Engineered cells for inducing tolerance

Abstract

To provide compositions, methods and uses for inducing immunosuppression and tolerance as defined by suppression of an immune response to an antigen.SOLUTION: Provided is cells that are engineered so as to increase the cellular level of cellular FLICE [Fas-associated death domain (FADD)-like IL-1β-converting enzyme]-inhibitory protein (CFLAR). The engineered cells have an ability to induce tolerance. Enhanced tolerogenicity is useful for prolonging survival of a foreign transplant and for treatment of autoimmune diseases.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to cells engineered to increase the cellular levels of FLICE [Fas-associated death domain (FADD)-like IL-1β-converting enzyme]-inhibitory protein (CFLAR). The engineered cells have the ability to induce tolerance. Enhanced tolerogenicity is useful for extending the survival of xenografts and for the treatment of autoimmune diseases. [Background technology]

[0002] The evolution of the immune system has provided vertebrates with a highly effective network based on two types of defense: innate and adaptive immunity. In contrast to the evolutionarily older innate immune system, which relies on invariant receptors that recognize common molecular patterns associated with pathogens, adaptive immunity is based on highly specific antigen receptors and clonal selection on B cells (B lymphocytes) and T cells (T lymphocytes). B cells trigger the humoral immune response by secreting antibodies, while T cells mediate the cellular immune response that leads to the destruction of recognized cells, playing a central role in cell-mediated immunity in humans and animals.

[0003] Mature T cells recognize and respond to antigens through their antigen-specific receptors (TCRs), which bind to major histocompatibility complex (MHC) molecules and interact with immunogenic peptides (epitopes) presented on the surface of target cells. For example, cytotoxic T cells respond to antigens presented in association with MHC-I proteins. Helper T cells recognize antigens presented on MHC-II proteins. The most immediate consequence of TCR activation is the initiation of signal transduction pathways that result in clonal expansion of T cells, upregulation of activation markers on the cell surface, differentiation into effector cells, induction of cytotoxic or cytokine secretion, and induction of apoptosis. The TCR is part of a complex signal transduction machinery that includes a heterodimeric complex of TCR α and β chains, co-receptors CD4 or CD8, and the CD3 signal transduction module. The CD3 chain transmits activation signals inside the cell, while the TCR α / β heterodimer is solely involved in antigen recognition.

[0004] In addition to the important role that T cells play in the immune response, dendritic cells (DCs) are equally important: they are professional antigen-presenting cells that have a key regulatory role in maintaining tolerance to self-antigens and in activating innate and adaptive immunity against foreign antigens.

[0005] By producing soluble factors such as cytokines, interleukins, and metabolites, tumors induce alternative hematopoiesis that alters normal myeloid cell differentiation and promotes the proliferation and expansion of cells with immunosuppressive functions called myeloid-derived suppressor cells (MDSCs) (Ugel S et al., 2015, J Clin Invest. 125:3365-76; Marvel D et al., 2015, J Clin Invest. 125:3356-64). MDSCs are characterized by their myeloid origin, heterogeneous cell composition, and ability to negatively regulate adaptive and innate immune responses. MDSC functional activity is associated with upregulation of immunosuppressive enzymes such as arginase 1 (ARG1), indoleamine 2,3-dioxygenase 1 (IDO1), NADPH oxidase (NOX) and inducible nitric oxide synthase (iNOS, also called NOS2), and increased production of nitric oxide (NO) and reactive oxygen and nitrogen species (ROS and RNS), such as the free radical peroxynitrite (ONOO-), which inhibit T cell vitality and fitness (Bronte V et al., 2005, Nat Rev Immunol. 5:641-54). MDSC composition is plastic, specific to each cancer type, and often changes according to the dynamics and development of the pathology: activated MDSCs are mostly present in primary tumors and metastatic sites, but some are also found circulating, suggesting that they can be distinguished from other non-suppressive myeloid cells using MDSC-specific activation markers (Gabrilovich DI et al., 2012, Nat Rev Immunol. 12:253-68; De Sanctis F et al, 2016, Biochim Biophys Acta. 1865:35-48). To date, human MDSCs have been classified into three main subsets: monocytic (MO)-MDSCs, polymorphonuclear / granulocytic (PMN)-MDSCs, and immature (i)-MDSCs. However, differences in sample preparation procedures and analysis of cytofluorometric data, combined with the lack of exclusive MDSC markers, have generated many discrepancies between studies and delayed the listing of MDSCs in clinical routine.Conversely, in the last two decades, the identification of MDSC-associated molecules and biological pathways and the partial classification of cellular networks in the tumor microenvironment by the use of innovative tumor mouse models has increased interest in MDSCs and their functions in cancer pathology. Recently, it has been reported that mouse MDSC heterogeneity between two main subsets results from differential activation of molecular pathways regulating the death-inducing signaling complex (DISC). The development of monocytic (MO)-MDSCs constitutively requires the presence of the antiapoptotic molecule FLICE [Fas-associated death domain (FADD)-like IL-1β-converting enzyme]-inhibitory protein (CFLAR, also known as c-FLIP)], which controls the extrinsic apoptosis pathway and caspase 8 activation. On the other hand, the expansion of polymorphonuclear (PMN)-MDSCs depends on MCL-1-mediated regulation of the intrinsic mitochondrial death pathway (Haverkamp JM et al, 2014, Immunity 41:947-59). CFLAR has 13 different splice variants, three of which are delivered as proteins: the 26 kDa short form (c-FLIPS), the 24 kDa Raji form (c-FLIPR, specifically expressed only in some cell lines and human primary T cells), and the 55 kDa long form (c-FLIPL). All three CFLAR variants contain two cell death effector domains (DEDs) that interact with the apoptotic adaptor protein FAAD. The gene encoding CFLAR is evolutionarily conserved in vertebrates, and the structure of c-FLIPS is similar to viral FLICE-inhibitory protein (v-FLIP), a component of viruses of the gammaherpesvirus class, e.g., human herpesvirus 8, Kaposi's sarcoma-associated virus (Safa AR, 2012, Exp Oncol. 34:176-84; Safa AR et al., 2008, Curr Cancer Drug Targets 8:37-46).

