RU2779844C2 - Veto-cells obtained from memory t-cells - Google Patents

Abstract

FIELD: cell biology; medicine.

SUBSTANCE: present invention relates to the field of cell biology and medicine and discloses methods for the production of an isolated population of cells not inducing a reaction of “a graft vs host disease” (hereinafter – GvHD), having a phenotype of central memory T-lymphocytes (hereinafter – Tcm), as well as a population produced by such methods, and a method for the treatment of a subject who needs the transplantation of cells or tissues. To implement the specified methods for the production of a population, first, a population of T-cells is provided, containing at least 50% of memory T-cells. Then, the specified population is brought into contact with a viral, bacterial and/or tumor antigen in the presence of IL-21 in order to provide its enrichment with antigen-reactive cells. After that, the specified cells obtained above are cultivated in the presence of IL-21, IL-15 and/or IL-7 in order to provide proliferation of cells having Tcm phenotype.

EFFECT: present invention allows for the reproduction of functional antigen-reactive cells capable of providing effective and specific anti-diseases impact in order to eliminate host-reactive clones capable of causing GvHD.

24 cl, 26 dwg, 2 tbl, 6 ex

Description

Field of technology and state of the art

The present invention, in some embodiments, relates to memory T cell-derived veto cells, and more particularly, but not exclusively, methods for their preparation and use in transplantation and disease management.

Based on accumulated data, it has been shown that CD45RA-depleted peripheral blood mononuclear cells (PBMC) in humans are characterized by reduced graft-versus-host (GvH) response. The premise of this approach is related to suppression of CD45RA expression on the surface of antigen-stimulated T cells, therefore, by depleting CD45RA ⁺ cells, naive T cells are eliminated, while functional antigen-stimulated cells, including memory T cells, remain. Consequently, the risk of graft-versus-host disease (GvHD) is markedly reduced, and engraftment, immune reconstitution, and graft-versus-leukemia/lymphoma (GvL) reactions are enhanced, compared to T-cell-depleting stem cell transplant-only approaches (TCD). In addition, regulatory T cells (Treg) also belong to ^the CD45RA population and may possibly contribute to the tolerogenic effects exhibited by this cell preparation. This approach is based on preclinical studies demonstrating that mouse CD4 memory T cells, as well as effector memory CD8 T cells, lack graft-versus-host (GvH) reactivity [Anderson BE et al. J Clin Invest (2003) 112(1): 101-8].

However, Zheng et al. [Zheng H. et al. Immunol. (2009) 182(10):5938-48] demonstrated that central CD8 ⁺ memory T cells (T _cm ) had a significant, albeit somewhat reduced, GvHD compared to naive T cells. Considering that this decrease in GvHD may be associated with a reduced frequency of alloreactive clones in the pool of antigen-stimulated memory T cells, Juchem et al. further investigated the possible internal differences between naive T-cells and memory T-cells, which express at the same levels a transgenic TCR directed against an antigenic peptide that is ubiquitously expressed in the recipient's body [Juchem KW et al. Blood. (2011) 118(23):6209-19]. This study demonstrated that although effector memory T cells (T _em ) exhibit low GvH reactivity, possibly due to homing patterns and/or differing ability of these cells to secrete INFγ, T _cm exhibit high GvH reactivity, according to compared with that of naive T cells [Juchem KW. (2011), supra].

Recently, two large studies have attempted to use hematopoietic stem cell transplantation (HSCT) with depletion of CD45RA ⁺ in patients with leukemia [Bleakley M. et al. J Clin Invest. (2015) 125(7):2677-89; Triplett, BM et al. Bone Marrow Transplant. (2015) 50(7):968-977], however GvHD cases have not been completely eliminated. This could be due to the large number of infused CD45RA ⁺ T cells and the fact that the CD45KA-depleted fraction contained both T _em and T _cm , without taking into account data from preclinical studies that clearly showed that T _cm are potential GvHD inductors.

Donor-derived central CD8 ⁺ memory T cells (veto-Tcm) have previously been shown to promote engraftment of allogeneic T-depleted bone marrow transplant (TDBMT) under reduced-intensity non-myeloablative conditioning, resulting in resulting in the induction of tolerance to donor-type organ transplants without causing GvHD [Ophir, E. et al. Blood. (2013) 121(7):1220-1228].

In addition, various approaches have been considered for obtaining tolerance-inducing cells (eg, veto cells) lacking GvH reactivity and their use as adjuvant therapy for graft transplantation, some of which are listed below.

PCT Publication No. WO 2001/49243 discloses a method for transplanting a graft derived from a donor into a recipient, the method comprising the steps of (a) transplanting the graft into the recipient; and (b) administering to the recipient a dose comprising non-alloreactive foreign antigen-reactive cytotoxic T lymphocytes (CTLs), wherein the non-alloreactive foreign antigen-reactive CTLs are obtained by targeting the donor T lymphocytes to the foreign antigen or antigens (in the absence of exogenous IL-2 ), while the dose is significantly depleted in relation to T-lymphocytes (for example, CD4 ⁺ T cells and/or CD56 ⁺ natural killer cells) capable of developing into alloreactive CTLs, thereby preventing or attenuating both transplant rejection recipient and graft-versus-host disease.

PCT Publication No. WO 2007/023491 discloses the use of tolerogenic cells to reduce or prevent transplant rejection in the case of a non-isogenic transplant in a subject. The disclosed tolerogenic regulatory T cells (eg, CD4 ⁺ CD25 ⁺ cells) can be derived from any donor that is not isogenic with respect to both the subject and the graft ("third party" tolerogenic cells). The graft (eg, bone marrow) may be from any graft donor that is allogeneic or xenogeneic to the subject.

PCT Publication No. WO 2010/049935 discloses an isolated cell population containing non-GvHD-inducing cells, reactive to a foreign antigen, with a central memory T-lymphocyte (Tcm) phenotype, the cells being tolerance-inducing cells and capable of homing to lymph nodes. after transplant. According to WO 2010/049935, cells are obtained by: (a) contacting non-isogenic peripheral blood mononuclear cells (PBMCs) with a foreign antigen or antigens under conditions that eliminate GVH-reactive cells (e.g. cytokine-deprived culture) ; and (b) culturing the cells obtained in step (a) in the presence of IL-15 under conditions that allow cells with the Tcm phenotype to proliferate (eg, in the presence of IL-7 and/or IL-21).

PCT Publication No. WO 2012/032526 describes a method for treating a disease in a subject, comprising: (a) transplanting a non-isogenic graft of cells or tissues into the subject; and (b) administering to the subject a therapeutically effective amount of an isolated population of cells comprising non-graft-versus-host (GvHD) cells reactive to a foreign antigen with a central memory T-lymphocyte (Tcm) phenotype, wherein the cells are tolerance-inducing cells and are capable of homing to lymph nodes after transplantation. According to WO 2012/032526, cells are obtained by: (a) bringing PBMCs into contact with a foreign antigen or antigens in the presence or absence of IL-21 under conditions that eliminate GVH-reactive cells (for example, culturing for 1-5 days); and (b) culturing the cells obtained in step (a) in the presence of IL-15 in an antigen-free environment under conditions that allow cells with the Tcm phenotype to proliferate (eg, additionally in the presence of IL-7).

PCT Publication No. WO 2013/035099 discloses new methods for obtaining an isolated population of cells containing cells reactive against a foreign antigen with a central memory T-lymphocyte (Tcm) phenotype, wherein the cells are tolerance-inducing cells and/or endowed with activity against diseases and are capable of homing to lymph nodes after transplantation. According to WO 2013/035099, cells are obtained by: (a) bringing PBMCs into contact with a foreign antigen or antigens in the presence of IL-21 to ensure enrichment in antigen-reactive cells; and (b) culturing the cells obtained in step (a) in the presence of IL-21, IL-15 and IL-7 in an antigen-free environment in order to ensure the proliferation of cells with the Tcm phenotype.

The essence of the invention

In accordance with one aspect of some embodiments of the present invention, a method is provided for obtaining an isolated population of non-graft-versus-host (GvHD)-inducing cells having a central memory T-lymphocyte (Tcm) phenotype, wherein the cells are tolerance-inducing cells and/or endowed with activity with respect to disease and are capable of homing to lymph nodes after transplantation, the method comprising: (a) providing a population of at least 70% memory T cells; (b) bringing the memory T cell population into contact with the antigen or antigens in order to enrich it with antigen-reactive cells; and (c) culturing the cells obtained in step (b) in the presence of cytokines to allow the proliferation of cells having the Tcm phenotype, thereby obtaining an isolated population of non-GvHD-inducing cells.

In accordance with one aspect of some embodiments of the present invention, a method is provided for obtaining an isolated population of non-GvHD-inducing cells having a central memory T-lymphocyte (Tcm) phenotype, wherein the cells are tolerance-inducing cells and/or are endowed with activity against a disease and are capable of homing to transplanted lymph nodes, the method comprising: (a) treating non-adherent peripheral blood mononuclear cells (PBMCs) with an agent capable of depleting CD4 ⁺ , CD56 ⁺ and CD45RA ⁺ cells to obtain a population of memory T cells having the CD45RA ^- CD8 phenotype ⁺ ; (b) bringing the memory T cell population into contact with the antigen or antigens in the presence of IL-21 in order to enrich it with antigen-reactive cells; and (c) culturing the cells obtained in step (b) in the presence of IL-21, IL-15 and/or IL-7 to allow the proliferation of cells having the Tcm phenotype, thereby obtaining an isolated population of non-GvHD-inducing cells.

In accordance with one aspect of some embodiments of the present invention, a method is provided for obtaining an isolated population of non-GvHD-inducing cells having a central memory T-lymphocyte (Tcm) phenotype, wherein the cells are tolerance-inducing cells and/or are endowed with activity against a disease and are capable of homing to transplanted lymph nodes, the method comprising: (a) treating non-adherent peripheral blood mononuclear cells (PBMCs) with an agent capable of depleting CD4 ⁺ , CD56 ⁺ and CD45RA ⁺ cells to obtain a population of memory T cells having the CD45RA ^- CD8 phenotype ⁺ ; (b) bringing the memory T cell population into contact with the viral antigen or antigens in the presence of IL-21 in order to enrich it with antigen-reactive cells; and (c) culturing the cells obtained in step (b) in the presence of IL-21, IL-15 and/or IL-7 to allow the proliferation of cells having the Tcm phenotype, thereby obtaining an isolated population of non-GvHD-inducing cells.

In accordance with one aspect of some embodiments, the present invention provides an isolated population of non-GvHD-inducing cells comprising cells with a central memory T-lymphocyte (Tcm) phenotype, wherein the cells are tolerance-inducing cells and/or are endowed with disease activity and are capable of homing to lymph nodes after transplantation, obtained in accordance with the method according to some embodiments of the present invention.

In accordance with one aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising, as an active ingredient, an isolated population of non-GvHD-inducing cells according to some embodiments of the present invention and a pharmaceutically acceptable carrier.

In accordance with one aspect of some embodiments of the present invention, there is provided a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated population of non-GvHD-inducing cells according to some embodiments of the present invention, thereby providing treatment for the disease in the subject.

In accordance with one aspect of some embodiments of the present invention, there is provided the use of an isolated population of non-GvHD-inducing cells according to some embodiments of the present invention for the manufacture of a medicament identified for the treatment of a disease in a subject in need thereof.

In accordance with one aspect of some embodiments of the present invention, there is provided a method of treating a disease in a subject in need thereof, the method comprising: (a) analyzing a biological sample from the subject for the presence of a disease-associated antigen or antigens; (b) obtaining an isolated population of non-GvHD-inducing cells in accordance with the method of some embodiments of the present invention, directed to the disease-associated antigen or antigens, in order to ensure its enrichment with antigen-reactive cells; and (c) administering to the subject a therapeutically effective amount of the isolated population of non-GvHD-inducing cells of (b), thereby providing treatment for the disease in the subject.

According to one aspect of some embodiments of the present invention, there is provided a method of treating a subject in need of cell or tissue transplantation, the method comprising: (a) transplanting a cell or tissue graft into the subject; and (b) administering to the subject a therapeutically effective amount of an isolated population of non-GvHD-inducing cells, according to some embodiments of the present invention, thereby providing treatment to the subject in need of cell or tissue transplantation.

In accordance with one aspect of some embodiments of the present invention, the use of an isolated population of non-GvHD-inducing cells according to some embodiments of the present invention is provided for the manufacture of a medicament identified as an adjuvant therapy for transplantation of cells or tissues in a subject, where the subject is in need of cell or tissue transplantation.

In accordance with some embodiments of the present invention, memory T cells lack CD45RA ⁺ cells.

In accordance with some embodiments of the present invention, memory T cells lack CD4 ⁺ and/or CD56 ⁺ cells.

In accordance with some embodiments of the present invention, bringing a population of memory T cells into contact with an antigen or antigens is carried out in the presence of IL-21.

According to some embodiments of the present invention, culturing the cells obtained in step (b) in the presence of cytokines involves culturing the cells in the presence of any of IL-21, IL-15 and/or IL-7.

In accordance with some embodiments of the present invention, the method further comprises subjecting the cell donor to treatment with an antigen or antigens until a population of at least 70% memory T cells is provided.

In accordance with some embodiments of the present invention, a population of at least 70% memory T cells is enriched for an antigen or antigens.

In accordance with some embodiments of the present invention, the antigen or antigens are selected from the group consisting of a viral antigen, a bacterial antigen, a tumor antigen, an antigen associated with an autoimmune disease, a protein extract, a purified protein, and a synthetic peptide.

In accordance with some embodiments of the present invention, the antigen or antigens are autologous antigen-presenting cells, non-autologous antigen-presenting cells, artificial carriers, or artificial antigen-presenting cells.

In accordance with some embodiments of the present invention, the antigen or antigens are represented by antigen-presenting cells of the same origin as memory T cells.

In accordance with some embodiments of the present invention, the viral antigen or antigens are autologous antigen-presenting cells, non-autologous antigen-presenting cells, artificial carriers, or artificial antigen-presenting cells.

In accordance with some embodiments of the present invention, the antigen or antigens are represented by antigen-presenting cells of the same origin as the PBMC.

According to some embodiments of the present invention, the antigen presenting cells are dendritic cells.

In accordance with some embodiments of the present invention, the viral antigen or antigens comprise two or more viral peptides.

In accordance with some embodiments of the present invention, the viral antigen or antigens include an EBV peptide, a CMV peptide, and/or an adenovirus (Adv) peptide.

In accordance with some embodiments of the present invention, the viral antigen or antigens include three EBV peptides, two CMV peptides, and two adenovirus (Adv) peptides.

In accordance with some embodiments of the present invention, the viral antigen or antigens are selected from the group consisting of EBV-LMP2, EBV-BZLF1, EBV-EBNA1, CMV-pp65, CMV-IE-1, Adv-penton, and Adv-hexon.

In accordance with some embodiments of the present invention, the viral antigen or antigens include two or more of EBV-LMP2, EBV-BZLF1, EBV-EBNA1, CMV-pp65, CMV-IE-1, Adv-penton, and Adv-hexon.

In accordance with some embodiments of the present invention, the viral antigen or antigens further comprise a bacterial antigen.

In accordance with some embodiments of the present invention, contacting a population of memory T cells with an antigen or antigens in the presence of IL-21 is carried out for 12 hours to 5 days.

In accordance with some embodiments of the present invention, contacting a population of memory T cells with an antigen or antigens in the presence of IL-21 is carried out for 3 days or more.

In accordance with some variants of implementation of the present invention, the cultivation obtained in stage (b) cells in the presence of IL-21, IL-15 and/or IL-7 is carried out for 12 hours to 10 days.

In accordance with some variants of implementation of the present invention, the cultivation obtained in stage (b) cells in the presence of IL-21, IL-15 and IL-7 is carried out for 4 days to 8 days.

In accordance with some variants of implementation of the present invention, the cultivation obtained in stage (b) cells in the presence of IL-21, IL-15 and IL-7 is carried out for 6 days.

In accordance with some embodiments of the present invention, the total duration of obtaining non-GvHD-inducing cells is 10 days.

In accordance with some embodiments of the present invention, the method further provides for the depletion of alloreactive cells after step (c).

In accordance with some embodiments of the present invention, depletion of alloreactive cells is accomplished by depleting CD137+ and/or CD25+ cells after bringing cells with the Tcm phenotype into contact with antigen presenting cells (APC) of the host.

In accordance with some embodiments of the present invention, the method is carried out ex-vivo.

In accordance with some embodiments of the present invention, the PBMCs are not isogenic with respect to the subject.

In accordance with some embodiments of the present invention, PBMCs are allogeneic with respect to the subject.

In accordance with some variants of implementation of the present invention, cells with the phenotype of the central T-memory lymphocytes have an antigenic profile of CD3 ⁺ , CD8 ⁺ , CD62L ⁺ , CD45RA ^- , CD45RO ⁺ .

In accordance with some embodiments of the present invention, the biological sample is selected from the group consisting of blood, blood plasma, blood serum, cerebrospinal fluid, lymphatic fluid, and tissue biopsy.

In accordance with some embodiments of the present invention, the antigen or antigens are selected from the group consisting of a viral antigen, a bacterial antigen, a tumor antigen, and an antigen associated with an autoimmune disease.

In accordance with some embodiments of the present invention, the method further comprises transplanting a graft of cells or tissues into a subject.

In accordance with some embodiments of the present invention, the drug further provides for a transplant of cells or tissues.

In accordance with some variants of implementation of the present invention, transplantation is carried out simultaneously with the introduction of an isolated population of non-GvHD-inducing cells, before or after it.

In accordance with some embodiments of the present invention, the disease is a malignant disease.

In accordance with some embodiments of the present invention, the disease is a disease that is not malignant.

In accordance with some embodiments of the present invention, the isolated population of non-GvHD-inducing cells is intended to be administered prior to, concurrently with, or after transplantation of cells or tissues.

In accordance with some embodiments of the present invention, (b) is carried out before (a) in a method according to some embodiments of the present invention.

In accordance with some embodiments of the present invention, (a) and (b) are carried out simultaneously in a method according to some embodiments of the present invention.

In accordance with some embodiments of the present invention, the method further comprises conditioning the subject under sub-lethal, lethal, or super-lethal conditions prior to transplantation.

In accordance with some embodiments of the present invention, the use of an isolated population of non-GvHD-inducing cells further provides for a conditioning protocol under sublethal, lethal, or superlethal conditions.

In accordance with some embodiments of the present invention, sublethal, lethal, or superlethal conditioning is selected from the group consisting of total body irradiation (TBI), partial body irradiation, myeloablative conditioning, non-myeloablative conditioning, costimulation blockade, chemotherapeutic agent, and antibody immunotherapy.

