany) supplemented with 1% human serum and penicillin / streptomycin + GM - CSF (800 IU / ml) and IL - 4 (20 ng / ml). After 48 hours of culture, 1.5 ml of medium (GM - CSF at 1600 IU / ml and IL - 4 at 20 ng / ml) was added. After 24 hours, non - adherent cells were harvested, and large cells were counted (mostly immature DCs), resuspended in fresh medium containing GM - CSF 800 IU / ml, IL - 4 20 ng / ml, 10 ng / ml of LPS from Escherichia coli O55:B5 (Sigma, Deisenhofen, Germany), and IFNγ (Peprotech, 100 IU / ml), and seeded at approximately 106 DCs per well in 2 ml and incubated overnight. The next day, non - adherent cells were discarded, and adherent DCs were gently removed using cold PBS / 1% HS after incubation on ice for 20 minutes. Large cells consisting of mature DCs were counted. Cells were irradiated with 30 Gy to avoid growth of a few potentially contaminating NK or memory T cells and then used for T - cell stimulation.

[0514] CD8 T cells for the first experiment were isolated from PBMCs

[0515] Using the CD8 negative selection kit (Miltenyi, Bergisch Gladbach, Germany) according to the manufacturer's instructions, CD8 T cells from the first experiment were isolated by initial negative selection. Then CD45RO-beads were used and antigen-experienced CD8+ T cells were depleted on an LD column.

[0516] Generation of anti-third-party central memory human CD8 T cells

[0517] CD8 T cells from the first experiment were isolated and resuspended in T cell medium supplemented with IL-21 (Peprotech, 30 ng / ml). Irradiated DCs were added to each well of a 48-well plate at a 1:4 DC:T cell ratio with 4 x 10 5 T cells. The total volume per well was 500 μl.

[0518] At 72 hours after the start of culture, 500 μl of T cell medium containing IL-7 and IL-15 (Peprotech, 5 ng / ml final concentration) was added and the cells were subsequently fed every 2 - 3 days as outlined in the results section.

[0519] Statistical analysis

[0520] Analysis of survival data was performed using Kaplan-Meier curves (log-rank test). Student t-tests were used for comparison of means.

[0521] Example 1

[0522] MHC-mismatched Tcm survive and exert specific veto activity in host mice in a syngeneic bone marrow environment

[0523] Considering that syngeneic bone marrow transplantation (BMT) is far safer in humans than allogeneic BMT even when given after lethal total body irradiation (TBI), the present inventors first sought to determine whether adoptively transferred F1-Tcm cells survive under host-versus-donor HTC attack when infused together with syngeneic TDBMT (Figure 1A - B). As can be seen in Figure 2A - B, at 60 days post-transplantation, F1-Tcm persisted in the peripheral blood. Tcm cells constituted approximately 13% ± 10 of the total CD8 + fraction (data not shown). Next, to evaluate the ability of Tcm to induce antigen-specific clonal deletion within a wild-type polyclonal HTC population and to verify that the remaining HTC retained their functionality, a chromium release killing assay was used. The results showed that H2 b CD8 + HTC from Tcm-treated mice showed significantly reduced killing of H-2 d targets and retained H-2k the killing ability of the target, while mice not treated with Tcm (i.e., the BM-alone group) showed similar killing levels against both cell types ( Figure 3 ). These results indicate that Tcm exerts specific veto activity against the polyclonal HTC population and confirm that clones not due to Tcm deletion retain their functionality. Subsequently, these experiments were repeated in mice pretreated with reduced-intensity conditioning (RIC), which is more suitable for clinical implementation. Thus, studies in Balb / c mice sub-lethally irradiated with 5.5 Gy TBI yielded similar results ( Figure 4 ), which were injected with syngeneic T cell-depleted bone marrow (TDBMT) and allogeneic (Balb×Black) F1 Tcm (described in Figure 1C ). Tcm cells were present in the peripheral blood of these mice for more than 15 months (when the experiment was terminated, data not shown). Thus, the survival of MHC-mismatched Tcm can be induced under a very safe protocol, which includes pretreatment with sub-lethal 5.5 Gy TBI and autologous BMT.