[0006] The immune system can also produce undesirable effects. For example, autoimmune disorders are characterized by the loss of tolerance to self-antigens, activation of lymphocytes reactive to "self" antigens (autoantigens), and pathological damage in target organs. In addition, the immune system can cause the rejection of cell, tissue, and organ transplants from unrelated donors. The immune system does not distinguish beneficial invaders, such as transplanted tissue, from harmful ones, and therefore the immune system rejects transplanted tissues or organs. Rejection of transplanted organs is usually mediated by alloreactive T cells present in the host that recognize the donor's allo- or xeno-antigens. On the other hand, when allogeneic immunologically active cells are introduced, transplanted, or grafted into a host, graft-versus-host disease can occur, which results in a severe, sometimes fatal, reaction due to immune incompatibility between the host and the graft, in which the graft's immunocompetent cells immunologically attack the host.

[0007] Thus, there is a need for agents that have tolerogenic or immunosuppressive properties, and in particular for agents that enhance the tolerogenic properties of the host. Summary of the Invention

[0008] Summary of the Invention The present invention encompasses compositions, methods and uses for inducing immunosuppression and tolerance, defined by the suppression of the immune response to an antigen.

[0009] The present invention is based on the concept that increasing the cellular level of cellular FLICE [Fas-associated death domain (FADD)-like IL-1β-converting enzyme]-inhibitory protein (CFLAR) in cells, particularly bone marrow cells or their precursor cells, increases the immunosuppressive activity of the cells. The cells in which the cellular level of CFLAR is increased can be present in vitro, i.e. isolated cells, or present in vivo, i.e. cells present within a subject or individual. Thus, the present invention provides a method for enhancing the tolerogenic or immunosuppressive capacity of cells, particularly bone marrow cells or their precursor cells.

[0010] The tolerogenic or immunosuppressive cells described herein are useful for extending the survival of foreign grafts in mammalian hosts and for ameliorating inflammation-related diseases, such as autoimmune diseases. The tolerogenic or immunosuppressive cells are also useful for suppressing immune responses in gene therapy of desired genes or foreign protein-based therapies. Thus, the present invention encompasses methods and compositions for reducing and / or eliminating immune responses to a graft in a recipient by treating the recipient with a quantity of the tolerogenic or immunosuppressive cells described herein to reduce or inhibit host rejection of the graft. A graft refers to any material administered to a host. For example, grafts include, but are not limited to, biocompatible lattices, tissues, organs, cells, nucleic acids, and polypeptides. Also encompassed are methods and compositions for reducing and / or eliminating host immune responses to a transplant foreign to the host, i.e., graft-versus-host disease, by treating the donor graft and / or the recipient of the graft with the tolerogenic or immunosuppressive cells described herein to inhibit or reduce adverse reactions of the donor graft to the recipient. In addition, the present invention encompasses methods and compositions for reducing and / or eliminating immune responses to exogenously delivered proteins in a recipient by treating the recipient with a quantity of the tolerogenic or immunosuppressive cells described herein to reduce or inhibit rejection of the protein.