In accordance with some embodiments of the present invention, the transplant of cells or tissues is not isogenic with respect to the subject.

In accordance with some embodiments of the present invention, the cell or tissue graft and the isolated population of non-GvHD-inducing cells are from the same donor.

In accordance with some embodiments of the present invention, the cell or tissue transplant is from a donor selected from the group consisting of an HLA-identical allogeneic donor, a non-HLA-identical allogeneic donor, and a xenogeneic donor.

In accordance with some embodiments of the present invention, the cell or tissue graft contains immature hematopoietic cells.

In accordance with some embodiments of the present invention, the cell or tissue graft is selected from the group consisting of a liver, pancreas, spleen, kidney, heart, lung, skin, intestine, brain, ovary, and lymphoid/hematopoietic cell or tissue cell or tissue.

In accordance with some variants of implementation of the present invention, a transplant of cells or tissues provides co-transplantation of several organs.

According to some embodiments of the present invention, co-transplantation involves the transplantation of immature hematopoietic cells and a parenchymal organ.

In accordance with some embodiments of the present invention, the immature hematopoietic cells and the parenchymal organ are obtained from the same donor.

In accordance with some embodiments of the present invention, immature hematopoietic cells are transplanted prior to, simultaneously with or after solid organ transplantation.

In accordance with some embodiments of the present invention, the subject has a malignant disease.

In accordance with some embodiments of the present invention, the cancer is a solid tumor or tumor metastasis.

In accordance with some embodiments of the present invention, the cancer is a malignant neoplasm of hematopoietic tissue.

In accordance with some embodiments of the present invention, the cancer is selected from the group consisting of leukemia, lymphoma, myeloma, melanoma, sarcoma, neuroblastoma, colon cancer, colon and rectal cancer, breast cancer, ovarian cancer, esophageal cancer, cancer of the synovial cells, liver cancer and pancreatic cancer.

In accordance with some embodiments of the present invention, the subject has a disease that is not malignant.

In accordance with some embodiments of the present invention, the non-cancerous disease is selected from the group consisting of organ dysfunction or failure, blood disease, transplant related disease, infectious disease, autoimmune disease, inflammation, allergy, injury, and injury.

In accordance with some embodiments of the present invention, the infectious disease is a viral disease or a bacterial disease.

In accordance with some embodiments of the present invention, the subject is a human subject.

Unless otherwise indicated, all technical and/or scientific terms used herein have the same meanings as are commonly understood by one of ordinary skill in the art to which the present invention pertains. Although methods and materials similar or equivalent to those described herein may be used to practice or test embodiments of the present invention, illustrative methods and/or materials are described below. In case of conflict, the description of the patent, including definitions, shall prevail. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

Brief description of the drawings

Some embodiments of the present invention are described herein by way of example only with reference to the accompanying drawings. Referring to the drawings in detail, it is emphasized that the information presented is given by way of example and for purposes of illustrative discussion of embodiments of the present invention. In this regard, the present description, together with the drawings, will enable those skilled in the art to understand how embodiments of the present invention may be practiced.

In drawings:

Fig. 1 is a schematic representation of a bone marrow transplant with T cell depletion (TDBMT) and reduced intensity conditioning using veto Tcm cells derived from CD44 ⁺ CD8 ⁺ memory T cells from OVA-immunized mice.

Fig. 2A is a graph showing that veto Tcm cells derived from a total population of antigen-stimulated cells (CD8 ⁺ CD44 ⁺ ) after immunization of OT1 mice with hen's egg albumin induce T-cell depleted tolerance (TCD) allogeneic stem cell transplant (SCT). Sublethally irradiated (5.25 Gy) Balb/c (H-2 ^d ) mice were transplanted with 20×10 ⁶ C57BL/6-nude (H-2 ^b ) BM cells with or without: 5×10 ⁶ allogeneic veto-Tcm C57BL/6 cells (H-2 ^b ) or 5×10 ⁶ CD8 ⁺ CD44 ⁺ cells derived from OVA-immunized OT-1 mice. Percentage of donor cells in peripheral blood was analyzed 45 days after transplantation by FACS using antibodies to host antigens (H-2D ^d ) to donor antigens (H-2K ^b ).

Figures 2B-C are graphs showing that CD8 ⁺ CD44 ⁺ Tcm cells do not induce GvHD in a stringent criteria mouse model. Sub-lethal dose (5 Gy) irradiated Balb/c (H-2d) mice were transplanted with 5×10 ⁶ or 10×10 ⁶ derived from allogeneic OVA-immunized C57BL/6 (H-2b) CD8 ⁺ CD44 ⁺ Tcm cells or fresh CD8 ⁺ CD44 ⁺ cells. CD8 ⁺ CD44 ^- naive cells were used as a positive control for GvHD. (FIG. 2B) Mean change in weight over 62 days after cell transfer. (FIG. 2C) The survival graph shows the duration of survival of mice in specific groups.

Fig. 2D is a schematic representation of a reduced intensity conditioning (RIC) model for testing tolerance induction by Tcm cells derived from naturally occurring memory cells (eg, OVA-reactive CD44 ⁺ CD8 ⁺ ).

Fig. 2E is a graph showing that veto Tcm cells derived from a population of naturally occurring memory cells (CD8 ⁺ CD44 ⁺ ) induce tolerance to TCD alloSCT. Sub-lethal (5 Gy) irradiated Balb/c (H-2 ^d ) mice were transplanted with 20×10 ⁶ BM C57BL/6-naked mice (H-2 ^b ) cells with or without: 5×10 ⁶ allogeneic veto-Tcm cells C57BL/6 (H-2 ^b ) or 5×10 ⁶ CD8 ⁺ CD44 ⁺ cells derived from OVA-immunized mice, or 5×10 ⁶ allogeneic freshly isolated CD8 ⁺ CD44 ⁺ C57BL/6 cells. Percentage of donor cells in peripheral blood was analyzed 55 days after transplantation by FACS using antibodies to host antigens (H-2D ^d ) to donor antigens (H-2K ^b ).

Figures 3A-D are graphs showing the generation of CD4 ^- CD56 veto ^- Tcm cells reactive against a foreign antigen using viral peptides. Human CD4 ^- CD56 responsive cells ^, which were obtained after depletion of CD4 ⁺ and CD56 ⁺ cells from the donor PBMC pool on day 0, were co-cultured with irradiated donor-derived DC stimulated with EBV, CMV and adenovirus viral peptides, with IL-21 to day +3, with the addition of IL-21, IL-15 and IL-7 from day +3-+9. (Figures 3A-B) FACS analysis of Tcm veto phenotype in CD4 ^- CD56 responsive cells ^on day 0 (FIG. 3A) and derived antiviral Tcm cells on culture day 9 (FIG. 3B). (Figures 3C-D) On day +9, cells were harvested and cultured with irradiated host PBMCs for 5 days (i.e., mixed culture) and then harvested and restimulated for 7 days with irradiated host PBMCs in a cut-off assay. dilution (LDA) in the presence of IL-2 to induce an effector phenotype. On day +21, a cytotoxicity assay was performed based on 358-methionine release against host-derived ConA blast cells. After 5 hours of mixed lymphocyte culture reaction (MLR), the supernatant was collected from the wells and counted based on radioactivity in a β-counter. (FIG. 3C) Is a graph of % responding cultures versus number of cells per culture. (FIG. 3D) This is a linear regression plot showing % non-responding cultures versus number of cells per culture. The frequency of occurrence (f) of host-reactive clones in a particular culture was calculated from the slope of the linear regression curve.

In FIG. 4A shows a schematic representation of a protocol for obtaining leukapheresis-derived antiviral human CD4-CD56-CD45RA-veto-Tcm cells and testing their host reactivity.

In FIG. 4B shows a schematic representation of the preparation of mature human dendritic cells loaded with a viral peptide.

Figures 5A-B are graphs showing antiviral veto-Tcm cells derived from CD4 ^- CD56 ^- CD45RA responsive cells using autologous DCs loaded with viral peptides as off ^- site stimulation. (FIG. 5A) Phenotype of responding CD4 ^- CD56 cells ^at day 0 and Tcm cells at day +9 (right panel). (FIG. 5B) Phenotype of CD4 ^- CD56 ^{- CD45RA} responsive cells on day 0 and Tcm cells on day +9 (right panel)

Fig. 5C is a graph showing the results of LDA analysis of the frequency of host-reactive CTL progenitors among antiviral Tcm cells derived from CD4 ^- CD56 ^{- CD45RA-} and CD4 ^- CD56 cell fractions ^, in compared with fresh CD4 ^- CD56 ^- CD19 ^- T cells. On day +9, a control population of fresh CD4 ^- CD56 ^- CD19 cells ^were bead sorted from freshly thawed donor cells. All three types of donor cell preparations (i.e. antiviral veto-Tcm CD4 ^- CD56 ^- , antiviral veto-Tcm CD4 ^- CD56 ^- CD45RA ^- and fresh CD4 ^- CD56 ^- CD19 ^- cells) were cultured with irradiated host PBMCs for 5 days in day +9 (ie mixed culture) and then harvested and restimulated for 7 days against irradiated host PBMCs in LDA in the presence of IL-2 to induce an effector phenotype. On day +21, a cytotoxicity assay was performed based on 358-methionine release against host-derived ConA blast cells. After 5 hours, the MLR supernatant was collected from the wells and counted based on radioactivity in a β-counter. A linear regression plot is presented, which shows the dependence of % non-responding cultures on the number of cells per culture. The frequency of occurrence (f) of host-reactive clones in a particular culture was calculated from the slope of the linear regression curve.

Fig. 6 is a table showing the results of the depletion of host-reactive T cells before and after the production of veto Tcm cells directed against viral peptides (which was done as in FIG. 4A and FIGS. 5A-C). It should be noted that the low frequency of host-reactive CTL-p and the overall levels of host-reactive CTL-p are based on LDA analysis (circled in the graph).

In FIG. 7 shows a schematic representation of an embodiment of a protocol for generating human Tcm veto cells derived from memory T cells and cultured with viral antigens in the context of autologous antigen presenting cells.

Fig. 8 is a table showing the results of 10 experiments in which veto Tcm cells were generated from memory T cells using the protocol shown in FIG. 7. It should be noted that the isolation and purification of the cells were quite reproducible.

Figures 9A-H are graphs showing the results of a typical FACS analysis within a single experiment, demonstrating the purity of veto Tcm cells derived from memory T cells using the protocol shown in FIG. 7. The figures show the results of the FACS analysis of each step shown in FIG. 8 as follows: Figures 9A-B show the results of a FACS analysis of peripheral blood mononuclear cells (PBMCs) before purification; figures 9C-D show the results of the post-purification FACS analysis for CD4 ^- CD56 ^- ; Figures 9E-F show the results of post-purification FACS analysis for CD4 ^- CD56 ^- CD45RA ^- (ie enrichment in CD4-CD56-CD45RO+ cells); figures 9G-H show the results of a FACS analysis of antiviral Tcm cells.

Detailed description of the present invention

The present invention, in some embodiments, relates to memory T cell-derived veto cells, and more particularly, but not exclusively, methods for their preparation and use in transplantation and disease management.

The principles and practice of the present invention can be better understood with reference to the drawings and the accompanying descriptions.

Before a detailed explanation of at least one embodiment of the present invention, it should be understood that the present invention is not necessarily limited in its application to the details set forth in the following description or illustrated in the examples. The present invention is capable of other embodiments or may be practiced or carried out in various ways. It should also be understood that the language and terminology used herein is for the purpose of description and should not be construed as limiting.

Bone marrow transplantation (BM) provides a definitive treatment for many patients with malignant neoplasms of the hematopoietic tissue and other disorders (eg, diseases of the hematopoietic system, functional organ failure). In addition, VMs can be co-transplanted with various other organs (eg, kidney or liver transplants from the same organ donor) to improve transplant success by inducing chimerism. However, the VM transplant contains donor T cells that are responsive to host antigens (Ag) and lead to generalized graft-versus-host disease (GvHD). GvHD, which is almost always fatal under these conditions, can be prevented by T-depleted bone marrow transplantation (TDBMT). However, the benefit of preventing GvHD may be offset by a significantly increased rate of graft rejection.

One approach to overcome allogeneic TDBMT rejection has been to use various veto cell based preparations as described in PCT Publications Nos. 035099. However, transplant rejection and GvHD still remain a major problem in adoptive cell transfer therapy, especially in allogeneic systems.

In the course of putting the present invention into practice, the present inventors have found an improved population of veto cells that also have disease activity (eg, antiviral activity) without inducing graft versus host (GvH). These new cells are obtained by depleting memory T cells for alloreactive clones through antigen activation.

As shown herein below and in the following "Examples" section, the inventors of the present invention have introduced novel methods for generating veto cells for HLA mismatch applications (eg, allogeneic transplantation), starting with memory T cells. In particular, as shown in FIG. 1, the present inventors used a mouse model to generate veto Tcm cells from naturally occurring memory T cells. Memory Tcm-derived Tcm cells induced tolerance in the T-cell-depleted bone marrow transplantation (TDBMT) model with reduced intensity conditioning, without graft-versus-host reactivity (Fig. 2A), and exhibited a marked increase in chimerism after performing a reduced intensity conditioning protocol (FIG. 2E). However, it was shown that fresh CD8 ⁺ CD44 ⁺ memory cells (which were not subjected to antigenic activation) induced a significant level of mortality and weight loss due to GvHD (figures 2B-C).

The viral antigens were then used to generate human veto Tcm cells from a population of CD4-CD56 cells. As illustrated in Figures 3A-B, cells cultured in the presence of viral antigens displayed on the surface of autologous dendritic cells exhibited a 93% Tcm phenotype (CD62 ⁺ CD45RO ⁺ cells) 9 days after the start of culture. In addition, these Tcm cells provided a 2-fold logarithmic depletion of host alloreactive clones compared to fresh CD4 ^- CD56 cells ⁽ Figures 3C-D and Table 2, below). Veto cells were then obtained from the human memory cell population by first depleting peripheral blood mononuclear cells (PBMCs) from a cell donor for CD4 ⁺ , CD56 ⁺ and CD45RA ⁺ cells (FIG. 4A). Accordingly, the rest of the cell population contained CD8+ donor memory T cells. Memory CD8+ T cells were co-cultured with dendritic cells (from the same cell donor) where the dendritic cells were engineered to express an antigen (eg, a cocktail of viral antigens including EBV, CMV and adenovirus). For the first 3 days, the cell culture was supplemented with IL-21, and then from day 3 until day 9, IL-21, IL-15 and IL-7 were added to the culture. The resulting cell population contained a Tcm phenotype and was not characterized by any host reactivity (as illustrated in Figures 5A-C and 6).

In general, by depleting alloreactive clones in the memory T cell pool by antigenic activation (eg, viral antigens, tumor antigens), the problem of residual GvHD caused by the memory T cell pool can be addressed. In addition, these results suggest that the new drug based on veto cells derived from a population of memory cells can be used in cell therapy to induce transplant tolerance without GvHD-related complications, as well as for the treatment of diseases (for example, for antiviral or anti-cancer applications).

Thus, in accordance with one aspect of the present invention, a method is provided for obtaining an isolated population of non-graft-versus-host (GvHD)-inducing cells having a central memory T-lymphocyte (Tcm) phenotype, wherein the cells are tolerance-inducing cells and/or endowed with activity with respect to disease and are capable of homing to lymph nodes after transplantation, the method comprising: (a) providing a population of at least 70% memory T cells; (b) bringing the memory T cell population into contact with the antigen or antigens in order to enrich it with antigen-reactive cells; and (c) culturing the cells obtained in step (b) in the presence of cytokines to allow the proliferation of cells having the central memory T lymphocyte (Tcm) phenotype, thereby obtaining an isolated population of non-GVHD-inducing cells.

The phrase "isolated population of cells" in the context of this document refers to cells that have been isolated from their natural environment (eg, the human body).

The term "non-graft-versus-host-inducing" or "non-GvHD-inducing" as used herein refers to having significantly reduced or absent graft-versus-host (GvH)-inducing reactivity. Thus, the cells of the present invention are produced as being substantially free of graft-versus-host disease (GvHD) as evidenced by the survival, weight, and general condition of the transplanted subject 30-120 days after transplantation. Methods for assessing reduced GvHD in a subject are well known to those skilled in the art.

In accordance with one embodiment, the cells of the present invention have host reactivity reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 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% compared to cells that were not obtained in accordance with the ideas of the present invention.

The phrase "central memory T lymphocyte (Tcm) phenotype" as used herein refers to a subpopulation of cytotoxic T cells that relocate back to the lymph nodes. Cells with the Tcm phenotype in humans usually have a CD3+/CD8+/CD62L+/CD45RO+/CD45RA antigenic profile. It is understood that Tcm cells may express all of the antigenic profile markers on an individual cell surface, or may express only a portion of the antigenic profile markers on an individual cell surface. Determination of the phenotype of a cell can be carried out using any method known to the person skilled in the art, such as, for example, fluorescence activated cell sorting (FACS) or labeling using capture ELISA.

According to one embodiment, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or even 100% of the isolated cell population have a Tcm antigenic profile.

In accordance with a particular embodiment, about 20-40%, about 30-50%, about 40-60%, about 50-70%, about 60-80%, about 70-90%, about 80-100%, or about 90 -100% of the isolated cell population are characterized by the Tcm antigenic profile.

The isolated population of non-GvHD-inducing cells of the present invention is also referred to herein as "Tcm cells".

As mentioned, Tcm cells usually go back to the lymph nodes after transplantation. According to some embodiments, the isolated population of cells of the present invention may revert back to any lymph nodes after transplantation, such as, for example, peripheral lymph nodes and mesenteric lymph nodes. The homing property of these cells allows them to exercise their veto effect in a fast and efficient manner.

The isolated population of Tcm cells of the present invention are tolerance-inducing cells.

The phrase "tolerance-inducing cells" in the context of this document refers to cells that cause reduced reactivity of recipient cells (for example, recipient T cells) when they come into contact with cells of the recipient, compared with the reactivity of the recipient cells in the absence of introduced tolerance-inducing cells . Tolerance-inducing cells include veto cells (i.e., T cells that lead to apoptosis of host T cells upon contact), as previously described in PCT Publication Nos. WO 2001/049243 and WO 2002/102971.

The term "veto activity" refers to immune cells (eg, donor-derived T cells) that result in inactivation of the recipient's T cells reactive against the donor's cells upon recognition and binding to veto cells. According to one embodiment, inactivation results in apoptosis of the recipient's T cells that are reactive with the donor's cells.

In addition, or alternatively, the isolated population of Tcm cells of the present invention has activity against the disease.