[0524] Example 2

[0525] MHC-mismatched Tcm survive in host mice in the absence of a bone marrow graft

[0526] In view of the above data, the ability of Tcm cells to induce tolerance alone in the absence of BM was evaluated. The scope of such a protocol would be much larger. Specifically, inducing immune tolerance by administering anti-third party Tcm cells alone under safe pretreatment is not only valuable for immunocompromised individuals, but may also allow the treatment of non-malignant blood diseases (such as anemia and thalassemia), autoimmune diseases, and provide a platform for the administration of cell therapy. First, the inventors attempted to define the minimum irradiation dose for engraftment of F1-derived Tcm cells to establish a model that could test host tolerance induction. For this purpose, Balb / c mice were exposed to a series of sub-lethal pretreatment doses with and without adoptive transfer of CB6 F1-Tcm cells. Analysis of H2 db positive Tcm cells in the whole peripheral blood showed that the minimum irradiation dose at which Tcm could be detected (i.e., where Tcm cells are not rejected) was 5.5 Gy TBI ( Figure 5 ). Thus, the sustainability of survival of fully allogeneic C57BL / 6-derived Tcm at the sub-lethal TBI dose of 5.5 Gy was tested (as in Figure 1Cas depicted in Figure 1C as depicted in

[0527] Thus, the sole application of Tcm can be used to create an opportunity window for administration of therapies such as cell therapy for at least several months.

[0528] Example 3

[0529] MHC-mismatched Tcm supports adoptive transfer of cells from the same donor

[0530] To test the hypothesis that Tcm cells can be used for adoptive cell therapy, the present inventors employed transgenic OT1 mice carrying a TCR specific for ovalbumin peptide. The motivation for using OT1 transgenic cells in this case stemmed from the idea that these cells can be used as a model for a cell therapy called donor lymphocyte infusion (DLI), where the donor lymphocyte infusion (DLI) has the entire donor T cell population or antigen-specific T cells against viral or tumor antigens.

[0531] First, 90 days after the initial adoptive transfer of Tcm, the CD8 + OT1 + CD45.1 + T cells of the first experiment were infused into Tcm chimeric mice. The main purpose was to determine whether the surviving Tcm population could promote engraftment of newly infused allogeneic cells. Before injecting the Tg cells of the first experiment, the Tcm population in the chimeric mice was analyzed by FACS. Thus, 2 / 5 and 9 / 11 mice that had received C57BL / 6 Tcm or CB6-Tcm, respectively, maintained their Tcm population (Figure 6A - B).

[0532] These mice were further pre-treated with 2 Gy TBI on day 90 after transplantation (to deplete some T cells to allow introduction of new T cells), and then the mice were infused with 2×10 6OT1 cells (H-2 b ). Interestingly, when evaluated at day 120 (30 days after OT1 cell transplantation), OT1 cells were only detectable in those mice that had exhibited a Tcm population prior to transplantation ( Figure 7 ). These preliminary results showed that the addition of cells from the same donor source was acceptable in mice presenting a Tcm population, further confirmed using transgenic OT-1 cells as follows: CD8+ OT-1 cells were co-transplanted with Tcm at day 0 to prevent the need for a second preconditioning (previously 2 Gy TBI at day 90), and the presence of OT1 cells in peripheral blood was monitored at different time points after cell infusion.

[0533] As shown in Figure 8 , the results of this experiment demonstrated that:

[0534] 1. C57BL / 6 Tcm and (BalbxC57) F1 Tcm can persist in allogeneic recipients.

[0535] 2. When co-injected, C57BL / 6 Tcm can confer protection to cells of the first experiment of CD8+ OT-1 engendered on a C57BL / 6 background (OT-1+CD45.1+RAG-), while the OT-1 cells withdraw from the circulation on their own.

[0536] 3. CB6 (F1) Tcm expressing the MHC haplotype of C57BL / 6 (H-2 b ) mice can also confer protection to naive OT-1 cells.