[0011] Various aspects of the invention contemplate genetic modification of cells to express or increase the expression level of CFLAR in vitro or in vivo. Different vector formats can be used for genetic modification of cells, such as RNA, DNA or viral vectors, which can be applied in vitro or in vivo. If the cells that increase the cellular level of CFLAR are present in vitro, a strategy can be adopted in which these cells are genetically modified in vitro and, optionally, expanded in vitro before transferring the cells into a recipient. The cells used for therapy can be autologous, allogeneic or syngeneic to the recipient. If the cells that increase the cellular level of CFLAR are present in vivo, the cells can be manipulated in situ in the patient, such as by transfection with nucleic acid, and a strategy based on genetically modifying the cells in vivo by applying the appropriate nucleic acid, optionally in a suitable carrier, to the recipient.

[0012] The strategies for inducing immune suppression and / or tolerance described herein can be used to suppress anticipated unwanted immune responses, for example, to extend gene therapy or repeated administration of a therapeutic protein expressed, e.g., via a vector in a mammalian host or exogenously applied to a mammalian host, to extend the survival of a foreign graft in a host, to treat or prevent graft-versus-host disease, to treat or prevent autoimmune diseases including, but not limited to, autoimmune arthritis, autoimmune diabetes, asthma, septic shock, pulmonary fibrosis, glomerulonephritis, and atherosclerosis.

[0013] In one aspect, the invention relates to a population of cells, including bone marrow cells or their progenitors, engineered to increase cellular levels of cellular FLICE [Fas-associated death domain (FADD)-like IL-1β-converting enzyme]-inhibitory protein (CFLAR). For example, the cellular levels of CFLAR can be increased by increasing expression of CFLAR in the cells.

[0014] In one embodiment, increasing the cellular level of CFLAR increases the immunosuppressive activity of the cell.

[0015] In one embodiment, bone marrow cells or their progenitor cells are engineered to increase cellular expression of CFLAR.

[0016] In one embodiment, bone marrow cells or its precursor cells are transfected with a nucleic acid encoding CFLAR. In one embodiment, the expression of CFLAR is increased in transfected cells compared to non-transfected cells. In one embodiment, transfection is stable or transient transfection. In one embodiment, bone marrow cells or its precursor cells are transfected with a viral vector as a carrier, such as a lentiviral vector. In one embodiment, said nucleic acid is RNA.

[0017] In one embodiment, CFLA R CFLARL and / or CFLAR S It is.

[0018] In one embodiment, the bone marrow cells or their progenitor cells are isolated and / or purified cells, in particular cells isolated and / or purified by magnetic cell separation.

[0019] In one embodiment, the bone marrow cells or their precursor cells are monocytes. In one embodiment, the bone marrow cells or their precursor cells are CD14+ monocytes. In one embodiment, the bone marrow cells or their precursor cells are CD14+ monocytes isolated from buffy coat.

[0020] In one embodiment, the bone marrow cells or their progenitor cells are stem cells. In one embodiment, the bone marrow cells or their progenitor cells are bone marrow CD34+ cells. In one embodiment, the bone marrow cells or their progenitor cells are bone marrow CD34+ cells isolated from bone marrow mononuclear cells (MNC).

[0021] In one embodiment, the cells have been treated to promote acquisition of immunosuppressive activity, hi one embodiment, the treatment to promote acquisition of immunosuppressive activity comprises in vitro differentiation in the presence of G-CSF and GM-CSF.

[0022] In one embodiment, the population of cells of the invention also includes bone marrow cells or their progenitors that are not engineered to elevate the cellular level of CFLAR. In one embodiment, the population of cells of the invention includes only bone marrow cells or their progenitors that are engineered to elevate the cellular level of CFLAR. In one embodiment, the population of cells of the invention includes cells other than bone marrow cells or their progenitors. In one embodiment, the population of cells of the invention includes only bone marrow cells or their progenitors.