The term "disease activity" refers to the function of Tcm cells in relation to a diseased cell. Activity against a disease may be directed directly to a disease-affected cell, for example, in the case of the ability to kill the disease-affected cell. This activity may be associated with TCR-independent killing mediated by LFA1-I/CAM1 binding [Arditti et al., Blood (2005) 105(8):3365-71. Epub 2004 Jul 6]. In addition, or alternatively, disease activity may be mediated, for example, by activating other cell types (e.g., CD4+ T cells, B cells, monocytes, macrophages, NK cells), resulting in the death of the diseased cells (eg, by killing, apoptosis, or by secretion of other factors, eg, antibodies, cytokines, etc.).

The diseased cell may include, for example, a virus-infected cell, a bacteria-infected cell, a cancer cell [e.g., a solid tumor cell or a leukemic/lymphoma cell, also referred to herein as Tcm-mediated graft-versus-leukemia (GVL)] , a cell associated with an autoimmune disease, a cell associated with an allergic reaction, or a cell altered due to stressors, exposure, or age.

In accordance with some embodiments, the Tcm cells of the present invention may be non-genetically modified or genetically modified cells (e.g., cells that have been engineered to express or repress the expression of specific genes, markers, or peptides). or to secrete or inhibit the secretion of specific cytokines). Any method known in the art can be used in the genetic engineering of cells, such as by inactivating the relevant gene/genes or by inserting an antisense RNA that prevents the expression of the polypeptide (see, for example, WO/2000/039294, which is incorporated herein by reference ).

In accordance with some embodiments of the present invention, a method is provided for obtaining an isolated population of cells, the method comprising (a) providing a population of memory T cells; (b) bringing the memory T cell population into contact with the antigen or antigens in order to enrich it with antigen-reactive cells; and (c) culturing the cells obtained in step (b) in the presence of cytokines to ensure the proliferation of cells having the phenotype of central memory T-lymphocytes (Tcm).

The term "memory T cells" in the context of this document refers to a subpopulation of T lymphocytes that have arisen previously and respond to an antigen, also called antigen-stimulated T cells.

According to one embodiment, memory T cells comprise at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or even 100% of the cell population.

According to one embodiment, memory T cells include cytotoxic T cells expressing the CD8 marker (ie, CD8 ⁺ T cells).

According to another embodiment, memory T cells contemplate the CD8 ⁺ CD45RO ⁺ phenotype.

According to another embodiment, memory T cells contemplate the CD8 ^{+ CD45RA} phenotype.

According to another embodiment, the memory T cells contemplate the CD8 ⁺ CD45RO ^{+ CD45RA} phenotype.

The selection of CD8+ memory T cells can be done by selecting cells co-expressing CD8 ⁺ and CD45RA ^- and/or cells co-expressing CD8 ⁺ and CD45RO ⁺ and can be performed using any method known in the art, such as using affinity purification (eg, by using MACS beads, FACS sorter and/or labeling using capture ELISA).

The selection of CD8+ memory T cells can further be carried out by selecting effector T cells and central memory T cells, the latter expressing, for example, CD62L, CCR7, CD27 and/or CD28.

According to one embodiment, memory T cells are derived from a population of peripheral blood mononuclear cells (PBMCs).

According to one embodiment, memory T cells are derived from lymphoid tissue, such as from lymph nodes or the spleen.

In order to obtain a cell population with a high degree of purity for memory T cells (for example, at least about 50-70% of memory T cells), or to increase the number of memory T cells, PBMC can be depleted in relation to naive cells eg, CD45RA ⁺ cells, adherent cells (eg, monocytes, macrophages), CD4 ⁺ cells (eg, helper T cells), CD56 ⁺ cells (eg, NK cells), or any other cell without a T cell phenotype memory.

Depletion of naive T cells (e.g., expressing CD45RA ⁺ cells), CD4 ⁺ and/or CD56 + cells can be performed using any method known in the art, such as using affinity purification (e.g., using beads for MACS, FACS sorter and/or labeling using capture ELISA).

Depletion of adherent cells can be accomplished using any method known in the art, for example by culturing PBMCs on a cell culture dish (eg for 2-6 hours) and harvesting non-adherent cells.

According to one embodiment, memory T cells lack CD45RA ⁺ cells.

In accordance with one embodiment, memory T cells lack CD4 ⁺ and/or CD56 ⁺ cells.

In order to deplete alloreactive clones from the pool of memory T cells, the memory T cells are brought into contact with an antigen or antigens.

As used herein, the phrase "antigen or antigens" refers to a soluble or insoluble (such as, for example, membrane-associated) molecule capable of inducing an immune response.

For example, the antigen or antigens can be whole cells (eg, live or dead cells), cell fractions (eg, lysed cells), cellular antigens (eg, cell surface antigens), protein extract, purified protein, or synthetic peptide. For example, an antigen or antigens according to a certain embodiment of the present invention include antigens associated with a malignant disease (i.e., tumor antigens), antigens associated with an autoimmune disease (i.e., autoimmune antigens), antigens associated with an allergic reaction (i.e., allergic antigens), viral antigens (i.e. viral antigens), bacterial antigens (i.e. bacterial antigens), or fungal antigens (i.e. fungal antigens).

In one embodiment, the antigen or antigens are derived from an infectious agent (eg, virus, bacterium, fungus) that typically infects immunocompromised subjects, such as transplant recipients. Exemplary infectious agents that may affect immunocompromised patients include, but are not limited to, viruses such as parvovirus (e.g. parvovirus B19), rotavirus, varicella zoster virus (VZV), herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein virus -Barr (EBV), polyomavirus (eg, VC virus); bacteria such as S pneumoniae, P aeruginosa, Legionella pneumophila, L monocytogenes, Nocardia spp., Mycobacterium spp., S aureus, Nocardia spp., P aeruginosa spp., Serratia spp., Chromobacterium, Streptococcus, Burkholderia, Mycobacterium (e.g. Mycobacterium avium-intracellulare complex) , encapsulated bacteria such as S pneumoniae, H influenzae and N meningitidis; fungi such as P jiroveci, Candida, and Aspergillus; and parasitic organisms such as Toxoplasma species, Cryptosporidium and Strongyloides species.

In one embodiment, the antigen is a viral antigen, such as, but not limited to, an antigen of Epstein-Barr virus (EBV), adenovirus (Adv), cytomegalovirus (CMV), rhinoviruses, viruses that cause acute respiratory illness, hepatitis A viruses, B and C, herpes simplex virus, HIV, influenza virus, Japanese encephalitis virus, measles virus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, variola virus, chickenpox virus, rotavirus, West Nile virus, polyomavirus ( such as the VC virus) or the Zika virus.

As further specific examples of viral antigens, adenovirus antigens include, without limitation, Adv-pentone or Adv-hexon; CMV antigens include, but are not limited to, envelope glycoprotein B, CMV IE-1, and CMV pp65; EBV antigens include, without limitation, LMP2 EBV, BZLF1 EBV, EBNA1 EBV, P18 EBV, and P23 EBV; hepatitis B antigens include, but are not limited to, HBV S, M, and L proteins, HBV pre-S antigen, HBCAG DELTA, HBV HBE, HCV RNA, HCV NS3, and HCV NS4; viral antigens of the herpes simplex virus include, without limitation, immediate early proteins and glycoprotein D; HIV antigens include, without limitation, gene products of the gag, pol, and env genes such as HIV gp32, HIV gp41, HIV gp120, HIV gp160, HIV P17/24, HIV P24, HIV P55 GAG, HIV P66 POL, HIV TAT, HIV GP36, Nef protein and reverse transcriptase; influenza virus antigens include, without limitation, hemagglutinin and neuraminidase; Japanese encephalitis virus viral antigens include, but are not limited to, the E, M-E, M-E-NS1, NS1, NS1-NS2A, and 80% E proteins; measles virus antigens include, without limitation, measles virus fusion protein; rabies virus antigens include, without limitation, rabies virus glycoprotein and rabies virus nucleoprotein; respiratory syncytial virus viral antigens include, but are not limited to, the RSV fusion protein and the M2 protein; rotavirus antigens include, without limitation, VP7sc; rubella virus antigens include, but are not limited to, the E1 and E2 proteins; and varicella-zoster virus viral antigens include, without limitation, gpl and gpll.

According to one embodiment, the antigen is a bacterial antigen, such as, without limitation, an anthrax antigen; gram-negative bacilli, chlamydia, diphtheria-causing bacteria, Haemophilus influenzae, Helicobacter pylori, malaria-causing plasmodium, Mycobacterium tuberculosis, pertussis toxin, pneumococcus, rickettsia, staphylococcus, streptococcus, and tetanus-causing bacteria.

As further specific examples of bacterial antigens, anthrax antigens include, without limitation, protective anthrax antigen; Gram-negative bacillus antigens include, without limitation, lipopolysaccharides; Haemophilus influenzae antigens include, without limitation, capsular polysaccharides; diphtheria-causing bacterium antigens include, without limitation, diphtheria toxin; Mycobacterium tuberculosis antigens include, but are not limited to, mycolic acid, heat shock protein 65 (HSP65), 30 kDa major secreted protein, and 85A antigen; whooping cough bacteria antigens include, but are not limited to, haemagglutinin, pertactin, FIM2, FIM3, and adenylate cyclase; pneumococcal antigens include, without limitation, pneumolysin and pneumococcal capsular polysaccharides; rickettsia antigens include, without limitation, rompA; streptococcal antigens include, but are not limited to, M proteins; and tetanus-causing antigens include, without limitation, tetanus toxin.

In accordance with one embodiment, the antigen is a superbug antigen (eg, multidrug-resistant bacteria). Examples of superbugs include, without limitation, Enterococcus faecium, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae (including Escherichia coli, Klebsiella pneumoniae, Enterobacter spp.).

According to one embodiment, the antigen is a fungal antigen. Examples of fungi include, but are not limited to, Candida, coccidiodes, cryptococcus, histoplasma, leishmania, plasmodium, protozoa, parasitic organisms, schistosomes, lichen organisms, toxoplasma, and trypanosoma cruzi.

As additional specific examples of fungal antigens, coccidiodes antigens include, without limitation, spherule antigens; cryptococcal antigens include, without limitation, capsular polysaccharides; histoplasma antigens include, without limitation, heat shock protein 60 (HSP60); leishmania antigens include, but are not limited to, gp63 and lipophosphoglycan; plasmodium falciparum antigens include, without limitation, merozoite surface antigens, sporozoite surface antigens, sporozoan antigens, gametocyte/gamete surface antigens, protozoan and other parasitic antigens, including the pf 155/RESA hematostage antigen; schistosome antigens include, without limitation, glutathione-S-transferase and paramyosin; lichen-causing fungal antigens include, but are not limited to trichophytosis; Toxoplasma antigens include, without limitation, SAG-1 and p30; and trypanosoma cruzi antigens include, but are not limited to, a 75-77 kDa antigen and a 56 kDa antigen.

In accordance with one embodiment, the antigen is an antigen expressed by cells associated with an undesired autoimmune or allergic condition. Exemplary autoimmune conditions include, without limitation, acute necrotizing hemorrhagic encephalopathy, allergic asthma, alopecia areata, anemia, aphthous ulcer, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), asthma, autoimmune thyroiditis, conjunctivitis, Crohn's disease, cutaneous erythematosus lupus, dermatitis (including atopic dermatitis and pruritic dermatitis), diabetes mellitus, diabetes mellitus, erythema nodosa leprosy, keratoconjunctivitis, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, Sjogren's syndrome, including keratoconjunctivitis sicca associated with Sjogren's syndrome, Stevens-Johnson syndrome , systemic lupus erythematosus, ulcerative colitis, vaginitis and Wegener's granulomatosis.

Examples of autoimmune antigens include, but are not limited to, glutamic acid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelin proteolipid protein, acetylcholine receptor components, thyroglobulin, and thyroid stimulating hormone (TSH) receptor.

Examples of allergic antigens include, but are not limited to, pollen antigens such as Cryptomeria japonica pollen antigens, ragweed pollen antigens, chaff pollen antigens, animal antigens (such as dust mite antigens and cat antigens), histocompatibility antigens, and penicillin and other drugs.

According to one embodiment, the antigen is an antigen (or a portion thereof, eg an antigen epitope) expressed by tumor cells. In one embodiment, the antigen (or part thereof) is derived from a protein expressed in hematopoietic tissue (e.g., in a malignant neoplasm of hematopoietic tissue, such as a leukemic antigen) or expressed in a solid tumor (e.g., in melanoma, pancreatic cancer). gland, liver cancer, gastrointestinal cancer, etc.).

Examples of tumor antigens include, but are not limited to, A33, BAGE, Bcl-2, B cell maturation antigen (BCMA), BCR-ABL, β-catenin, cancer/testis antigens (STA, e.g. MAGE-1, MAGE-A2/A3 and NY-ESO-1), CA 125, CA 19-9, CA 50, CA 27.29 (BR 27.29), CA 15-3, CD5, CD19, CD20, CD21, CD22, CD33, CD37, CD45, CD123, CEA, c-Met, CS-1, cyclin B1, DAGE, EBNA, EGFR, ELA2, ephrin B2, estrogen receptor, FAP, ferritin, folate-binding protein, GAGE, G250/CA GC, GD-2, GM2, gp75, gp100 (Pmel 17), HA-1, HA-2, HER-2/neu, HM1.24, HPV E6, HPV E7, hTERT, Ki-67, LRP, mesothelin, mucin-like cancer antigen (MCA), MUC1, p53, PR1, PRAME, PRTN3, RHAMM (CD 168), WT-1. Additional tumor antigens are provided in Molldrem J. Biology of Blood and Marrow Transplantation (2006) 12:13-18; Alatrash G. and Molldrem J., Expert Rev Hematol. (2011) 4(1): 37-50; Renkvist et al., Cancer Immunol Immunother (2001) 50:3-15; van der Bruggen P, Stroobant V, Vigneron N, Van den Eynde B. Peptide database: T cell-defined tumor antigens. Cancer Immun (2013), www(dot)cancerimmunity(dot)org/peptide/; Rittenhouse, Manderino and Hass, Laboratory Medicine (1985) 16(9) 556-560; all of which are incorporated herein by reference.

Below is a list of tumor antigens that can be used in accordance with the ideas of some embodiments of the present invention.

In accordance with one embodiment, the antigen includes a single antigen (eg, a viral, bacterial, or tumor antigen).

In accordance with one embodiment, the antigen or antigens comprise two or more antigens (e.g., a mixture of antigens from one antigen group, e.g., viral antigens, tumor antigens, etc.; or a mixture of antigens from different antigen groups, e.g., viral and bacterial antigens, viral and tumor antigens, viral and autoimmune antigens, tumor and autoimmune antigens, or autoimmune and allergic antigens).

In accordance with one embodiment, the antigen or antigens include two, three, four, five or more antigens (eg, in one composition or in several compositions).

In accordance with one embodiment, the antigen or antigens include two, three, four, five or more tumor antigens (eg, in one formulation or in multiple formulations).

In accordance with one embodiment, the antigen or antigens include two, three, four, five or more viral antigens (eg, in one composition or in several compositions).

In a specific embodiment, the antigen or antigens include three viral antigens, for example, an EBV peptide, a CMV peptide, and an Adv peptide.

In accordance with a specific embodiment, the antigen or antigens include two or more of EBV-LMP2, EBV-BZLF1, EBV-EBNA1, CMV-pp65, CMV-IE-1, Adv-penton, and Adv-hexon (e.g., two, three, four, five, six, or all seven antigens).

In a particular embodiment, the antigen or antigens comprise a collection of peptide mixtures that are libraries of overlapping peptides (e.g., 15-mers overlapping at 11 amino acids) spanning the entire protein sequence of three viruses: CMV, EBV, and Adeno (such peptide mixtures can be purchased, for example, from JPT Technologies, Berlin, Germany).

In accordance with another specific embodiment, the antigen or antigens include a set of seven peptide mixtures, covering EBV-LMP2, EBV-BZLF1, EBV-EBNA1, CMV-pp65, CMV-IE-1, Adv-pentone and Adv-hexon, at a concentration, for example, 100 ng/peptide or 700 ng/mixture of seven peptides.

In accordance with a particular embodiment, the viral antigens further comprise a bacterial antigen or antigens.

Additional stimulatory antigens such as, but not limited to, hen's egg albumin, DNP (dinitrophenyl), KLH (Megathura crenulata mollusc hemocyanin) can be used to stimulate the memory T cell immune response.

In accordance with one embodiment, the antigen or antigens are "foreign antigen or antigens", i. soluble or insoluble (such as membrane-associated) antigen or antigens that are absent from either the donor or the recipient. For example, the foreign antigen may be a foreign cell.

Third-party cells can be either allogeneic or xenogenic with respect to the donor and recipient (explained in more detail later in this document). In the case of allogeneic foreign cells, such cells have HLA antigens different from those of the donor, but which are not cross-reactive with HLA antigens of the recipient, so that foreign cells formed against such cells are not reactive with the graft or recipient antigens. .

In accordance with one embodiment of the present invention, the allogeneic or xenogeneic foreign cells are stimulatory cells selected from the group consisting of cells isolated from a population of peripheral blood lymphocytes (PBL), spleen or lymph nodes, cytokine-mobilized PBL, expanded in vitro antigen presenting cells (ARC), in vitro propagated dendritic cells (DC) and artificial antigen presenting cells.

The antigens of the present invention may be present on the surface of cells, viruses, fungi or bacteria, or may be derived from and/or purified from them. In addition, the viral, fungal or bacterial antigen may be presented on the surface of the infected cell, or the cellular antigen may be presented in an artificial carrier (eg, liposome) or on the surface of an artificial antigen-presenting cell (eg, a cell line transfected with the antigen or antigens). Thus, viral, bacterial or fungal antigens can be represented by cells infected with them, or otherwise obtained with the ability to express viral/bacterial/fungal peptides. Similarly, tumor antigens, autoimmune antigens, or allergic antigens can be represented by cells that are capable of expressing such proteins.

The use of cells, virus-infected cells, bacteria-infected cells, viral cell peptides, or bacterial cell peptides as antigens is particularly promising in view of the fact that such antigens include a diverse array of antigenic determinants, and thus direct the formation of Tcm cells of a heterogeneous population. , which may additionally provide faster recovery of T cells in cases where such recovery is necessary, for example, after lethal or sublethal radiation exposure or a chemotherapeutic procedure (as discussed in detail below), or for disease control (as discussed in detail below) .

Thus, antigen-presenting cells (autologous or non-autologous, as discussed below), cell lines, artificial carriers (such as a liposome), or artificial antigen-presenting cells (for example, a line of leukemic cells or fibroblasts transfected with an antigen or antigens), can be used to represent short synthetic peptides fused to or loaded on them, or to represent protein extracts or purified proteins. Such short peptides, protein extracts or purified proteins may be peptides derived from a viral, bacterial, fungal, tumor, autoimmune or allergic antigen, or peptides representing any other antigen.