[0537] Example 4

[0538] Implantation and in vivo survival of anti-third party Tcm veto cells prepared from OT-1 mouse cells

[0539] Experiments were conducted in mice preconditioned with reduced intensity conditioning (RIC), which is suitable for clinical implementation. Thus, Balb / c mice sub-lethally irradiated with 5.5 Gy TBI were injected with different concentrations of allogeneic Tcm cells from OT-1 mouse sources (described in Figure 9 ). Tcm cells persisted in the peripheral blood of these mice for at least 30 days. Thus, the survival of MHC-mismatched Tcm cells can be induced under a safe RIC protocol.

[0540] Although the present invention has been described in conjunction with its specific embodiments, it will be apparent that many alternatives, modifications, and variations will be obvious to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0541] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety into the specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein. Additionally, the citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

1. An isolated cell having a central memory T lymphocyte (Tcm) phenotype, wherein the Tcm phenotype comprises CD3 + 、CD8 + 、CD62L + 、CD45RA - 、CD45RO + The invention relates to a method for treating a T cell of the present invention, wherein the cell is an anti-third party cell that is not induced by graft-versus-host disease (GVHD), is tolerance-induced and can home to lymph nodes after transplantation, and the cell is transduced to express a cell surface receptor, wherein the cell surface receptor comprises a T cell receptor signaling module and an extracellular domain for a disease antigen, wherein the cell having the Tcm phenotype and being an anti-third party cell that is not induced by GVHD, is tolerance-induced and can home to lymph nodes after transplantation is produced by a method comprising the following steps: (a) Contacting peripheral blood mononuclear cells (PBMCs) with one or more third-party antigens in the presence of IL-21 so as to enable enrichment of antigen-reactive cells; and (b) Culturing the cells generated from step (a) in the presence of IL-21, IL-15, and IL-7 so as to enable proliferation of anti-third-party cells comprising the central memory T lymphocyte (Tcm) phenotype.

2. An isolated cell having a central memory T lymphocyte (Tcm) phenotype, wherein the Tcm phenotype comprises CD3 + , CD8 + , CD62L + , CD45RA - , CD45RO + characteristics, the cell being a non-graft-versus-host disease (GVHD)-induced anti-third party cell, being tolerance-induced and capable of homing to lymph nodes after transplantation, the cell being transduced to express a chimeric antigen receptor (CAR) comprising an extracellular domain directed against a disease antigen, wherein the cell having the Tcm phenotype and being a non-GVHD-induced anti-third party cell, tolerance-induced and capable of homing to lymph nodes after transplantation is produced by a method comprising the following steps: (a) Contacting peripheral blood mononuclear cells (PBMCs) with one or more third-party antigens in the presence of IL-21 so as to enable enrichment of antigen-reactive cells; and (b) Culturing the cells generated from step (a) in the presence of IL-21, IL-15, and IL-7 so as to enable proliferation of anti-third-party cells comprising the central memory T lymphocyte (Tcm) phenotype.

3. An isolated cell having a central memory T lymphocyte (Tcm) phenotype, wherein the Tcm phenotype comprises CD3 + , CD8 + , CD62L + , CD45RA - , CD45RO + characteristics, the cell being a non-graft-versus-host disease (GVHD)-induced anti-third-party cell, being tolerance-inducing and capable of homing to lymph nodes after transplantation, the cell being transduced to express a chimeric antigen receptor (CAR), wherein the CAR comprises a co-stimulatory domain and an extracellular domain comprising an antigen against a disease, wherein the cell having the Tcm phenotype and being a non-GVHD-induced anti-third-party cell, tolerance-inducing and capable of homing to lymph nodes after transplantation is produced by a method comprising the following steps: (a) Contacting peripheral blood mononuclear cells (PBMCs) with one or more third-party antigens in the presence of IL-21 so as to enable enrichment of antigen-reactive cells; and (b) Culturing the cells generated from step (a) in the presence of IL-21, IL-15, and IL-7 so as to enable proliferation of anti-third-party cells comprising the central memory T lymphocyte (Tcm) phenotype.