[0023] In a further aspect, the invention relates to a population of cells as described herein for use in therapy. In one embodiment, the therapy requires immunosuppression.

[0024] In a further aspect, the invention relates to a population of cells as described herein for treating a patient in need of immunosuppression. In one embodiment, the population of cells is autologous to the patient.

[0025] In a further aspect, the invention relates to a pharmaceutical composition comprising a population of cells as described herein. The pharmaceutical composition of the invention can include a pharma- ceutically acceptable carrier, and can optionally include one or more adjuvants, stabilizers, and the like.

[0026] In a further aspect, the invention relates to a pharmaceutical composition as described herein for use in a method of therapy. In one embodiment, the therapy requires immunosuppression.

[0027] In a further aspect, the invention relates to a pharmaceutical composition as described herein for treating a patient in need of immunosuppression, hi one embodiment, the population of cells is autologous to the patient.

[0028] In a further aspect, the invention relates to the use of a population of cells as described herein for the manufacture of a medicament for treatment. In one embodiment, the treatment requires immunosuppression.

[0029] In a further aspect, the invention relates to the use of a population of cells as described herein for the manufacture of a medicament for treating a patient in need of immunosuppression. In one embodiment, the population of cells is autologous to the patient.

[0030] In a further aspect, the invention relates to a method for treating a patient in need of immunosuppression comprising administering a population of cells as described herein, hi one embodiment, the population of cells is autologous to the patient.

[0031] In a further aspect, the invention relates to a method for treating a patient in need of immunosuppression comprising increasing cellular levels of CFLAR in bone marrow cells or progenitor cells thereof in the patient.

[0032] In one embodiment, increasing the cellular level of CFLAR increases the immunosuppressive activity of the cell.

[0033] In one embodiment, increasing the cellular level of CFLAR in bone marrow cells or their progenitors of the patient comprises administering a nucleic acid encoding CFLAR. In one embodiment, the nucleic acid encoding CFLAR transfects bone marrow cells or their progenitors. In one embodiment, said transfection is stable or transient transfection. In one embodiment, bone marrow cells or their progenitors are transfected using a viral vector as a carrier, such as a lentiviral vector. In one embodiment, said nucleic acid is RNA.

[0034] In one embodiment, the CFLAR L and / or CFLAR S It is.

[0035] In one embodiment, the bone marrow cells or their progenitor cells are monocytes. In one embodiment, the bone marrow cells or their progenitor cells are CD14+ monocytes.

[0036] In one embodiment, the bone marrow cells or progenitor cells thereof are stem cells. In one embodiment, the bone marrow cells or progenitor cells thereof are bone marrow CD34+ cells.

[0037] In a further aspect, the invention relates to a nucleic acid encoding CFLAR for use in therapy, hi one embodiment, the therapy requires immunosuppression.

[0038] In a further aspect, the invention relates to a nucleic acid encoding CFLAR for the treatment of a patient in need of immunosuppression.

[0039] In a further aspect, the invention relates to a pharmaceutical composition comprising a nucleic acid encoding CFLAR.

[0040] In a further aspect, the invention relates to a pharmaceutical composition as described herein for use in a method of therapy. In one embodiment, the therapy requires immunosuppression.

[0041] In a further aspect, the invention relates to a pharmaceutical composition as described herein for treating a patient in need of immunosuppression.

[0042] In a further aspect, the invention relates to the use of a nucleic acid encoding CFLAR for the manufacture of a medicament for treatment. In one embodiment, the treatment requires immunosuppression.

[0043] In a further aspect, the invention relates to the use of a nucleic acid encoding CFLAR for the manufacture of a medicament for treating a patient in need of immunosuppression.

[0044] In one embodiment of all aspects described herein, the therapy requiring immunosuppression is a transplant and the patient requiring immunosuppression is the recipient of the transplant. Thus, the invention also encompasses treatment of a transplant recipient to reduce the recipient's immune response to the transplant and / or to treat or prevent rejection of the transplant by the recipient. In different embodiments, the transplant is selected from the group consisting of tissues, organs, cells, nucleic acids, proteins, biocompatible lattices, and any combination thereof. In some aspects, the therapy described herein for inducing immunosuppression and tolerance is administered to the recipient prior to transplantation. In other aspects, the therapy described herein for inducing immunosuppression and tolerance is administered to the recipient simultaneously with transplantation. In yet another aspect, the therapy described herein for inducing immunosuppression and tolerance is administered to the recipient as part of the transplantation. In yet another aspect, the therapy described herein for inducing immunosuppression and tolerance is administered to the recipient after transplantation.