To analyze the sequences of a viral, bacterial, fungal, tumor, autoimmune or allergic antigen in order to identify immunogenic short peptides, i.e. peptides presented in the context of a major histocompatibility complex (MHC) class I or MHC class II, specialized software can be used.

Additionally, the artificial carriers or artificial APCs of the present invention can be designed to display MHC without being primed by an exogenous peptide. Thus, according to one embodiment, the artificial APC comprises K562 tumor cells transfected with an MHC determinant (e.g., autologous to a memory T cell) and with a stimulatory molecule [as previously described, e.g., Suhoski MM et al., Mol Ther. (2007) 15(5): 981-8], or fibroblasts transfected with them.

In one embodiment, the antigen or antigens are represented by antigen presenting cells (e.g., DCs) autologous to memory T cells, e.g., of the same origin (e.g., from the same donor), in order to provide their recognition by memory T cells in the context of MHC class I or MHC class II.

In one embodiment, the antigen or antigens are genetically modified antigen presenting cells or artificial antigen presenting cells characterized by the presence of MHC antigens (also referred to as human leukocyte antigens (HLA)), recognized by memory T cells.

According to one embodiment, the antigen presenting cells include human cells.

According to one embodiment, the antigen presenting cells include dendritic cells (DC).

According to one embodiment, the antigen presenting cells include mature dendritic cells.

According to one embodiment, the antigen presenting cells include irradiated dendritic cells.

Thus, in accordance with one embodiment, about 5-10 Gy, about 10-20 Gy, about 20-30 Gy, about 20-40 Gy, about 20-50 Gy, about 10-50 Gy are irradiated with DC. In accordance with a specific embodiment, the DC is irradiated with approximately 10-50 Gy (eg, 30 Gy).

Methods for using dendritic cells as APCs are known in the art. Thus, as a non-limiting example, peripheral blood mononuclear cells (PBMCs) can be obtained from a cell donor [eg, the same cell donor as memory T cells]. PBMCs are plated in a culture dish and incubated for 1-5 hours (eg 3 hours) at 37° C. at 5% CO ₂ /O ₂ using DC cell medium (eg DC Cellgro medium) supplemented with serum human (eg, 1% human serum) and penicillin/streptomycin (eg, 1% penicillin/streptomycin). The cell supernatant (containing T cells) is discarded and the remaining cells (i.e., adherent cells) are further incubated for 48-96 (e.g., 72 hours) under the same culture conditions supplemented with GM-CSF cytokines (e.g., 800- 1600 IU/ml) and IL-4 (eg 750 IU/ml) (available from eg Peprotech, Hamburg, Germany). Two to four days later (e.g., three days), non-adherent cells (i.e., comprising mostly immature dendritic cells) are harvested and seeded with DC maturation cytokines, e.g., GM-CSF (e.g., 800 IU/mL), IL- 4 (eg 750 IU/ml), LPS (eg from E. coli 055:B5 at eg 40 ng/ml) and IFN-γ (eg 200 IU/ml) (available from eg Peprotech, Hamburg, Germany) and incubated overnight. The next day, non-adherent cells can be discarded and adherent mature DCs can be gently isolated using, for example, chilled PBS containing, for example, 2 mM EDTA and 1% HS, after incubation on ice for 10-60 minutes (e.g., 30 minutes ), thereby obtaining large cells consisting of their mature DCs.

In order to present the antigen or antigens on the surface of the APC (eg, mature DC), the antigen or antigens are co-cultured with the APC (eg, DC) for about 30 minutes to 3 hours (eg, 1 hour) at 37° C. at 5%. CO ₂ /O ₂ . For example, DC can be loaded with a cocktail of peptide mixtures (viral peptides) by incubation for approximately 1 hour at 37°C at 5% CO ₂ /O ₂ . The antigen-loaded APCs (eg, DCs) are then ready to be used to generate Tcm cells from a population of memory T cells, in accordance with some embodiments of the present invention.

The Tcm cells of the present invention are typically produced by first contacting a population of memory T cells with an antigen or antigens (such as those described above) in a culture supplemented with IL-21 (e.g., in another non-cytokine culture, i.e. without adding any additional cytokines). This step is typically carried out for about 12-24 hours, about 12-36 hours, about 12-72 hours, 12-96 hours, 12-120 hours, about 24-36 hours, about 24-48 hours, about 24-72 hours. hours, approximately 36-48 hours, approximately 36-72 hours, approximately 48-72 hours, approximately 48-96 hours, approximately 48-120 hours, 0.5-1 days, 0.5-2 days, 0.5- 3 days, 0.5-5 days, 1-2 days, 1-3 days, 1-5 days, 1-7 days, 1-10 days, 2-3 days, 2-4 days, 2-5 days, 2-6 days, 2-8 days, 3-4 days, 3-5 days, 3-7 days, 4-5 days, 4-8 days, 5-7 days, 6-8 days or 8-10 days, and it provides enrichment for antigen-reactive cells.

According to a specific embodiment, contacting a population of memory T cells with an antigen or antigens (such as those described above) in an IL-21 supplemented culture (another cytokine-free culture) is performed for 1-5 days (e.g., 3 days) .

Contacting a population of memory T cells with an antigen or antigens (such as those described above) in IL-21 supplemented culture is typically done 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 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. In accordance with a specific embodiment, the concentration of IL-21 is 50-500 IU/ml (eg, 100 IU/ml).

In a particular embodiment, exposure of a population of memory T cells to an antigen or antigens is carried out in a cytokine-free culture (e.g., supplemented with IL-21 only), such a culture condition ensuring survival and enrichment of only those cells that are stimulated. and activation by an antigen or antigens (ie, antigen-reactive cells), since these cells secrete cytokines (eg, IL-2) that ensure their survival (all other cells die under such culture conditions).

The ratio of antigen or antigens (for example, those displayed on the surface of APCs, such as antigen-primed dendritic cells) and memory T cells is typically from about 1:2 to about 1:10, such as about 1:4, about 1: 5, approximately 1:6, approximately 1:8 or approximately 1:10. In a particular embodiment, the ratio of antigen or antigens (eg, those displayed on the surface of APCs) to memory T cells is from about 1:2 to about 1:8 (eg, 1:5).

The resulting memory T cells (i.e., after culturing with IL-21) are then cultured in the presence of IL-21, IL-15, and/or IL-7 in an antigen-free environment (i.e., no added antigen or antigens) to ensure the proliferation of cells with the Tcm phenotype. This step is typically carried out for about 12-24 hours, about 12-36 hours, about 12-72 hours, about 12-96 hours, about 12-120 hours, about 12-240 hours, 24-36 hours, 24-48 hours. hours, approximately 24-72 hours, 24-96 hours, 24-120 hours, 24-240 hours, approximately 48-72 hours, approximately 48-120 hours, approximately 48-240 hours, approximately 96-240 hours, approximately 120- 144 hours, approximately 120-240 hours, approximately 144-240 hours, 0.5-1 days, 0.5-2 days, 0.5-3 days, 0.5-5 days, 0.5-10 days, 1-2 days, 1-3 days, 1-4 days, 1-6 days, 1-8 days, 1-10 days, 1-15 days, 2-3 days, 2-4 days, 2-5 days, 2-6 days 2-8 days 2-10 days 4-5 days 4-6 days 4-8 days 4-10 days 5-6 days 5-7 days 5-8 days 5-10 days, 5-15 days, 6-7 days, 6-8 days, 6-10 days, 7-8 days, 7-9 days, 7-10 days, 7-13 days, 7-15 days, 8-10 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 resulting memory T cells (i.e., after cultured with IL-21) are cultured in the presence of IL-21, IL-15, and IL-7 in an antigen-free environment for approximately 4-8 days (for example, 6 days).

This step is usually carried out in the presence of IL-21 at a concentration of approximately 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. In accordance with a specific embodiment, the concentration of IL-21 is 50-500 IU/ml (eg, 100 IU/ml).

This step is further carried out in the presence of IL-15 at a concentration of approximately 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 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-15. In accordance with a specific embodiment, the concentration of IL-15 is 50-500 IU/ml (for example, 125 IU/ml).

This step is further carried out in the presence of IL-7 at a concentration of approximately 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 IL-7. In accordance with a specific embodiment, the concentration of IL-7 is 1-100 IU/ml (30 IU/ml).

It is understood that in the cell culture after culturing with IL-21 (i.e., during the Tcm proliferation step, involving, for example, the addition of IL-21, IL-15 and IL-7), residual antigen or antigens may be present, and thus , antigen-free environment refers to a cell culture without the addition of additional antigen or antigens.

An additional step that can be carried out in accordance with the teachings of the present invention comprises culturing the resulting memory T cells (i.e. after culturing with IL-21) with the antigen or antigens in the presence of IL-21, IL-15 and IL- 7 (i.e., before creating an antigen-free environment). This step is typically 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 carried out at the same doses of IL-21, IL-15 and IL-7 as above. In accordance with a specific embodiment, the cultivation of memory cells with antigen or antigens in the presence of IL-21, IL-15 and IL-7 is carried out for 12 hours to 4 days (for example, 1-2 days). In accordance with one embodiment, the total duration of obtaining Tcm cells is approximately 7, 8, 9, 10, 11, or 12 days (eg, 9 days).

An additional step that can be carried out in accordance with the ideas of the present invention includes the selection and removal of activated cells. This selection step ensures the removal of potentially host-reactive T cells.

Isolation of activated cells can be carried out in a two-step approach. In the first step, activated cells are selected before cell culture in the presence of IL-21, IL-15 and IL-7. The first step is usually performed after first bringing the memory T cells into contact with the antigen or antigens in the presence of IL-21. This selection process selects only those cells that have been activated by the antigen or antigens (e.g., express activation markers as described below) and is typically performed after about 12-24 hours, about 24-36 hours, about 12-36 hours, approximately 36-48 hours approximately 12-48 hours approximately 48-60 hours approximately 12-60 hours approximately 60-72 hours approximately 12-72 hours approximately 72-84 hours approximately 12-84 hours approximately 84 -96 hours, approximately 12-96 hours after initial exposure of the memory T cells to the antigen or antigens. In a specific embodiment, the selection process is carried out approximately 12-24 hours (eg, 14 hours) after the memory T cells are first exposed to the antigen or antigens.

Isolation of activated cells can be performed by affinity purification (e.g., using MACS beads, FACS sorter, and/or labeling using capture ELISA) and can be performed against any activation markers, including cell surface markers such as, but not limited to CD69, CD44, CD25, CFSE, CD137, or markers other than cell surface markers such as, but not limited to, IFN-γ and IL-2. Isolation of activated cells can also be accomplished by morphologically-based purification (eg, large cell isolation) using any method known in the art (eg, FACS). Typically, activated cells are also selected for expression of CD8 ⁺ cells. In addition, any combination of the above methods can be used to efficiently isolate activated cells.

In accordance with one embodiment of the present invention, the selection of activated cells is carried out by selecting CD137+ and/or CD25+ cells.

The second step of isolation of activated cells is usually carried out at the end of the culture (ie after culture in an antigen-free environment with IL-21, IL-15 and IL-7). This step provides for the depletion of alloreactive cells by depleting those cells that were activated after central memory T lymphocytes (Tcm) were brought into contact with irradiated host antigen presenting cells (APCs, eg, dendritic cells). As mentioned above, isolation of activated cells can be done by affinity purification (e.g., using MACS beads, FACS sorter, and/or labeling using capture ELISA) and can be done against any activation markers, including cell surface markers, such as, but not limited to, CD69, CD44, CD25, CFSE, CD137, or markers other than cell surface markers such as, but not limited to, IFN-γ and IL-2.

In accordance with one embodiment of the present invention, the depletion of alloreactive cells is carried out by the depletion of CD137 + and/or CD25 + cells.

According to one embodiment of the present invention, depletion of alloreactive cells is accomplished by culturing Tcm cells with irradiated host antigen presenting cells (APCs, e.g., dendritic cells) for, e.g., 12-24 hours (e.g., 16 hours), approximately 6 -9 days (eg day 8) from the start of culture (ie day 0 is the first day of culture of memory T cells with antigen or antigens).

In accordance with one embodiment, the isolation of activated cells is carried out using only the first stage, as discussed above.

In accordance with another embodiment, the isolation of activated cells is carried out using only the second stage, as discussed above.

In accordance with one embodiment, the non-GvHD-inducing cells with a central memory T-lymphocyte (Tcm) phenotype of the present invention do not occur naturally and are not a natural product. These cells are usually obtained by ex-vivo manipulation (ie exposed to an antigen or antigens in the presence of certain cytokines).

In accordance with one embodiment of the present invention, a method is provided for obtaining an isolated population of non-GvHD-inducing cells having a central memory T-lymphocyte (Tcm) phenotype, wherein the cells are veto-cells and/or endowed with disease activity and are capable of homing to lymph nodes. after transplantation, the method comprising: (a) treating non-adherent peripheral blood mononuclear cells (PBMCs) with an agent capable of depleting CD4 ⁺ , CD56 ⁺ and CD45RA ⁺ cells to generate a population of memory T cells having the CD45RA ^- CD8 ⁺ phenotype; (b) bringing the memory T cell population into contact with the antigen or antigens in the presence of IL-21 in order to enrich it with antigen-reactive cells; and (c) culturing the cells obtained in step (b) in the presence of IL-21, IL-15 and/or IL-7 to allow the proliferation of cells having the Tcm phenotype, thereby obtaining an isolated population of non-GvHD-inducing cells.

In accordance with one embodiment of the present invention, a method is provided for obtaining an isolated population of non-GvHD-inducing cells having a central memory T-lymphocyte (Tcm) phenotype, wherein the cells are veto-cells and/or endowed with disease activity and are capable of homing to lymph nodes. after transplantation, the method comprising: (a) treating non-adherent peripheral blood mononuclear cells (PBMCs) with an agent capable of depleting CD4 ⁺ , CD56 ⁺ and CD45RA ⁺ cells to generate a population of memory T cells having the CD45RA ^- CD8 ⁺ phenotype; (b) bringing the memory T cell population into contact with the viral antigen or antigens in the presence of IL-21 in order to enrich it with antigen-reactive cells; and (c) culturing the cells obtained in step (b) in the presence of IL-21, IL-15 and/or IL-7 to allow the proliferation of cells having the Tcm phenotype, thereby obtaining an isolated population of non-GvHD-inducing cells.

According to one embodiment, in order to obtain memory T cells specific for an antigen or antigens, the antigen/antigens (e.g., tumor antigen, viral antigen) are administered to a memory T cell donor prior to obtaining memory T cells (e.g., to provide a population of at least 70% memory T cells). Any method of immunizing a cell donor against an antigen can be used to stimulate an immune response (eg, production of memory T cells).

The antigen can be administered as a composition or as part of a composition containing an adjuvant (eg complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA)). In one embodiment, the antigen is administered to the memory T cell donor once. In one embodiment, the memory T cell donor receives at least one additional (eg, as part of a booster) antigen administration (eg, 2, 3, 4 or more administrations). Such additional introduction can be carried out after 1, 3, 5, 7, 10, 12, 14, 21, 30 days or more after the first introduction of the antigen.

Additional methods for immunizing a subject with a tumor antigen that can be used in some embodiments of the present invention (e.g., cell-based vaccines such as peptide-specific DC-based vaccines, DC-based vaccines against indeterminate epitopes, use of leukemia-derived DCs for vaccination, ^GVAX® platform) are described in Alatrash G. and Molldrem J., Expert Rev Hematol. (2011) 4(1): 37-50, incorporated herein by reference.

In order to further enrich memory T cells for a particular antigen/antigens and to deplete alloreactive clones from the memory T cell pool, memory T cells can be additionally contacted with the same antigen or antigens (e.g., the same antigen that was administered to a cell donor) as described herein above.

The protocols described above are typically used for applications involving non-isogenic systems, and hence the memory T cells or PBMCs used are typically allogeneic to the subject (eg, from an allogeneic donor). Similarly, in cases where applications involving xenogenic systems may be beneficial, the memory T cells or PBMCs used may be of xenogeneic origin, as discussed below.

However, in cases where applications involving isogenic systems may be beneficial, the memory T cells or PBMCs used may be autologous to the subject (eg, from the subject). Such definitions are within the skill of the art, especially in view of the disclosure provided.

Thus, as mentioned, memory T cells or PBMCs may or may not be isogenic with respect to the subject.

As used herein, the term "isogenic" cells refers to cells that are substantially genetically identical with respect to a subject or substantially all of the subject's lymphocytes. Examples of isogenic cells include cells derived from a subject (also referred to in the art as "autologous"), a clone of the subject, or an identical twin of the subject.

As used herein, the term "non-isogenic" cells refers to cells that are not substantially genetically identical to the subject or substantially all of the subject's lymphocytes, such as allogeneic cells or xenogeneic cells.

As used herein, the term "allogeneic" refers to cells that are derived from a donor that is of the same species as the subject, but that are not substantially clones of the subject's cells. Typically, outbred mammals that are fraternal twins of the same species are allogeneic to each other. It is understood that an allogeneic cell may be HLA-identical, partially HLA-identical, or may not be HLA-identical (ie, characterized by the presence of one or more different HLA determinants) with respect to the subject.

In accordance with one embodiment, the cell donor is a human.

In the context of this document, the term "xenogenic" refers to a cell that, in fact, expresses antigens of a species different from the species to which a significant proportion of the subject's lymphocytes belong. Usually outbred mammals of different species are xenogenic with respect to each other.

The present invention provides that xenogeneic cells are derived from different species. Thus, in accordance with one embodiment, the cells may be from any mammal. Suitable types of cell origin include basic domesticated animals or livestock and primates. Such animals include, without limitation, members of the porcine family (e.g., pig), members of the bovine family (e.g., cow), members of the equine family (e.g., horse), members of the goat family (e.g., goat, sheep), felines (e.g., Felis domestica), canines (eg Canis domestica), rodents (eg mouse, rat, rabbit, guinea pig, gerbil, hamster) and primates (eg chimpanzee, rhesus monkey, macaque, marmoset).

Cells of xenogeneic origin (eg porcine origin) are preferably obtained from a source known to be free of zoonoses, eg caused by porcine endogenous retroviruses. Similarly, cells or tissues of human origin are preferably obtained from sources that are substantially free of pathogenic organisms.

Thus, the source of memory T cells or PBMCs will be determined according to the intended use of the cells (see more details later in this document) and is within the skill of the art, especially in light of the detailed disclosure provided herein.

The use of veto cells is particularly advantageous in situations where there is a need to eliminate transplant rejection and overcome graft-versus-host disease (GvHD), such as in transplantation of allogeneic or xenogeneic cells or tissues.