4. An isolated cell having a central memory T lymphocyte (Tcm) phenotype, wherein the Tcm phenotype comprises CD3 + , CD8 + , CD62L + , CD45RA - , CD45RO + characteristics, the cell being a non-graft-versus-host disease (GVHD)-induced anti-third-party cell, being tolerance-inducing and capable of homing to lymph nodes after transplantation, the cell being transduced to express a chimeric antigen receptor (CAR), wherein the CAR comprises at least two co-stimulatory domains and an extracellular domain comprising an antigen against a disease, wherein the cell having the Tcm phenotype and being a non-GVHD-induced anti-third-party cell, tolerance-inducing and capable of homing to lymph nodes after transplantation is produced by a method comprising the following steps: (a) Contacting peripheral blood mononuclear cells (PBMCs) with one or more third-party antigens in the presence of IL-21 so as to enable enrichment of antigen-reactive cells; and (b) Culturing the cells generated from step (a) in the presence of IL-21, IL-15, and IL-7 so as to enable proliferation of anti-third-party cells comprising the central memory T lymphocyte (Tcm) phenotype.

5. The isolated cell of claim 1, wherein the cell surface receptor comprises a transgenic T cell receptor (tg-TCR) or a chimeric antigen receptor (CAR).

6. The isolated cell of any one of claims 2-5, wherein the CAR comprises an antigen-binding domain that is an antibody or an antigen-binding fragment.

7. The isolated cell of claim 6, wherein the antigen-binding fragment is a Fab or an scFv.

8. The isolated cell of any one of claims 2-5, wherein the CAR comprises CD3ζ.

9. The isolated cell of any one of claims 2-5, wherein the CAR comprises at least one co-stimulatory domain selected from CD28, CD134 / OX40, CD137 / 4-1BB, Lck, ICOS, and DAP10.

10. The isolated cell of any one of claims 2-5, wherein the CAR comprises at least two co-stimulatory domains selected from CD28, CD134 / OX40, CD137 / 4-1BB, Lck, ICOS, and DAP10.

11. The isolated cell of any one of claims 1-5, wherein the disease antigen is selected from tumor antigens, viral antigens, bacterial antigens, fungal antigens, protozoan antigens, and parasite antigens.

12. The isolated cell of claim 11, wherein the tumor antigen is associated with a solid tumor.

13. The isolated cell of claim 11, wherein the tumor antigen is associated with a hematological malignancy.

14. The isolated cell of claim 11, wherein the tumor antigen is selected from CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, Her2, GD2, gp100, p53, carcinoembryonic antigen (CEA), MART-1, telomerase reverse transcriptase (TERT), Caudin-6, extracellular domain of receptor tyrosine protein kinase (ErbB2-ECD), intracellular domain of receptor tyrosine protein kinase (ErbB2-ICD), histone H1.2, histone H4, tyrosinase, alpha-fetoprotein (AFP), MAGE A3, AIM-2a, AFP, ART-4, CLCA2, Cyp-B, EphA2, hTERT, iCE, FGF-5, G250, GnT-V, HST-2 (FGF-6), Livin (ML-IAP), MUC1, MUC2, PRAME, PSMA, P15, RAGE, RU1, RU2, SART-1, SART-3, SART-2, SOX10, survivin, survivin-2Bg, TRG, Neo-PAP, CAMEL, and NY-ESO-1.

15. The isolated cell of claim 11, wherein the viral antigen belongs to a virus selected from the group consisting of human immunodeficiency virus (HIV), cytomegalovirus (CMV), T-cell leukemia virus type 1 (TAX), influenza virus, herpes virus, papillomavirus, hepatitis virus, varicella virus, encephalitis virus, Ebola virus, human T-lymphotropic virus (HTLV), rubella virus, measles virus, rabies virus, lymphocytic choriomeningitis (LCM), rotavirus, mumps virus, adenovirus, BK polyomavirus (BKV), Epstein-Barr virus (EBV), and varicella-zoster virus (VZV).

16. The isolated cell of any one of claims 1-5, wherein the cell is further genetically modified to inhibit the expression of at least one endogenous immune checkpoint gene in the cell.

17. The isolated cell of claim 16, wherein the immune checkpoint gene is selected from the PD or CTLA genes.

18. The isolated cell of any one of claims 1-5, wherein the method of generating the cell having the Tcm phenotype and being non-GVHD-inducing against third-party cells, tolerance-inducing, and capable of homing to lymph nodes after transplantation further comprises: (c) separating the cells generated from step (b) into a single cell suspension.