[0045] In one embodiment of all aspects described herein, the therapy requiring immunosuppression is the treatment or prevention of graft-versus-host disease, and the patient requiring immunosuppression is a patient who has graft-versus-host disease or is at risk of developing graft-versus-host disease.

[0046] In one embodiment of all aspects described herein, the therapy requiring immunosuppression is the treatment or prevention of an autoimmune disease, and the patient requiring immunosuppression is a patient who has an autoimmune disease or is at risk of developing an autoimmune disease.

[0047] In one aspect, the invention provides agents and compositions, such as the cells and pharmaceutical compositions described herein, for use in the methods of treatment described herein.

[0048] In one aspect, the present invention provides a viral vector comprising a nucleic acid encoding CFLAR.In one embodiment, the viral vector is a lentiviral vector.The viral vector is preferably useful for manipulating or transfecting bone marrow cells or their progenitor cells in vivo or in vitro.

[0049] Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief description of the drawings]

[0050] [Figure 1] The gene sequences of the CFLAR long (designated CFLARL) containing plasmid used to generate lentivirus encoding CFLARL (A) and the CFLAR short (designated CFLARS) containing plasmid used to generate lentivirus encoding CFLARS (B) are shown. [Diagram 2] FIG. 1 shows the protocol and results of an immunosuppression assay for infected human CD34+ cells that express CFLARL and CFLARS and inhibit activated T cells, compared to luciferase-expressing CD34+ cells used as a negative control (A), and the protocol and results of an immunosuppression assay for infected human CD14+ cells that express CFLARL and CFLARS and inhibit activated T cells, compared to luciferase-expressing CD14+ cells used as a negative control (B). [Diagram 3] Heatmap of up- and down-regulated genes mediated by CFLAR forced expression on CD14+ cells compared to luciferase-infected CD14+ cells. CD14+ cells were obtained from four different buffy coats of healthy donors (HD). [Figure 4-1] List of genes significantly upregulated by lentivirus-induced CFLAR expression on CD14+ cells compared to luciferase-infected CD14+ cells. [Figure 4-2]List of genes significantly upregulated by lentivirus-induced CFLAR expression on CD14+ cells compared to luciferase-infected CD14+ cells. [Figure 4-3] List of genes significantly upregulated by lentivirus-induced CFLAR expression on CD14+ cells compared to luciferase-infected CD14+ cells. [Figure 4-4] List of genes significantly upregulated by lentivirus-induced CFLAR expression on CD14+ cells compared to luciferase-infected CD14+ cells. [Figure 4-5] List of genes significantly upregulated by lentivirus-induced CFLAR expression on CD14+ cells compared to luciferase-infected CD14+ cells. [Diagram 5] Validation of several gene targets regulated by forced CFLAR expression in CD14+ cells: upregulation of CFLARS, CFLARL and IDO1 genes by Real Time PCR (A); production of IL-10 by ELISA assay (B); enhanced expression of surface markers CD273, CD274, CD80, CD163, CD44, CD38 and CD124 by flow cytometry (C). [Figure 6A-B] Shown is the protocol for treating GvHD-affected mice engrafted with human PBMCs (A) (the immunomodulatory approach was based on repeated administration of CFLAR-expressing monocytes) and the outcome of this treatment in extending the survival time of treated mice compared to mice treated with non-transduced or transduced with a vector encoding luciferase (used as controls) (B). [Figure 6C] Pathological assessment by histopathological score (top panel); and enumeration of tissue-infiltrating human lymphocytes (defined as hCD3+ cells) by immunohistochemistry (bottom panel). [Figure 7]Immune evaluation of GvHD-affected mice treated with human CD14+ cells expressing CFLAR or luciferase as reported in the protocol scheme (A): it shows that mice treated with CFLAR-expressing cells present a lower frequency of circulating hCD3+ T cells in the blood (B, panel i), spleen (B, panel ii) and bone marrow (B, iii); furthermore, GvHD-affected mice treated with CFLAR-expressing CD14+ cells have a lower frequency of IFN-γ releasing hCD8+ T cells compared to GvHD-affected mice treated with luciferase-expressing CD14+ cells after in vitro stimulation with PMA (50 ng / ml) + ionomycin (1 μg / ml); finally, mice treated by repeated injections of CFLAR-expressing CD14+ cells exhibited a higher amount of regulatory T lymphocytes (Tregs) compared to mice treated with luciferase-expressing CD14+ cells. [Figure 8] Shown are the results of immunosuppression assays of both bone marrow- and spleen-derived CD11B+ Ly6G- Ly6Chigh monocytes (Panel A) and CD11B+ Ly6G+ Ly6Clow granulocytes (Panel B) (black) isolated from Rosa26.vFLIP;LysMcre