As mentioned above, the veto cells of the present invention are further endowed with activity against a disease and, therefore, are beneficial in situations in which a subject, for example, a transplanted subject, has a disease or condition (for example, malignant, viral, bacterial, fungal , an autoimmune or allergic disease or condition), before or after transplantation (for example, before immunorehabilitation therapy).

Thus, in accordance with another aspect of the present invention, there is provided a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated population of non-GvHD-inducing cells according to some embodiments of the present invention (i.e., Tcm cells), thereby providing treatment of the disease in the subject.

In accordance with another aspect of the present invention, there is provided a method for treating a subject in need of cell or tissue transplantation, the method comprising: (a) transplanting a cell or tissue graft into the subject; and (b) administering to the subject a therapeutically effective amount of an isolated population of non-GvHD-inducing cells according to some embodiments of the present invention (i.e., Tcm cells), thereby providing treatment to the subject in need of cell or tissue transplantation.

In the context of this document, the term "treatment" includes the elimination, significant inhibition, slowing or reversal of the progression of a condition, a significant reduction in the clinical or visual symptoms of a condition, or a significant prevention of the onset of clinical or visual symptoms of a condition.

As used herein, the term “subject” or “subject in need” refers to a mammal, preferably a human, male or female of any age, in need of cell or tissue transplantation or suffering from a disease that can be treated with Tcm cells. . Typically, the subject is in need of a cell or tissue transplant (also referred to herein as a recipient) due to a disorder or pathological or undesirable condition, status or syndrome, or physical, morphological or physiological abnormality that is treatable by cell or tissue transplantation. Examples of such violations are further provided below.

In the context of this document, the term "therapeutically effective amount" is the amount of Tcm cells effective for tolerization (ie, veto effect), disease effect, antitumor effect, and/or immune reconstitution without inducing GvHD. Because the Tcm cells of the present invention are recycled back to the lymph nodes after transplantation, lower cell numbers (compared to previously used dose of cells, see, for example, WO 2001/049243). It is understood that lower levels of immunosuppressive drugs (discussed below) may be needed in combination with the Tcm cells of the present invention (such as excluding rapamycin from the therapy protocol).

Determination of a therapeutically effective amount is within the skill of the art, especially in light of the detailed disclosure provided herein.

For any drug used in the methods of the present invention, a therapeutically effective amount or dose can be initially established using in vitro assays and cell culture assays. For example, the dose required to achieve a desired concentration or titer can be determined in animal models. Such information can be used to more accurately determine useful doses in humans.

For example, in the case of cell transplantation, the number of Tcm cells administered to the recipient by infusion should exceed 1×10 ⁴ /kg of body weight. The number of Tcm cells administered to the recipient by infusion should generally be in the range from 1×10 ³ /kg body weight to 1×10 ⁴ /kg body weight, in the range from 1×10 ⁴ /kg body weight to 1×10 ⁵ /kg body weight, in the range from 1×10 ⁴ /kg body weight to 1×10 ⁶ /kg body weight, in the range from 1×10 ⁴ /kg body weight to 10×10 ⁷ /kg body weight, in the range from 1×10 ⁴ /kg body weight to 1×10 ⁸ /kg body weight, ranging from 1×10 ³ /kg body weight to 1×10 ⁵ /kg body weight, ranging from 1×10 ⁴ /kg body weight up to 1×10 ⁶ /kg of body weight, in the range from 1×10 ⁶ /kg of body weight to 10×10 ⁷ /kg of body weight, in the range from 1×10 ⁵ /kg of body weight to 10×10 ⁷ /kg of body weight body, in the range from 1×10 ⁶ /kg of body weight to 1×10 ⁸ /kg of body weight. In accordance with a specific embodiment, the number of Tcm cells administered to the recipient by infusion should be in the range from 1×10 ⁵ /kg of body weight to 10×10 ⁷ /kg of body weight.

Thus, the method of the present invention can be used to treat any disease, such as, but not limited to, malignant disease, transplant-associated disease (e.g., transplant rejection, graft-versus-host disease), infectious disease (e.g., viral disease, fungal disease or bacterial disease), an inflammatory disease, an autoimmune disease, and/or an allergic disease or condition.

In accordance with one embodiment, the subject has a malignant disease.

Malignancies (also referred to as cancers) that can be treated using the method according to some embodiments of the present invention can be any solid or non-solid tumor and/or tumor metastasis.

Examples of cancer include, without limitation, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific 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, adenocarcinoma of the lung and squamous cell lung carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer. or malignant tumor of the stomach (including cancer of the gastrointestinal tract), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colon and rectal cancer, cancer rectal, endometrial or uterine carcinoma, carcinoid tumor, salivary gland carcinoma, kidney malignancy or cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, mesothelioma, multiple myeloma, post-transplant lymphoproliferative disease (PTLD), and various types of head and neck cancer (for example, a brain tumor). Cancer conditions treatable according to the present invention include metastatic cancers.

According to one embodiment, the cancer is a malignant neoplasm of hematopoietic tissue. Exemplary hematopoietic malignancies include, without limitation, leukemia [e.g., acute lymphocytic, acute lymphoblastic, acute lymphocytic progenitor B-cell leukemia, acute lymphoblastic T-cell leukemia, acute megakaryoblastic, monocytic, acute myelogenous, acute myeloid, acute myeloid with eosinophilia, B-cell, basophilic, chronic myeloid, chronic B-cell, eosinophilic leukemia, Freudian leukemia, granulocytic or myelocytic, hairy cell, lymphocytic, megakaryoblastic, monocytic, monocytic-macrophage, myeloblastic, myeloid, myelomonocytic, plasma cell leukemia, progenitor leukemia B -cell, promyelocytic, subacute, T-cell leukemia, lymphoid tissue tumor, predisposition to myeloid tissue malignancy, acute non-lymphocytic leukemia, T-cell acute lymphocytic leukemia (T-ALL), and B-cell chronic lymphocytic leukemia (B-CLL) ] and lymphoma [eg, Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt's lymphoma, cutaneous T-cell, histiocytic, lymphoblast, T-cell, thymic, B-cell lymphoma, including low-grade NHL/follicular NHL; small cell lymphocytic (SL) NHL; NHL intermediate grade/follicular NHL; diffuse NHL of intermediate grade; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; NHL with high grade uncleaved nucleus cells; NHL with massive lymph node involvement; marginal zone cell lymphoma; AIDS-associated lymphoma; and Waldenström's macroglobulinemia].

In a specific embodiment, the cancer is leukemia, lymphoma, myeloma, melanoma, sarcoma, neuroblastoma, colon cancer, colon and rectal cancer, breast cancer, ovarian cancer, esophageal cancer, synovial cell cancer, liver cancer, and pancreas cancer.

In accordance with one embodiment, the subject has a disease that is not malignant.

In one embodiment, the non-malignant disease is an organ dysfunction or failure, a blood disease, a transplant-related disease, an infectious disease, an inflammatory disease, an autoimmune disease, an allergic disease, trauma, and injury.

Inflammatory diseases - includes, without limitation, chronic inflammatory diseases and acute inflammatory diseases.

Inflammatory diseases associated with hypersensitivity

Examples of hypersensitivity include, without limitation, type I hypersensitivity, type II hypersensitivity, type III hypersensitivity, type IV hypersensitivity, immediate type hypersensitivity, antibody-mediated hypersensitivity, immune complex-mediated hypersensitivity, T-lymphocyte-mediated hypersensitivity, and DTH.

Type I hypersensitivity or immediate type hypersensitivity such as asthma.

Type II hypersensitivity includes, without limitation, rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 Jul;15(3):791), spondylitis, ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001 ; 3 (3): 189), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998;17 (1-2):49), sclerosis, systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol 1999 Mar 6(2):156); Chan FROM. et al., Immunol Rev 1999 Jun; 169:107), glandular disease, autoimmune glandular disease, autoimmune pancreatic disease, diabetes mellitus, type I diabetes mellitus (Zimmet P. Diabetes Res Clin Pract 1996 Oct; 34 Suppl:S125), thyroid disease, autoimmune thyroid disease, diffuse toxic goiter (Orgiazzi J. Endocrinol Metab Clin North Am 2000 Jun; 29(2):339), thyroiditis, 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), myxedema, idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug; 57 (8): 1759); autoimmune diseases of the reproductive system, diseases of the ovary, autoimmune disorders of the ovaries (Garza KM. et al., J Reprod Immunol 1998 Feb; 37(2):87), autoimmune infertility associated with antisperm antibodies (Diekman AB. et al., Am J Reprod Immunol. 2000 Mar;43(3):134), recurrent fetal loss (Tincani A. et al., Lupus 1998;7 Suppl 2:S107-9), neurodegenerative diseases, neurological diseases, neurological autoimmune diseases, multiple sclerosis ( Cross AH et al., J Neuroimmunol 2001 Jan 1;112 (1-2): 1), Alzheimer's disease (Oron L. et al., J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ . And Kraig E, Int Rev Immunol 1999;18(1-2):83), motor neuropathies (Kornberg AJ. J Clin Neurosci. 2000 May; 7(3):191), Guillain-Barré syndrome, neuropathies and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 Apr; 319 (4): 234), myasthenia gravis, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr; 319 (4): 204), paraneoplasty neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy, non-paraneoplastic muscle stiffness syndrome, cerebellar atrophy, progressive cerebellar atrophy, encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydenham's chorea, Tourette's disease, polyendocrinopathy, autoimmune polyendocrinopathy (JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan; 156(1):23); neuropathies, disimmune neuropathies (Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl 1999;50:419); neuromyotonia, acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13;841:482), cardiovascular disease, autoimmune cardiovascular disease, 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), granulomatosis , Wegener's granulomatosis, arterial inflammation, Takayasu syndrome and Kawasaki syndrome (Praprotnik S. et al., Wien Klin Wochenschr 2000 Aug 25; 112(15-16):660); autoimmune disease associated with antibodies to factor VIII (Lacroix-Desmazes S. et al., Semin Thromb Hemost. 2000; 26 (2): 157); vasculitis, necrotizing vasculitis of small vessels, microscopic polyangiitis, Churg-Strauss syndrome, glomerulonephritis, malimmune focal necrotizing glomerulonephritis, 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); heart failure, β-adrenergic agonist-like antibodies in 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); hemolytic anemia, autoimmune hemolytic anemia (Efremov DG. et al., Leuk Lymphoma 1998 Jan; 28(3-4):285), gastrointestinal disease, autoimmune gastrointestinal disease, intestinal disease, 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), autoimmune muscle diseases, myositis, autoimmune myositis, Sjögren's syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 Sep; 123(1):92); smooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother 1999 Jun; 53(5-6):234), liver disease, autoimmune liver disease, autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33(2): 326) and primary biliary cirrhosis (Strassburg CP. et al., Eur J Gastroenterol Hepatol. 1999 Jun; 11(6):595).

Type IV hypersensitivity or T-cell mediated hypersensitivity includes, but is not limited to, rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevitt HO. Proc Natl Acad Sci U S A 1994 Jan 18;91(2):437), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Datta SK., Lupus 1998;7 (9): 591), glandular disease, autoimmune glandular disease, pancreatic disease, autoimmune pancreatic disease, type 1 diabetes mellitus (Castano L. and Eisenbarth GS. Ann. Rev. Immunol 8:647); thyroid diseases, autoimmune thyroid diseases, diffuse toxic goiter (Sakata S. et al., Mol Cell Endocrinol 1993 Mar; 92(1):77); ovarian disease (Garza KM. et al., J Reprod Immunol 1998 Feb;37(2):87), prostatitis, autoimmune prostatitis (Alexander RB. et al., Urology 1997 Dec;50(6):893), polyglandular syndrome , autoimmune polyglandular syndrome, autoimmune polyglandular syndrome type I (Naga T. et al., Blood. 1991 Mar 1; 77 (5): 1127), neurological diseases, autoimmune neurological diseases, multiple sclerosis, neuritis, optic neuritis (Soderstrom M et al., J Neurol Neurosurg Psychiatry 1994 May;57(5):544), myasthenia gravis (Oshima M. et al., Eur J Immunol 1990 Dec; 20(12):2563), muscle stiffness syndrome (Hiemstra HS et al., Proc Natl Acad Sci USA 2001 Mar 27;98(7):3988), cardiovascular disease, cardiac autoimmune response in Chagas disease (Cunha-Neto E. et al., J Clin Invest 1996 Oct 15 ;98 (8): 1709), autoimmune thrombocytopenic purpura (Semple JW. et al., Blood 1996 May 15;87 (10):4245), caused by antibodies to helper T-lymphocytes autoimmune reaction (Caporossi AR. et al., Viral Immunol 1998;11(1):9), hemolytic anemia (Sallah S. et al., Ann Hematol 1997 Mar; 74(3):139), liver disease, autoimmune liver disease, hepatitis, chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol 1990 Mar;54(3):382), biliary cirrhosis, primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov;91(5):551), disease kidney disease, autoimmune kidney disease, nephritis, interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;1(2):140), rheumatic diseases, auditory system diseases, autoimmune rheumatic diseases, autoimmune auditory system disease (Yoo TJ. et al ., Cell Immunol 1994 Aug;157(1):249), inner ear disease (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec 29;830:266), skin diseases, skin diseases, skin diseases, bullous diseases skin, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.

Examples of delayed hypersensitivity include, without limitation, contact dermatitis and drug rash.

Examples of types of hypersensitivity mediated by T cells include, without limitation, hypersensitivity mediated by helper T lymphocytes and cytotoxic T lymphocytes.

Examples of hypersensitivity mediated by helper T lymphocytes include, without limitation, hypersensitivity mediated by T _h 1 lymphocytes and hypersensitivity mediated by T _h 2 lymphocytes.

Autoimmune diseases

Autoimmune diseases include, without limitation, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, skin diseases, liver diseases, neurological diseases, muscle diseases, kidney diseases, reproductive diseases, rheumatic diseases, and systemic diseases.

Examples of autoimmune diseases of the cardiovascular system include, without limitation, 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 syndrome, Kawasaki syndrome (Praprotnik S. et al., Wien Klin Wochenschr 2000 Aug 25; 112 (15-16):660) , an autoimmune disease associated with antibodies to factor VIII (Lacroix-Desmazes S. et al., Semin Thromb Hemost. 2000;26 (2): 157); necrotizing small vessel vasculitis, microscopic polyangiitis, Churg-Strauss syndrome, malimmune 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 autoimmune reaction in Chagas disease (Cunha-Neto E. et al., J Clin Invest 1996 Oct 15;98(8):1709) and anti-helper T-lymphocyte antibody-mediated autoimmune reaction (Caporossi AP. et al., Viral Immunol 1998;11(1):9).

Examples of autoimmune rheumatoid diseases include, without limitation, rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 Jul; 15 (3): 791; Tisch R, McDevitt HO. Proc Natl Acad Sci units S A 1994 Jan 18; 91 (2): 437) and ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3(3):189).

Examples of autoimmune glandular diseases include, without limitation, pancreatic disease, type I diabetes mellitus, thyroid disease, diffuse toxic goiter, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, autoimmune ovarian disorders, autoimmune infertility associated with antisperm antibodies, autoimmune prostatitis and autoimmune polyglandular syndrome type I. Diseases include, without limitation, autoimmune diseases of the pancreas, type 1 diabetes mellitus (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 Oct; 34 Suppl:S125), autoimmune thyroid diseases glands, diffuse toxic goiter (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), autoimmune ovarian disorders (Garza KM. et al., J Reprod Immunol 1998 Feb;37(2):87), autoimmune infertility associated with antisperm antibodies (Diekman AB et al., Am J Reprod Immunol.

2000 Mar; 43(3):134), autoimmune prostatitis (Alexander R.B. et al., Urology 1997 Dec; 50(6):893), and type I autoimmune polyglandular syndrome (Naga T. et al., Blood. 1991 Mar 1;77 (5):1127).

Examples of autoimmune diseases of the gastrointestinal tract include, without limitation, 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.

Examples of autoimmune skin diseases include, without limitation, autoimmune bullous skin diseases such as, but not limited to, pemphigus vulgaris, bullous pemphigoid, and pemphigus foliaceus.

Examples of autoimmune liver diseases include, without limitation, 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).

Examples of autoimmune neurological diseases include, but are not limited to, multiple sclerosis (Cross AH et al., J Neuroimmunol 2001 Jan 1;112(1-2):1), Alzheimer's disease (Oron L. et al., J Neural Transm Suppl. 1997; 49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999;18(l-2):83; Oshima M. et al., Eur J Immunol 1990 Dec;20(12):2563), neuropathies, motor neuropathies (Kornberg AJ. J Clin Neurosci. 2000 May;7(3):191); Guillain-Barré syndrome and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 Apr;319 (4):234), myasthenia gravis, myasthenic Lambert-Eaton syndrome (Takamori M. Am J Med Sci. 2000 Apr;319 (4) :204); a couple of plastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy and stiffness syndrome (Hiemstra HS. et al., Proc Natl Acad Sci units S A 2001 Mar 27;98(7):3988); non-paraneoplastic stiffness syndrome, progressive cerebellar atrophy, encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydenham's chorea, Tourette's disease and autoimmune polyendocrinopathy (Antoine JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan;156(1):23); disimmune neuropathies (Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl 1999;50:419); acquired neuromyotonia, arthrogryposis multiplex congenita (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.

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

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).

Examples of reproductive-related autoimmune diseases include, without limitation, recurrent fetal loss (Tincani A. et al., Lupus 1998;7 Suppl 2:S107-9).

Examples of autoimmune rheumatic diseases include, without limitation, diseases of the auditory system, autoimmune diseases of the auditory system (Yoo TJ. et al., Cell Immunol 1994 Aug;157 (1):249) and autoimmune diseases of the inner ear (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec 29;830:266).

Examples of autoimmune systemic diseases include, without limitation, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998;17(1-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol. 1999 Mar; 6(2):156); Chan FROM. et al., Immunol Rev 1999 Jun; 169:107).

Infectious diseases

Examples of infectious diseases include, without limitation, chronic infectious diseases, subacute infectious diseases, acute infectious diseases, viral diseases, bacterial diseases, protozoal diseases, parasitic diseases, fungal diseases, mycoplasma diseases, and prion diseases.

Specific types of viral pathogens that cause infectious diseases treatable in accordance with the ideas of the present invention include, without limitation, retroviruses, circoviruses, parvoviruses, papovaviruses, adenoviruses, herpesviruses, iridoviruses, poxviruses, hepadnaviruses, picornaviruses, caliciviruses, togaviruses, flaviviruses, reoviruses, orthomyxoviruses , paramyxoviruses, rhabdoviruses, buniaviruses, coronaviruses, arenaviruses and filoviruses.