19. The isolated cell of any one of claims 1-5, wherein the method of generating the cell that has the Tcm phenotype and is non-GVHD-inducing, anti-third-party cell, tolerance-inducing and capable of homing to lymph nodes after transplantation further comprises selecting activated cells after step (a) and before step (b).

20. The isolated cell of claim 19, wherein the selection of activated cells is achieved by selecting CD137+ and / or CD25+ cells.

21. The isolated cell of any one of claims 1-4, wherein at least 50% of the isolated cells are CD3+CD8+ cells, and at least 50% of the CD3+CD8+ cells have the said characteristics.

22. A method of generating the isolated cells of any one of claims 1-17, the method comprising transducing cells having a central memory T lymphocyte (Tcm) phenotype with a polynucleotide encoding the cell surface receptor comprising a T cell receptor signaling module or the chimeric antigen receptor (CAR), wherein the Tcm phenotype comprises CD3 + , CD8 + , CD62L + , CD45RA - , CD45RO + Characterized in that the cells are non-graft-versus-host disease (GVHD)-induced against third-party cells, are tolerance-induced and capable of homing to lymph nodes after transplantation, and are generated by a method comprising the following: (a) contacting peripheral blood mononuclear cells (PBMCs) with one or more third-party antigens in the presence of IL-21 so as to enable enrichment of antigen-reactive cells; and (b) culturing the cells generated from step (a) in the presence of IL-21, IL-15 and IL-7 so as to enable proliferation of anti-third-party cells comprising the central memory T lymphocyte (Tcm) phenotype.

23. The method of claim 22, wherein the method is implemented ex vivo.

24. The method of claim 22, wherein the cells are transduced with a vector comprising the polynucleotide.

25. The method of claim 22 or 24, wherein the polynucleotide encodes a transgenic T cell receptor (tg-TCR) or a chimeric antigen receptor (CAR).

26. A cell population comprising the isolated cell of any one of claims 1-21.

27. A pharmaceutical composition comprising the cell population of claim 26 and a pharmaceutically active carrier.

28. A therapeutically effective amount of the cell population of claim 26 for treating a disease in a subject in need thereof.

29. The therapeutically effective amount of the cell population for use of claim 28, wherein the disease is selected from malignant diseases, viral diseases, bacterial diseases, fungal diseases, protozoal diseases and parasitic diseases.

30. The therapeutically effective amount of the cell population for use of claim 29, wherein the malignant disease is a solid tumor or tumor metastasis.

31. The therapeutically effective amount of the cell population for use of claim 29, wherein the malignant disease is a hematological malignancy.

32. The therapeutically effective amount of the cell population for use of claim 31, wherein the hematological malignancy includes leukemia or lymphoma.

33. The therapeutically effective amount of the cell population for use of claim 29, wherein the malignant disease is selected from leukemia, lymphoma, myeloma, melanoma, sarcoma, neuroblastoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, esophageal cancer, synovial cell carcinoma and pancreatic cancer.

34. The therapeutically effective amount of the cell population for use of claim 29, wherein the viral disease is selected from human immunodeficiency virus (HIV), influenza, cytomegalovirus (CMV), T cell leukemia virus type 1 (TAX), hepatitis C virus (HCV) and hepatitis B virus (HBV). A therapeutically effective amount of a cell population for use according to any one of claims 28-34, wherein the cell population is non-homologous to the subject. A therapeutically effective amount of a cell population for use according to any one of claims 28-34, further comprising a sub-lethal, lethal or supra-lethal preconditioning regimen. A therapeutically effective amount of a cell population for use according to claim 36, wherein the sub-lethal, lethal or supra-lethal preconditioning is selected from total body irradiation (TBI), regional irradiation, myeloablative preconditioning, non-myeloablative preconditioning, co-stimulatory blockade, chemotherapeutic agents and antibody immunotherapy. A therapeutically effective amount of a cell population for use according to any one of claims 28-34, wherein the subject is a human subject.

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