Specific examples of viral infections that can be treated in accordance with the ideas of the present invention include, without limitation, those caused by the human immunodeficiency virus (HIV) that induces acquired immunodeficiency syndrome (AIDS), influenza, rhinovirus infection, Epstein-Barr virus meningitis ( EBV) hepatitis A, B, or C infection, measles, papillomavirus infection/warts, cytomegalovirus (CMV) infection, herpes simplex virus infection, yellow fever, Ebola virus infection, adenovirus rabies (Adv ) infection, rhinovirus infection, acute respiratory viral infection, Japanese encephalitis, poliomyelitis, respiratory syncytial virus infection, rubella, smallpox, chickenpox, rotavirus infection, West Nile virus infection and Zika virus infection.

Specific examples of bacterial infections that can be treated in accordance with the ideas of the present invention include, without limitation, those caused by the bacterium that causes anthrax; gram-negative bacilli, chlamydia, diphtheria-causing bacteria, Haemophilus influenzae, Helicobacter pylori, malaria-causing plasmodia, Mycobacterium tuberculosis, pertussis toxin, pneumococcus, rickettsiae, staphylococcus, streptococcus, and tetanus-causing bacteria.

Specific examples of infections caused by superbugs (for example, multidrug-resistant bacteria) that can be treated in accordance with the ideas of the present invention include, without limitation, those caused by Enterococcus faecium, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae (including Escherichia coli, Klebsiella pneumoniae, Enterobacter spp.).

Specific examples of fungal infections that can be treated in accordance with the teachings of the present invention include, without limitation, those caused by Candida, coccidiodes, cryptococcus, histoplasma, leishmania, plasmodium, protozoa, parasitic organisms, schistosomes, lichen-causing organisms, toxoplasma, and trypanosoma cruzi.

Diseases associated with transplant rejection

According to another embodiment, the disease is associated with transplantation. Examples of diseases associated with transplantation include, without limitation, transplant rejection, chronic transplant rejection, subacute transplant rejection, hyperacute transplant rejection, acute transplant rejection, allograft rejection, xenograft rejection, and graft versus host disease (GVHD).

Allergic diseases

Examples of allergic diseases include, without limitation, asthma, hives, hives, hay fever, dust mite allergy, insect venom allergy, cosmetics allergy, latex allergy, chemical allergy, drug allergy, insect sting allergy, animal dander, stinging plant allergy, toxicodendron rooting allergy, and food allergy.

Blood disease that is not cancerous

Examples of blood disorders that are not malignant include, but are not limited to, anemia, bone marrow disorders, deep vein thrombosis/pulmonary embolism, congenital hypoplastic anemia, hemochromatosis, hemophilia, immune-mediated hematopoietic disorders, iron metabolism disorders, sickle cell anemia , thalassemia, thrombocytopenia and von Willebrand disease.

In order to enhance the activity of Tcm cells against a disease, it is advantageous to select an antigen or antigens associated with the disease to be treated and obtain antigen-specific Tcm cells for treatment.

Thus, in accordance with one embodiment, the method comprises: (a) analyzing a biological sample from a subject for the presence of a disease-associated antigen or antigens; (b) obtaining an isolated population of non-GvHD-inducing cells in accordance with the method of some embodiments of the present invention, directed to the disease-associated antigen or antigens, in order to ensure its enrichment with antigen-reactive cells; and (c) administering to the subject a therapeutically effective amount of the isolated population of non-GvHD-inducing cells of (b), thereby providing treatment for the disease in the subject.

As used herein, a "biological sample" refers to a fluid sample or tissue sample derived from a subject. Examples of "biological specimens" include, without limitation, whole blood, blood serum, blood plasma, cerebrospinal fluid, urine, lymph fluid, tissue biopsy and various respiratory, gastrointestinal, and genitourinary extrinsic products, tears, saliva, milk, and leukocytes. , tissue, cell culture, for example, primary culture.

Methods for obtaining such biological samples are known in the art, including, without limitation, standard blood sampling procedures, urine collection, and lumbar puncture.

Determination of the presence of an antigen or antigens in a biological sample can be carried out using any method known in the art, for example, using serological testing (testing for the presence of a pathogen), bacterial testing, determining the sensitivity of bacteria to antimicrobials, tests for the detection of fungi, viruses, mycobacteria (AFB testing) and/or parasitic organisms, electrophoresis, enzyme immunosorbent assay (ELISA), Western blot analysis and fluorescence-activated cell sorting (FACS).

Once the assay is performed, an antigen or antigens are selected and Tcm cells are generated from the memory T cell population as discussed above using the disease specific antigen or antigens (e.g., tumor antigens, viral antigens, bacterial antigens, etc.) and administered to a subject for treatment.

As discussed above, the Tcm cells of the present invention are endowed with veto activity. Accordingly, the Tcm cells of the present invention can be used as adjuvant therapy for cell or tissue transplantation. Since the Tcm cells of the present invention are also endowed with activity against a disease, the method of the present invention can additionally be successfully used to treat a disease in a subject while promoting engraftment of a cell or tissue graft.

As used herein, the phrase "transplantation of cells or tissues" refers to a whole cell (eg, a single cell or group of cells) or tissue (eg, solid tissues/parenchymal organs or soft tissues, which can be transplanted in whole or in part). Exemplary tissues or organs that may be transplanted in accordance with the teachings of the present invention include, but are not limited to, the liver, pancreas, spleen, kidney, heart, lung, skin, intestine, and lymphoid/hematopoietic tissues (e.g., lymph node, Peyer's patches, thymus or bone marrow). Illustrative cells that can be transplanted in accordance with the ideas of the present invention include, without limitation, immature hematopoietic cells, including stem cells, cardiomyocytes, liver cells, pancreatic cells, spleen cells, lung cells, brain cells, kidney cells, intestinal cells / intestines, ovarian cells, skin cells (for example, a selected population of any of these cells). In addition, the present invention also provides for the transplantation of whole organs, such as, for example, kidney, heart, liver or skin.

Depending on the application, the method can be carried out using a cell or tissue that is isogenic or non-isogenic with respect to the subject.

In accordance with one embodiment of the present invention, both the subject and the donor are human.

Depending on the application and available sources, the cells or tissues of the present invention may be obtained from a prenatal organism, a postnatal organism, an adult or a cadaveric donor. Moreover, depending on the application, the desired cells or tissues may be intact or genetically modified. Such definitions are not beyond the competence of a person skilled in the art.

Any method known in the art can be used to obtain a cell or tissue (eg, for transplantation).

Transplantation of a cell or tissue to a subject can be done in a variety of ways, depending on various parameters such as, for example, the type of cells or tissues; the type, stage, or severity of the recipient's disease (eg, functional organ failure); physical or physiological parameters specific to the subject; and/or the desired therapeutic outcome.

The transplantation of a cell or tissue graft of the present invention can be accomplished by transplanting a cell or tissue graft to any of a variety of anatomical locations, depending on the application. The graft of cells or tissues can be transplanted into a homotopic anatomical location (normal anatomical location for a graft) or an ectopic anatomical location (atypical anatomical location for a graft). Depending on the application, a cell or tissue graft can be successfully implanted under the renal capsule or in the kidney, testicular fat, subcutaneous layer, omentum, portal vein, liver, spleen, heart cavity, heart, chest cavity, lung, skin, pancreas, and / or intraperitoneal space.

For example, liver tissue in accordance with the ideas of the present invention can be transplanted into the liver, portal vein, renal capsule, subcutaneous layer, omentum, spleen and intraperitoneal space. Liver transplantation at various anatomical locations, such as those indicated, is widely practiced in the art for the treatment of diseases treatable by liver transplantation (eg, liver failure). Similarly, pancreatic tissue grafting in accordance with the present invention can be successfully performed by grafting tissue into the portal vein, liver, pancreas, testicular fat, subcutaneous layer, omentum, intestinal loop (subserous layer of the U-loop of the small intestine) and/or inside the peritoneal space. Pancreatic tissue transplantation can be used to treat diseases treatable by pancreatic transplantation (eg, diabetes mellitus). Similarly, tissue transplantation, such as kidney, heart, lung, or skin tissue, can be performed at any of the anatomical sites described above for the treatment of recipients suffering from, for example, kidney failure, heart failure, pulmonary failure, or skin injury (e.g., burns). ). In cases where isolated cells are transplanted, such cells can be administered, for example, by the intravenous route, the intratracheal route, the intraperitoneal route, or the intranasal route.

The method of the present invention can also be used, for example, to treat a recipient suffering from a disease that requires transplantation of immature hematopoietic cells.

In the latter case, immature autologous, allogeneic, or xenogeneic hematopoietic cells (including stem cells), which may be derived, for example, from bone marrow, mobilized peripheral blood (eg, by leukapheresis), fetal liver, yolk sac, and/or umbilical cord blood of a donor, may be transplanted into a recipient suffering from a disease. In one embodiment, the immature hematopoietic cells are CD34+ T cell depleted immature hematopoietic cells. Such disease includes, without limitation, leukemia [e.g., acute lymphocytic, acute lymphoblastic leukemia (ALL), progenitor B-cell acute lymphoblastic leukemia, acute lymphoblastic T-cell leukemia, acute megakaryoblastic, monocytic, acute myelogenous, acute myeloid, acute myeloid with eosinophilia , B-cell, basophilic, chronic myeloid, chronic, B-cell, eosinophilic leukemia, Freud's leukemia, granulocytic or myelocytic, acute myelocytic leukemia (AML) or chronic myelocytic leukemia (CML), hairy cell, lymphocytic, megakaryoblastic, monocytic, monocytic- macrophage, myeloid, myeloid, myelomonocytic, plasma cell leukemia, progenitor B-cell leukemia, promyelocytic, subacute, T-cell, lymphoid tissue tumor, predisposition to myeloid tissue malignancy, acute non-lymphocytic leukemia, acute non-lymphoblastic leukemia (ANLL), T-cell acute lymphocytic leukemia (T-ALL) and B-cell chronic lymphocytic leukemia (B-CLL)], lymphoma (eg, Hodgkin's disease, non-Hodgkin's lymphoma, B-cell lymphoma, Burkitt's lymphoma, cutaneous T-cell, histiocytic, lymphoregional, T -cellular, thymic lymphoma), forms of severe combined immunodeficiency syndrome (SCID), including adenosine deaminase (ADA) deficiency, marbled disease, aplastic anemia, Gaucher's disease, thalassemia and other hereditary or genetically determined disorders of the hematopoietic system.

It is understood that the immature autologous, allogeneic, or xenogeneic hematopoietic cells of the present invention may be transplanted into a recipient using any cell transplantation method known in the art, such as, without limitation, cell infusion (e.g., I.V.) or intraperitoneal route.

Optionally, when transplanting a cell or tissue graft of the present invention into a subject with a damaged organ, it may be advantageous to first at least partially remove the affected organ from the subject to ensure optimal development of the graft and its structural/functional integration with the anatomy/physiology of the subject.

In accordance with one embodiment, the transplant of cells or tissues comes from an allogeneic donor. According to one embodiment, the cell or tissue graft is from an HLA-identical allogeneic donor or from a non-HLA-identical allogeneic donor. In accordance with one embodiment, the transplant of cells or tissues comes from a xenogeneic donor.

In one embodiment, the cell or tissue transplant and the isolated Tcm cell population are from the same (eg, non-isogenic) donor.

In accordance with one embodiment, the cell or tissue transplant and the isolated population of Tcm cells are from different (eg, non-isogenic) donors. Accordingly, a transplant of cells or tissues may not be isogenic with respect to Tcm cells.

In accordance with one embodiment, the immature hematopoietic cells and the isolated population of Tcm cells are from the same (eg, non-isogenic) donor.

In accordance with one embodiment, the immature hematopoietic cells and the isolated population of Tcm cells are from different (eg, non-isogenic) donors. Accordingly, immature hematopoietic cells may not be isogenic to Tcm cells.

The method of the present invention also contemplates co-transplantation of multiple organs (eg, heart and lung tissues) in case such a procedure may have a beneficial effect on the subject.

According to one embodiment, co-transplantation involves the transplantation of immature hematopoietic cells and solid tissue/parenchymal organ or a number of solid tissues/parenchymal organs.

According to one embodiment, the immature hematopoietic cells and the parenchymal organ are from the same donor.

According to another embodiment, the immature hematopoietic cells and the parenchymal organ/solid tissue or organs/tissue are from different (eg, non-isogenic) donors.

According to one embodiment, immature hematopoietic cells are transplanted prior to, simultaneously with, or after solid organ transplantation.

In one embodiment, hematopoietic chimerism is first induced in a subject by transplantation of immature hematopoietic cells in combination with the Tcm cells of the present invention, resulting in tolerance of other tissues/organs transplanted from the same donor.

In accordance with one embodiment, the Tcm cells of the present invention are used per se to reduce the rejection of transplanted tissues/organs transplanted from the same donor.

Following transplantation of a cell or tissue graft to a subject in accordance with the teachings of the present invention, it is recommended, in accordance with standard medical practice, to monitor the growth of organ performance and immunocompatibility in accordance with any of a number of standard techniques in the art. For example, the performance of a pancreatic tissue graft can be monitored post-transplant using standard pancreatic function tests (eg, assays of serum insulin levels). Similarly, characteristics of a liver tissue graft can be monitored post-transplant using standard liver function tests (eg, serum albumin, total protein, ALT, AST, and bilirubin assays and clotting time assays). Structural development of cells or tissues can be monitored by computed tomography or ultrasound imaging.

Depending on the context of transplantation, in order to promote engraftment of a graft of cells or tissues, the method may further advantageously include conditioning the subject under sub-lethal, lethal, or super-lethal conditions prior to transplantation.

As used herein, the terms "sub-lethal", "lethal" and "super-lethal" in relation to the conditioning of subjects of the present invention refer to myelotoxic and/or lymphocytotoxic types of treatment which, when applied to a representative population of subjects, respectively, are usually: for all members of the population; lethal to some but not all members of the population; or lethal to basically all members of the population under normal conditions of sterility.

In accordance with some embodiments of the present invention, conditioning under sublethal, lethal, or superlethal conditions includes total body irradiation (TBI), total lymphoid tissue irradiation (TLI, i.e. exposure to all lymph nodes, thymus and spleen), partial body irradiation ( for example, specific effects on the lungs, kidney, brain, etc.), myeloablative conditioning, and/or non-myeloablative conditioning, for example, using various combinations, including, without limitation, costimulation blockade, a chemotherapeutic agent, and/or antibody immunotherapy. In accordance with some embodiments of the present invention, conditioning involves a combination of any of the above-described conditioning protocols (eg, chemotherapeutic agent and TBI, costimulation blockade and chemotherapeutic agent, antibody immunotherapy and chemotherapeutic agent, etc.).

In accordance with one embodiment, TBI provides for a single or divided dose of radiation within 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, 1-1.5 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.

In accordance with a specific embodiment, TBI provides for a single or divided dose of radiation within the range of 1-7.5 Gy.

According to one embodiment, conditioning is accomplished by conditioning the subject under superlethal conditions, such as myeloablative conditions.

Alternatively, conditioning can be accomplished by conditioning the subject under lethal or sublethal conditions, such as by conditioning the subject under myeloreductive conditions or non-myeloablative conditions.

In one embodiment, conditioning is accomplished by conditioning the subject with a myeloablative drug (eg, busulfan or melphalan) or a non-myeloablative drug (eg, cyclophosphamide and/or fludarabine).

Examples of conditioning agents that can be used to condition a subject include, without limitation, radiation, drugs, and tolerance-inducing cells (as described herein).

Examples of drugs include myelotoxic drugs, lymphocytotoxic drugs and immunosuppressants (discussed in detail below).

Examples of myelotoxic drugs include, but are not limited to, busulfan, dimethylmyleran, melphalan, and thiotepa.

In addition, or alternatively, the method may further comprise conditioning the subject using an immunosuppression regimen prior to, concurrently with or after transplantation of cells or tissues.

Examples of suitable types of immunosuppression regimens include the administration of immunosuppressive weed drugs, populations of tolerance-inducing cells (eg, Tcm cells, as detailed herein above), and/or the use of immunosuppressive radiation.

Detailed guidelines for the selection and use of suitable immunosuppression regimens for transplantation are provided in the prior art literature (see, for example: Kirkpatrick CH. and Rowlands DT Jr., 1992. JAMA. 268, 2952; Higgins RM. et al., 1996. Lancet 348, 1208; Suthanthiran M. and Strom TV., 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).

Preferably, the immunosuppression regimen consists of administering at least one immunosuppressant to the subject.

Examples of immunosuppressive agents include, without limitation, tacrolimus (also called FK-506 or fujimycin, trade names: Prograf, Advagraf, Protopic), mycophenolate mofetil, sodium mycophenolate, prednisone, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine (sulfasalazopyrine ), sodium hydrochloride, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, TNF-alpha blockers, an inflammatory cytokine-targeting biologic, and non-steroidal anti-inflammatory drugs (NSAIDs). Examples of NSAIDs include, without limitation, acetylsalicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, tolmetindac , acetaminophen, ibuprofen, Cox-2 inhibitors, tramadol, rapamycin (sirolimus) and rapamycin analogs (such as CCI-779, RAD001, AP23573). These agents may be administered alone or in combination.

Regardless of the type of transplant, novel Tcm cells (as detailed herein above) are used in the method of the present invention to avoid graft rejection and graft versus host disease.

According to the method of the present invention, such Tcm cells are administered simultaneously with, before or after transplantation of a cell or tissue graft.

Tcm cells can be administered via any cell transplantation method known in the art, such as, without limitation, cell infusion (eg, intravenously) or intraperitoneally.

Tcm cells according to some embodiments of the present invention can be administered per se or as a pharmaceutical composition, where they are mixed with suitable carriers or excipients.

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

As used herein, the term "active ingredient" refers to the Tcm cells responsible for the biological effect.

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 in the body and does not neutralize the biological activity and properties of the administered compound. Adjuvant is meant by these phrases.

As used herein, the term "inert excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the active ingredient. Examples of excipients include, without limitation, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

Methods for formulating and administering drugs can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pennsylvania, latest edition, which is incorporated herein by reference.

Suitable routes of administration may include, for example, oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections, as well as intrathecal, direct intraventricular, intracardial, for example, into the cavity of the right or left ventricle, in common coronary artery, intravenous, intraperitoneal, intranasal or intraocular injections.

Traditional drug delivery approaches to the central nervous system (CNS) include: neurosurgical strategies (eg, intracerebral injection or intracerebroventricular infusion); manipulation of the agent molecule (for example, obtaining a chimeric fusion protein that contains a transport peptide that has affinity for an endothelial cell cell surface molecule, in combination with an agent that is itself unable to pass through the BBB) in order to use one of the endogenous BBB transport pathways ; pharmacological strategies designed to increase the lipid solubility of the agent (eg, conjugation of water-soluble agents to lipid or cholesterol transporters); and temporary disruption of the integrity of the BBB by hyperosmotic destruction (as a result of infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as angiotensin peptide). However, each of these strategies has limitations such as the inherent risks associated with an invasive surgical procedure, the size limitation imposed by the limitation inherent in endogenous transport systems, the potential undesirable biological side effects associated with systemic administration of a chimeric molecule composed of a carrier motif, which may be active outside the CNS, and a possible risk of brain damage within areas of the brain where BBB disruption occurs, making it a suboptimal delivery method.

Alternatively, the pharmaceutical composition may be administered locally rather than systemically, for example by injecting the pharmaceutical composition directly into a tissue site of the patient.

Pharmaceutical compositions according to some embodiments of the present invention can be made using methods well known in the art, for example, through conventional mixing, dissolving, granulating, panning, triturating, emulsifying, encapsulating, coating, or lyophilization processes.

Pharmaceutical compositions for use in accordance with some embodiments of the present invention may thus be formulated in the conventional manner using one or more physiologically acceptable carriers, including excipients and excipients that facilitate formulation of the active ingredients into formulations that can be used pharmaceutically. The proper composition depends on the chosen route of administration.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution or physiological saline buffer. For transmucosal administration, the formulation employs penetration agents appropriate to the barrier to be permeated. Such penetration agents are generally known in the art.

For oral administration, the pharmaceutical composition can be readily formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers make it possible to formulate the pharmaceutical composition in the form of tablets, pills, dragees, capsules, solutions, gels, syrups, pastes, suspensions, etc. for oral administration by the patient. Pharmacological preparations for oral use can be obtained using a solid inert filler, optionally by grinding the resulting mixture and processing the mixture of granules after adding suitable excipients, if necessary, to obtain tablets or dragee cores. Suitable inert fillers are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulosic preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If necessary, disintegrating agents such as cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate can be added.

The dragee cores are coated with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer coating solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to tablets or dragee shells for the purpose of identifying or characterizing different combinations of active compound doses.

Pharmaceutical compositions that can be taken orally include hard capsules made from gelatin as well as soft sealed capsules made from gelatin and a plasticizer such as glycerol or sorbitol. Hard capsules may contain the active ingredients in admixture with an excipient such as lactose, binders such as starches, lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids such as fixed oils, liquid paraffin or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in the conventional manner.

For administration by nasal inhalation, the active ingredients for use in accordance with some embodiments of the present invention are conveniently delivered in the form of a spray aerosol delivery from a pressurized pack or nebulizer using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges, for example of gelatin, for use in a dispenser may be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Compositions for injection may be presented in unit dosage form, for example in ampoules or multi-dose containers, optionally with an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain adjuvants such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active drug in water-soluble form. In addition, suspensions of the active ingredients may be prepared as suitable oily or aqueous injectable suspensions. Suitable lipophilic solvents or carrier vehicles include fatty oils such as sesame oil or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to enable highly concentrated solutions to be obtained.

Alternatively, the active ingredient may be in the form of a powder for formulation prior to use with a suitable carrier vehicle, for example a sterile, pyrogen-free water solution.

The pharmaceutical composition according to some embodiments of the present invention may also be formulated into rectal compositions such as suppositories or retention enemas using, for example, traditional suppository bases such as coconut oil or other glycerides.

Pharmaceutical compositions suitable for use in the context of some embodiments of the present invention include compositions wherein the active ingredients are present in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (Tcm cells) effective to prevent, alleviate, or alleviate the symptoms of a disorder (eg, cancer or non-cancerous disease) or to prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is within the skill of the art, especially in light of the detailed disclosure provided herein.

For any drug used in the methods of the present invention, a therapeutically effective amount or dose can be initially established using in vitro assays and cell culture assays. For example, the dose required to achieve a desired concentration or titer can be determined in animal models. Such information can be used to more accurately determine useful doses in humans.

The toxicity and therapeutic efficacy of the active ingredients described herein can be determined using standard pharmaceutical procedures in vitro, in cell cultures, or using experimental animals. Data derived from such in vitro assays and cell culture assays and animal studies can be used in formulating dosages for human use. The dosage may vary depending on the dosage form used and the route of administration used. The exact composition, route of administration and dosage can be chosen by the attending physician, taking into account the condition of the patient. (See, for example, Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", chapter 1, page 1).

The dosage amount and interval between doses can be adjusted individually to provide sufficient levels of the active ingredient that are sufficient to induce or suppress the biological effect (minimum effective concentration, MEC). The MEC will vary for each drug but can be determined based on in vitro data. The dosages required to achieve the MEC will depend on individual characteristics and the route of administration. Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to be treated, dosing may also refer to single or multiple administrations, with a course of treatment lasting from several days to several weeks, or until a cure or alleviation of the course of the disease is achieved.

Depending on the severity and responsiveness of the condition to be treated, dosing may also refer to single or multiple administrations, with a course of treatment lasting from several days to several weeks, or until a cure or alleviation of the course of the disease is achieved.

The amount of composition administered will depend, as already mentioned, on the subject to be treated, the severity of the disease, the route of administration, the opinion of the attending physician, and the like.

Compositions according to some embodiments of the present invention may optionally be presented in a package or dispenser device, such as in an FDA-approved kit, which may contain one or more unit dosage forms containing the active ingredient. The packaging may include, for example, a metal foil or a plastic film, such as in the case of a blister pack. Instructions for use may be included with the packaging or dispenser device. The package or dispenser may be provided with information associated with the container in the form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals, which information reflects the authority's approval of the form of the compositions or use in human or veterinary medicine. Such information may, for example, be in the form of a US Food and Drug Administration-approved drug information form for prescription drugs or in the form of an approved package insert. Formulated in a compatible pharmaceutical carrier, compositions containing a preparation of the present invention may also be prepared, placed in an appropriate container, and provided with information about the preparation for the treatment of said condition, as described in more detail above.

In the context of this document, the term "approximately" refers to ± 10%.

The terms "provides", "providing", "having", "includes", "including", "having" and their related words in root and meaning mean "including without limitation".

The term "consisting of" means "including and limited to".

The term "consisting primarily of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not significantly change the essential and new characteristics of the claimed composition, method or structure. .

In the context of this document, the singular forms include their plural, unless the context clearly indicates otherwise. For example, the term "compound" or "at least one compound" can include a variety of compounds, including mixtures thereof.

Throughout the application, various embodiments of the present invention may be presented in range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the present invention. Accordingly, a description of a range should be construed as specifically disclosing all possible subranges as well as individual numerical values within the range. For example, a description of a range such as 1 to 6 should be considered as specifically disclosing subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6. and so on, as well as individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This principle applies regardless of the width of the range.

Whatever numerical range is specified herein, it is intended to include any referenced number (fractional or integer) within the specified range. The phrases "ranging between/ranging between" the first specified number and the second specified number and "ranging from/ranging from" the first specified number "to" the second specified number are used interchangeably herein, and it is understood that they include the first and second numbers specified, and all fractional and integer numbers in between.

In the context of this document, the term "method" refers to methods, means, techniques and procedures for performing a given task, including, without limitation, such methods, means, techniques and procedures that are known, or that are easily developed based on known methods, tools, techniques. and procedures by practitioners in the fields of chemistry, pharmacology, biologists, biochemistry and medicine.

It is understood that certain features of the present invention, which, for clarity, are described in the context of separate embodiments, may also be provided in combination within a single embodiment. Conversely, various features of the present invention, which, for brevity, are described in the context of one embodiment, may also be provided separately or in any suitable subcombination, or as suitable in any other described embodiment of the present invention. Certain features described in the context of various embodiments should not be construed as primary features of such embodiments if the embodiment is unrealizable without such elements.

Various embodiments and aspects of the present invention, as described herein above and as stated in the Claims section below, are experimentally confirmed in the following examples.

Examples

Below are the following examples, which together with the above descriptions illustrate the present invention in a non-limiting manner.

In general, the terminology used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are described in detail 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, MD (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 set forth in US Pat. Nos. 4,666,828; 4683202; 4801531; 5192659 and 5272057; "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 immunological assays are described in detail in patents and scientific literature, see, for example, US patent No. 3791932; 3839153; 3850752; 3850578; 3853987; 3867517; 3879262; 3901654; 3935074; 3984533; 3996345; 4034074; 4098876; 4879219; 5011771 and 5281521; "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 which are incorporated by reference as if they were set forth herein in their entirety. Other general references are given in the text of this document. It is assumed 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.

General Materials and Experimental Procedures

Animals

Female BALB/c, FVB, C57BL/6 and BALB/c-NUDE mice, 6-12 weeks old, were obtained from Harlan Laboratories. B6.SJL, C57BL/6-Tg(CAG-OVA)916Jen/J congenic mice (Mice expressing OVA) and OT1 mice (Expressing a transgenic (Tg) TCR engineered to recognize hen egg albumin residues 257-264 (OVA) in the context of H2Kb MHC-I) and OT1/Rag-/CD45.1 were bred at the Weizmann Institute Animal Center. All mice were kept in small cages (5 animals in each cage) and fed with sterile food and acidified water. These studies were approved by The Weizmann Institute of Science Institutional Animal Care and Use Committee.

Obtaining Veto Cells from Mouse Memory I Cells

OT1 mice were used expressing a transgenic (Tg) TCR designed to recognize hen egg albumin residues 257-264 in the context of H2Kb MHC-I. These mice acted as donors of veto Tcm cells. Prior to harvesting these OT1 CD8 T cells, mice were immunized with OVA peptide mixed with Complete Freund's Adjuvant (CFA) and boosted 14 days after primary challenge with OVA peptide + Incomplete Freund's Adjuvant (IFA). Seven to fourteen days after immunization, mice were sacrificed, their spleens and lymph nodes were removed and minced, and cell sorting with magnetic beads was used to isolate memory cells (CD8 ⁺ CD44 ⁺ ) from the total pool of CD8 ⁺ cells. The resulting population was subjected to "side" stimulation by co-cultivation with irradiated splenocytes derived from mouse spleens expressing OVA under cytokine deficient conditions. Six hours after the start of co-culture, hIL-15 (10 ng/mL) was added to the culture to stimulate cells to express a Tcm-like phenotype as described below for wild type (WT) Tcm, i.e. previously described veto-Tcm cells that were obtained from whole spleen or peripheral blood mononuclear cells, as previously confirmed [Ophir, E. (2013), supra]).

Preparation of human dendritic cells loaded with viral peptide

Human dendritic cells (DC) loaded with the viral peptide were prepared as illustrated in FIG. 4B. Briefly, donor peripheral blood mononuclear cells (PBMCs) were seeded in a culture dish and incubated for 3 hours at 37° C. at 5% CO ₂ /O ₂ . The cell supernatant (containing T cells) was discarded and the adherent cells were further incubated for 72 hours with the addition of cytokines IL-4 and GM-CSF (under the same culture conditions). Three days later, non-adherent cells were harvested (immature dendritic cells), seeded and incubated overnight followed by the addition of cytokines INF-γ, IL-4, GM-CSF and LPS to mature the DC. On day 4, adherent cells (mature DC, ie mDC) were detached, loaded with a cocktail of viral peptides and incubated for 1 hour at 37°C. The viral peptide cocktail provides 7 peptide mixtures of EBV, CMV and adenovirus (Adv). The peptide mixtures are 15-mers overlapping 11 amino acids of the complete protein sequence of the antigen of interest, purchased from JPT Technologies (Berlin, Germany). Peptide mixtures were used covering EBV (LMP2, BZLF1, EBNA1), Adv-(pentone, hexon) and CMV-(pp65, IE-1). Human mDCs loaded with viral peptide were then irradiated with 30 Gy and added to the T cell fraction as discussed below.

Generation of human Tcm cells reactive to a third-party antigen using viral peptides

Human veto cells reactive for foreign antigen were obtained by first depleting peripheral blood mononuclear cells (PBMCs) from a cell donor for CD4 ⁺ and CD56 ⁺ cells (using magnetic cell sorting using magnetic beads obtained from Milteni Biotec.

The rest of the cell population was co-cultured with irradiated (30 Gy) dendritic cells (from the same cell donor) where the dendritic cells were stimulated to express a third party antigen (eg, a cocktail of viral antigens including EBV, CMV and adenovirus). For the first 3 days of cultivation, cells were supplemented only with IL-21, then, from day +3 to day +9, IL-21, IL-15 hIL-7 were added to the culture.

Generation of human veto Tcm cells from memory I cells using viral peptides

Veto cells were obtained from human memory cells by first depleting peripheral blood mononuclear cells (PBMCs) obtained from a cell donor against CD4 ⁺ , CD56 ⁺ and CD45RA ⁺ cells (using magnetic cell sorting using magnetic beads obtained from Milteni Biotec ). The rest of the cell population contained CD8 ⁺ CD45RO ⁺ memory T cells. Next, CD8 ⁺ CD45RO ⁺ memory T cells were co-cultured with irradiated (30 Gy) dendritic cells (from the same cell donor) where the dendritic cells were stimulated to express an antigen (e.g. a cocktail of viral antigens including EBV, CMV and adenovirus) . For the first 3 days of culture, cells were supplemented with IL-21 only, then IL-21, IL-15 and IL-7 were added to the culture from day +3 to day +9.

Generation of human veto Tcm cells from memory I cells using tumor peptides

Veto cells are derived from human memory cells by first immunizing a cell donor with a tumor peptide (e.g., BCR-ABL, ELA2, O250/carbonic anhydrase IX, HA-1, HA-2, hTERT, MAGE-1, MUC1, NY-ESO-1 , PRAME, PR1, PRTN3, RHAMM and WT-1, or combinations thereof, as discussed in Molldrem J. Biology of Blood and Marrow Transplantation (2006) 12:13-18; Alatrash G. and Molldrem J., Expert Rev Hematol.( 2011) 4(1): 37-50) with complete Freund's adjuvant (CFA) and boosted 14 days after primary challenge with tumor peptide + incomplete Freund's adjuvant (IFA). Then, peripheral blood mononuclear cells (PBMCs) were obtained and depleted of CD4 ⁺ , CD56 ⁺ and CD45RA ⁺ cells (using magnetic cell sorting using magnetic beads obtained from Milteni Biotec). The rest of the cell population contained CD8 ⁺ CD45RO ⁺ memory T cells. Then, CD8 ⁺ CD45RO ⁺ memory T cells were co-cultured with irradiated (30 Gy) dendritic cells (from the same cell donor), where dendritic cells were stimulated to express tumor antigen. For the first 3 days of culture, cells were supplemented with IL-21 alone, and then, from day +3 to day +9, IL-21, IL-15, and IL-7 were added to the culture.

Analysis using flow cytometry

Fluorescence activated cell sorting (FACS) analysis was performed using a Becton Dickinson FACScanto II to determine the purity level and phenotype of the resulting Tcm cells, ie. CD8+CD45RO+CD62L+ and CD4-CD56-CD45RA-. Cells were stained in two panels using the following labeled antibodies:

Panel 1: CD8-fluorescein isothiocyanate (FITC), CD45RO-phycoerythrin (PE), CD62L-allophycocyanin (APC), CD56-APC-Cy7, CD3-brilliant violet 711, CD16-PE-Cy7 and 7AAD-PerCp.

Panel 2: CD3-Brilliant Violet 711, CD8-FITC, CD45RA-PE-Cy7, CD45RO-APC-Cy7, CD20-PE and 7AAD-PerCp.

Limiting Dilution Assay (LDA) and Cytotoxicity Assay Based on ³⁵ S-Methionine Release

To evaluate the frequency of host-reactive cells in Tcm cultures that are reactive for foreign antigens derived from PBMCs at different stages of purification, such as CD4 ^- CD56 ^- (i.e. enriched in CD8 ⁺ cells) or CD4 ^- CD56 ^- CD45RA ^- (ie memory cells) was analyzed using limiting dilution (LDA) in comparison with fresh CD4 ^- CD56 ^- CD19 ^- cells (serving as an allogeneic positive control). The three test cell preparations were cultured with irradiated host PBMCs in MLR culture for 5 days (ie, mixed culture) to ensure induction of host activity. After 5 days, effector cells were harvested from the MLR culture and separated in a Ficoll gradient. Effector cells were plated at different dilutions (range 1-40,000 cells per well) in 96-well round bottom well plates (16 replicates per input number). Irradiated (30 Gy) host stimulator cells that were used in mixed MLR were also added to each well. Cultures at limiting dilutions were used to provide signal endpoint titration against the host and maintained for 7 days in the presence of IL-2 to amplify the effector signal.

After 7 days, cytotoxic activity was measured in a standard 5-hour assay against ³⁵ S-methionine labeled cells. Briefly, concanavalin A-derived blast cells (Sigma, St. Louis, MO) from the host that were used as target cells were labeled with ³⁵ S-methionine and seeded along with various dilutions of test induced effector cells. After a 5-hour incubation, the average radioactivity in the supernatants of the samples in 16 replicates was calculated, and the percentage of specific lysis was calculated using the following equation: 100 × (average experimental release - average spontaneous release) / (average total release - average spontaneous release). The level of ³⁵ S-methionine released by the target cells corresponds to the level of destruction, which corresponds to the level of reactivity towards the host.

(которое представляет собой член нулевого порядка распределения Пуассона), в котором у представляет собой процентную долю неотвечающих культур, х представляет собой число отвечающих клеток на культуру, ƒ представляет собой частоту встречаемости отвечающих клеток-предшественников и а представляет собой отрезок y, теоретически равный 100%. Среднее плюс 3 стандартных отклонения для 16 лунок, содержащих только целевые клетки, было определено как пороговое значение фоновой радиоактивности. Экспериментальные лунки были оценены как положительные в отношении лизиса в случае превышения порогового значения. Процент отвечающих культур определяли путем расчета процента положительных культур. Частоту встречаемости CTL-p (f) и стандартную ошибку (SE) определяли из наклона кривой, построенной на основе линейного регрессионного анализа данных.The following equation was used to calculate the frequency of occurrence from crop values at limiting dilutions:

(which is the zero-order term of the Poisson distribution), in which y is the percentage of non-responding cultures, x is the number of responding cells per culture, ƒ is the frequency of responding progenitors, and a is the interval y, theoretically equal to 100% . The mean plus 3 standard deviations for 16 wells containing only target cells was defined as the threshold for background radioactivity. Experimental wells were scored positive for lysis if the threshold was exceeded. The percentage of responding cultures was determined by calculating the percentage of positive cultures. The frequency of occurrence of CTL-p (f) and standard error (SE) was determined from the slope of the curve, built on the basis of a linear regression analysis of the data.

IFN-γ Elispot Analysis

An ELISpot assay, a highly sensitive immunoassay that measures the frequency of cytokine-secreting cells at the individual cell level, was used. In particular, the INF-γ-(interferon-gamma)-ELISpot assay was used to evaluate the frequency of residual host-reactive cells in Tcm cultures that are reactive to third-party antigens, since IFN-γ is predominantly produced by activated T cells and NK cells. .

Briefly, the membrane surfaces of a 96-well microtiter plate with PVDF membranes were coated with an immobilized antibody (purified anti-human INF-γ antibody) that binds to the specific epitope of the cytokine (IFN-γ) to be analyzed. During the 16 hours of MLR and the stimulation stage, various dilutions (in the range of 1-40,000 cells/well) of test cells were plated in wells of the plate along with irradiated host-reactive stimulator cells. They formed a monolayer on the well membrane surface. After activation of specific host-reactive cells, IFN-γ is released, which is captured directly on the membrane surface by the immobilized antibody. IFN-γ is thus "trapped" in the region immediately surrounding the secreting cell before diffusing into the culture medium or being degraded by proteases and binding to receptors on the surface of the "background" cells. Subsequent detection steps allow the immobilized IFN-γ to be visualized as an ImmunoSpot. After washing the wells to remove cells, debris, and media components, a biotinylated antibody specific for human IFN-γ was added to the wells. This antibody is reactive for a single epitope of the IFN-γ cytokine and thus is used to detect the captured cytokine. After washing to remove any unbound biotinylated antibody, the detected cytokine was visualized using horseradish peroxidase (HRP)-conjugated streptavidin and a precipitating substrate (eg, ABS, BCIP/NBT). The stained end product (red spot, (in the case of HRP) usually corresponds to a single IFN-γ-producing cell. Spots were counted manually (for example, using a dissecting microscope) or using an automated reader to capture images from microwells and analyze the number and size of spots.

Example 1

TCR-transgenic CD8 memory T cells expanded in response to their cognate antigen markedly enhance engraftment of fully allogeneic T-depleted BM

The possibility that memory T cells may be associated with a reduced risk of developing GvHD has been discussed over the past decade. In general, the memory cell pool is enriched in virus-reactive clones and therefore may contain a reduced level of alloreactive T cells. More recently, however, two large studies attempting to use CD45RA ⁺ depleted HSCT in patients with leukemia have confirmed significant levels of GvHD, even with post-transplant GvHD prophylaxis [Bleakley M. et al. J Clin Invest. (2015) 125(7):2677-89; Triplett, BM et al. Bone Marrow Transplant. (2015) 50(7):968-977]. Thus, the depletion of allore active clones from the pool of memory T cells by activating and expanding T cells reactive to a foreign antigen may solve the problem of residual GvHD that remains after depletion of CD45RA cells. Moreover, the use of common viral antigen peptides as off-site stimulation could potentially generate veto-Tcm cells that are both depleted in alloreactivity and endowed with antiviral activity.

To this end, the authors of the present invention modified the previous protocol for obtaining veto-Tcm cells reactive against a third-party antigen by conducting stimulation using specific peptides, in relation to which the TCR of the existing pool of memory T cells is directed. Thus, proof-of-concept experiments were performed in mouse models using OT1 mice expressing a transgenic (Tg) TCR engineered to recognize hen albumin residues 257-264 in the context of H2Kb MHC-I. These mice acted as donors of veto Tcm cells. Prior to harvesting these OT1 CD8 T cells, mice were immunized with OVA peptide mixed with Complete Freund's Adjuvant (CFA) and boosted 14 days after primary challenge with OVA peptide + Incomplete Freund's Adjuvant (IFA) (See Figure in Fig. 1). Seven to fourteen days after immunization, mice were sacrificed, their spleens and lymph nodes were removed and minced, and cell sorting with magnetic beads was used to isolate memory cells (CD8 ⁺ CD44 ⁺ ) from the total pool of CD8 ⁺ cells. The resulting population was subjected to "side" stimulation by co-cultivation with irradiated splenocytes derived from mouse spleens expressing OVA under cytokine deficient conditions. Six hours after the start of co-culture, hIL-15 (10 ng/mL) was added to the culture to stimulate the cells to express a Tcm-like phenotype as described above for Tcm WT (i.e. "regular" Tcm cells as discussed below).

As shown in FIG. 2A, OT-1 Tcm cells derived from the original memory cell population (CD8 ⁺ CD44 ⁺ ) were able to enhance the engraftment of allogeneic T-depleted bone marrow transplant, similar to the chimerism induced by "regular" Tcm reactive Tcm cells. against a foreign antigen (i.e. previously described veto-Tcm cells, which were obtained from whole spleen or peripheral blood mononuclear cells, as previously confirmed [Ophir, E. (2013), supra]). Taken together, these results strongly demonstrate that CD8 ⁺ CD44 ⁺ -derived Tcm cells expanded in response to their cognate peptides can indeed induce tolerance without GvHD.

Example 2

Veto Tcm cells derived from CD8 memory T cells have strong veto activity with a reduced risk of developing GVHD

Then, the present inventors attempted to obtain Tcm cells from CD8 B6-WT memory T cells after OVA immunization. To this end, after immunization of C57BL/6 mice, CD8 ⁺ CD44 ⁺ memory T cells were subjected to magnetic sorting and the same protocol to obtain Tcm using all OVA stimulators. Initially, the authors of the present invention wanted to test the ability of these CD8 ⁺ CD44 ⁺ Tcm cells to induce GvHD in comparison with that of the initial population of CD8 ⁺ CD44 ⁺ cells, which was previously used in the clinic. It was expected that CD8 ⁺ CD44 ⁺ Tcm cells would be depleted of alloreactive clones by antigen stimulation using the OVA peptide, which selectively activates only those T cell clones that have the corresponding TCR.

Indeed, CD8 ⁺ CD44 ⁺ Tcm cells have not been shown to induce observable GvHD symptoms in animal models, while fresh CD8 ⁺ CD44 ⁺ memory cells that have not undergone third-party activation induced significant GvHD mortality and weight loss (Figures 2B). -FROM). Next, the present inventors wanted to evaluate whether these cells were capable of inducing tolerance in a reduced intensity conditioning (RIC) model (illustrated in FIG. 2D). As can be seen in FIG. 2E, CD8 ⁺ CD44 ⁺ Tcm showed a marked increase in chimerism after transplantation of naked C57BL BM into Balb/c recipients conditioned with 5 Gy TBI.

Example 3

Generation of human Tcm cells reactive to foreign antigen using viral antigens

The present inventors have previously developed a protocol for obtaining human-derived foreign antigen reactive Tcm cells from an initial population of CD4 ^- CD56 cells with minimal risk of developing GvHD using a two ^- step approach using magnetic sorting to deplete allore activity. This protocol was developed using three cell donors, where a third-party donor was selected to ensure that none of its HLA class I alleles are shared with the host's HLA class I alleles, in order to prevent GvHD.

In the present experiments, similarly to what was described above for the mouse proof-of-concept experiments, the present inventors used naturally occurring CD8 memory cells as a starting material to obtain veto Tcm cells, since these cells were reported to have a reduced propensity to induce GvHD compared to naive cells. This option has recently been made feasible by the market launch of GMP-level anti-CD45RA antibody magnetic beads to deplete naïve T cells. As described above and in the Field and Prior Art section of this document above, the CD45RA cell infusion approach ^was tested in two clinical trials in patients with leukemia [Bleakley M. et al. (2015) above; Triplett, V.M. et al. (2015) above], however, prevention of GvHD did not occur, with some patients developing severe forms of GvHD, even when treated with immune suppression after transplantation. These data prompted the present inventors to evaluate the possibility that stimulation of CD45RA-depleted CD8 T cells in response to specific antigens could completely deplete these cells of GvH reactivity. Since the TCRs of most cells in this pool of memory cells are directed against common viral and bacterial antigens and therefore naturally less prone to allorasis actively towards the host, the present inventors hypothesized that viral antigen peptides (e.g., CMV, EBV, and adenovirus ) loaded on donor DCs (ie, the same cell donor as in the case of Tcm cells) could be successfully used as stimulants. Using this approach, one can take advantage of the possible antiviral activity of these cells in addition to their veto activity, which is a particularly promising feature for transplantation, in which repeated antiviral activation is a common adverse event.

To confirm this assumption, a preliminary experiment was initiated in which the same culture conditions for veto-Tcm cells were used, except that stimulation against antigen was performed against donor DCs stimulated with mixtures of viral peptides from three common viruses (EBV, CMV, and adenovirus ), instead of a third HLA-incompatible donor for third-party DCs. It should be noted that this experiment was started with the previously described CD4 ^- CD56 population ⁽ ie CD45RA ⁺ cells were not removed). As can be seen in Figures 3A-3B, veto Tcm cells grew well in response to this antiviral challenge, with a 10-fold multiplication by day +9 observed with a high percentage (93.2%) of cells exhibiting the veto phenotype. tcm.

Host reactivity assayed by limiting dilution assay (LDA) is shown in Figures 3C-D and the corresponding parameters are summarized in Table 2 below. It can be seen from the results that this method provided a 2-fold logarithmic depletion of host alloreactive clones, similar to previous results using "normal" third-party activation against an HLA-incompatible donor. These results were further confirmed in the IFNγ Elispot assay (performed after mixed culture), in which fresh cells (CD4 ^- CD56 ^- CD19 ^- ) formed approximately 25,000 spots/10 ⁶ T cells after activation of host cells could not be detected. any stains in veto-Tcm cell culture (data not shown).

Example 4

Generation of Human Veto Cells from Memory T Cells Using Viral Antigens

Next, the present inventors tested the reactivity of Tcm cells grown from a CD45RA-depleted population activated against viral antigens presented on the surface of a donor DC (i.e. the same cell donor as in the case of Tcm cells) (as illustrated in figures 4A and 4B).

The reactivity of veto Tcm cells grown from this initial population (CD4 ^- CD56 ^{- CD45RA} cells) was tested using an LDA kill assay. As can be seen from the results, it is clear that memory cell-derived veto cells do not show any host reactivity (Figures 5A-C and Figure 6).

Taken together, these results strongly demonstrate that the viral peptide-stimulator approach can be applied to an initial population of reactive cells that are CD45RA-depleted (e.g., CD4 ^- CD56 ^- CD45RA ^- cells) known to have a relatively low tendency to induce GvHD. As shown, this cell population can further be depleted of putative host-reactive clones upon antiviral challenge, which can provide a highly safe, non-GvHD-inducing cell preparation.

Example 5

Generation of human veto cells from memory T cells using non-viral antigens

The present inventors have obtained veto cells from an initial population of memory cells ( ^CD45RA cells) stimulated for peptides other than viral ones, including peptides identified in cancer (eg, solid tumor or hematopoietic malignancy).

As described above, veto cells were generated by exposing memory T cells (ie, a CD45RA depleted population) obtained from a donor to tumor antigens displayed on the surface of the donor DC.

Alternatively, veto cells were generated by first immunizing a cell donor with the tumor peptide (as discussed in the General Materials and Experimental Procedures section above) with Complete Freund's Adjuvant (CFA) and performing a boost 14 days after the primary challenge with the tumor peptide. + incomplete Freund's adjuvant (IFA). Subsequently, peripheral blood mononuclear cells (PBMCs) were obtained from the subject and depleted of CD4 ⁺ CD56 ⁺ CD45RA ⁺ . The rest of the cell population (ie, CD8 ⁺ CD45RO ⁺ memory T cells) were co-cultured with tumor antigens displayed on the surface of the donor DC.

These cells can additionally be used for therapeutic elimination of residual cancer cells.

Example 6

Good manufacturing practice (GMP) protocol for large-scale production of antiviral central CD8 memory veto-T cells (Tcm)

The present inventors successfully repeated the protocol for generating human veto cells from memory T cells using viral antigens displayed on the surface of autologous antigen presenting cells 10 times (FIG. 7). As can be seen in Figures 8 and 9A-H, the new cell isolation and purification protocol was quite reproducible.

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

All publications, patents, and patent applications referred to in this specification are incorporated herein to the same extent as if each individual publication, patent, or patent application were specifically and individually designated for inclusion herein by reference. In addition, the citation or indication of any reference in this application should not be construed as an admission that such a reference exists as a prior art of the present invention. In the sense in which section headings are used, they are not necessarily to be construed as limiting.

Claims (50)

1. A method for obtaining an isolated population of non-graft-versus-host (GvHD)-inducing antiviral, antibacterial and/or antitumor cells with the phenotype of central memory T-lymphocytes (Tcm), wherein these cells are tolerance-inducing and capable of homing to the lymph nodes after transplantation, wherein the method includes: (a) providing a T cell population containing at least 50% memory T cells; (b) bringing said population of T cells into contact with a viral, bacterial and/or tumor antigen or antigens in the presence of IL-21 in order to enrich it with antigen-reactive cells; and (c) culturing said cells obtained in step (b) in the presence of IL-21, IL-15 and/or IL-7 in order to ensure the proliferation of cells having said Tcm phenotype, thereby obtaining an isolated population of non-GvHD-inducing antiviral, antibacterial and/or antitumor cells. 2. The method of claim 1, wherein said memory T cells depleted of CD45RA ⁺ cells and/or depleted in relation to CD4 ⁺ and/or CD56 ⁺ cells. 3. The method of claim 1, wherein: said T cell population comprising at least 50% memory T cells is enriched for an antigen or antigens. 4. The method of claim 1, further comprising treating the cell donor with said antigen or antigens until said provision of said population of T cells containing at least 50% memory T cells. 5. A method for obtaining an isolated population of non-GvHD-inducing antiviral, antibacterial and/or antitumor cells having the phenotype of central memory T-lymphocytes (Tcm), wherein said cells are tolerance-inducing and/or endowed with antiviral, antibacterial and/or antitumor activity and are capable of homing to lymph nodes after transplantation, wherein the method includes: (a) treating peripheral blood mononuclear cells (PBMCs) with an agent capable of depleting CD4 ⁺ , CD56 ⁺ and CD45RA ⁺ cells or an agent capable of selecting CD45RO ⁺ , CD8 ⁺ cells to obtain a cell population enriched in memory T cells, having the phenotype CD45RA-CD8 ⁺ ; (b) bringing said cell population enriched in memory T cells into contact with a viral, bacterial and/or tumor antigen or antigens in the presence of IL-21 in order to enrich it with antigen-reactive cells; and (c) culturing said cells obtained in step (b) in the presence of IL-21, IL-15 and/or IL-7 in order to ensure the proliferation of cells having said Tcm phenotype, thereby obtaining an isolated population of non-GvHD-inducing antiviral, antibacterial and/or antitumor cells. 6. The method according to p. 1 or 5, in which these viral, bacterial and/or tumor antigen or antigens: represented by autologous antigen-presenting cells that are not autologous antigen-presenting cells, artificial carriers or artificial antigen-presenting cells; and/or represented by antigen-presenting cells of the same origin as these memory T-cells. 7. The method according to claim 1 or 5, wherein said viral, bacterial and/or tumor antigen or antigens: include two or more viral peptides; and/or include EBV peptide, CMV peptide, adenovirus (Adv) peptide and/or BK virus peptide; and/or include three EBV peptides, two CMV peptides, and two adenovirus (Adv) peptides; and/or selected from the group consisting of EBV-LMP2, EBV-BZLF1, EBV-EBNA1, CMV-pp65, CMV-IE-1, Adv-penton and Adv-hexon; and/or include two or more of EBV-LMP2, EBV-BZLF1, EBV-EBNA1, CMV-pp65, CMV-IE-1, Adv-penton, and Adv-hexon. 8. A method according to any one of claims 1 or 5, wherein said contacting said population of memory T cells with said viral, bacterial and/or tumor antigen or antigens in the presence of said IL-21 carried out within 12 hours to 5 days or carried out within 3 days. 9. A method according to any one of claims 1 or 5, wherein said culturing said cells obtained in step (b) in the presence of IL-21, IL-15 and/or IL-7: carried out within 12 hours to 15 days; or carried out within 4 days to 10 days; or carried out within 6 to 9 days. 10. The method according to any one of paragraphs 1 or 5, in which the specified total duration of obtaining not inducing GvHD cells is 9-12 days; and/or where said method is carried out ex-vivo; and/or further providing for the depletion of alloreactive cells after the implementation of stage (c), and optionally said depletion of said alloreactive cells is carried out by depleting CD137 ⁺ and/or CD25 ⁺ cells after bringing said cells with the indicated Tcm phenotype into contact with antigen-presenting cells ( APC) of the host. 11. The method of claim 5, wherein said PBMCs are not isogenic with respect to the subject. 12. The method according to any one of claims 1 or 5, wherein said cells with said Tcm phenotype have a CD3 ⁺ , CD8 ⁺ , CD62L ⁺ , CD45RA- ^, CD45RO ⁺ antigenic profile. 13. An isolated population of non-graft-versus-host (GvHD) antiviral, antibacterial and/or antitumor cells containing cells with a central memory T-lymphocyte (Tcm) phenotype, said cells being derived from memory T-cells according to the method according to any one of paragraphs. 1-12 for use in the induction of transplantation tolerance and/or for the treatment of bacterial disease, viral disease, or cancer in the absence of GvHD. 14. A method of treating a subject in need of cell or tissue transplantation, the method comprising: (a) transplanting a graft of cells or tissues into a subject; and (b) administering to the subject a therapeutically effective amount of an isolated population of non-GvHD-inducing antiviral, antibacterial, and/or antitumor cells, said cells being derived from memory T cells in accordance with the method of any one of claims. 1-12, thereby providing treatment to a subject in need of cell or tissue transplantation. 15. The method according to claim 14, in which (b) is carried out before (a) or (a) and (b) are carried out simultaneously. 16. The method of claim 14, further comprising conditioning the subject under sub-lethal, lethal, or super-lethal conditions prior to said transplant. 17. The method of claim 14, wherein said cell or tissue graft is not isogenic with respect to the subject. 18. The method of claim 14, wherein said cell or tissue graft and said isolated population of non-GvHD-inducing cells are from the same donor. 19. The method of claim 14, wherein said cell or tissue graft: selected from the group consisting of a cell or tissue of the liver, pancreas, spleen, kidney, heart, lung, skin, intestine, brain, ovary, and lymphoid/hematopoietic cell or tissue; or provides for co-transplantation of several organs, and optionally said co-transplantation provides for transplantation of immature hematopoietic cells and a parenchymal organ. 20. The method of claim 14, wherein said subject has a malignant disease. 21. The method of claim 20, wherein said malignant disease is a solid tumor or tumor metastasis. 22. The method of claim 20, wherein said malignant disease is a malignant neoplasm of hematopoietic tissue. 23. The method of claim 14, wherein said subject has a disease that is not malignant. 24. The method of claim 23, wherein said non-cancerous disease is selected from the group consisting of organ dysfunction or failure, transplant related disease, and blood disease.

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