mice compared to CD11B+ Ly6G- Ly6Chigh cells (white) isolated from control mice Rosa26.vFLIP. [Figure 9] c-FLAR induces an immunosuppressive program independent of its anti-apoptotic function. a) In vitro viability assessment of CD14+ cells infected with luciferase (control) or c-FLAR expressing lentivirus (monocytes isolated from four different buffy coats). b) shows that only c-FLAR forced expression induced immunosuppressive function. Indeed, monocytes infected with BCL-2 or BCL expressing lentivirus did not exhibit immunosuppressive capacity. All these data demonstrated that cFLAR drives an immunosuppressive function independent of apoptotic protection. [Figure 10a]Enforced c-FLAR expression in monocytes drives an MDSC-associated molecular program. a) Enrichment of GO BP terms of 486 unique upregulated genes in c-FLAR overexpressing samples. Enrichment was determined using a GO over-representation test implemented in the Cluster Profiler and the GO Biological Process category. p-values ​​were corrected using the BH procedure. [Figure 10b] Enforced c-FLAR expression in monocytes drives MDSC-associated molecular programs. b) Signaling pathway enrichment for upregulated genes in c-FLAR overexpressing samples (Figures 3, 4a and 4b). Differentially expressed genes were identified using SAM. By setting the q-value to <1%, 486 unique genes were upregulated in c-FLAR overexpressing samples (c-FLAR signature). Enrichment was determined using Fisher's test on all 267 signaling pathways in GSEA. p-values ​​were corrected using the BH procedure. FDR q-val, false discovery rate q-value. [Figure 11]c-FLAR activates the canonical NF-κB pathway in bone marrow cells. a) THP1 cells transfected with c-FLAR RNA acquired immunosuppressive function compared to controls (THP1 cells transfected with GFP-encoding RNA). Suppressive activity was measured by enumeration of absolute numbers of CD3+ T cells collected after co-culture with transfected THP1 cells. Data are presented as mean ± sem of three independent experiments. b) Immunofluorescence confocal microscopy of nuclear translocation of p65, p50 and p52 in transfected THP1 cells. Nuclei were counterstained with DAPI and slides were visualized using a confocal microscope. Original images ×800 for all panels. c) v-FLIP-expressing Ly6C+ cells were purified and transfected with scrambled, IKKα and IKKβ siRNA for at least 18 h. Bone marrow cells were then incubated with CellTrace-labeled OT-I cells and co-cultured for three days in the presence of SIINFEKL peptide. Suppressive activity was measured by enumeration of absolute numbers of CD8+ T cells harvested after coculture. Data are presented as the mean±sem of four independent experiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] The present invention is described in detail below, but it should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, and may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only, and are not intended to limit the scope of the present invention, which is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0052] The elements of the present invention are described below. Although the elements are enumerated using specific embodiments, it is understood that they can be combined in any manner and in any number to produce further embodiments. The various described examples and preferred embodiments do not limit the invention to only the embodiments explicitly described. Such descriptions are understood to support and encompass embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described herein should be considered disclosed by the description herein, unless the content is inconsistent.

[0053] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel, and H. Kolbl, Eds., (1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0054] The practice of the present invention employs, unless otherwise indicated, conventional methods of biochemistry, cell biology, immunology, and recombinant DNA technology described in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0055] Throughout this specification and the claims, unless the content is inconsistent, the word "comprise" and its grammatical variations mean the inclusion of a stated member, integer or step, or group of members, integers or steps, but not the exclusion of any other member, integer or step, or group of members, integers or steps. However, in some embodiments, such other member, integer or step, or group of members, integers or steps may be excluded, i.e., the subject matter consists of including a previously described member, integer or step, or group of members, integers or steps. As used in the context of describing the invention (particularly in the claims), "a," "the," and similar referents are intended to mean both the singular and the plural, unless otherwise indicated in the description herein or clearly contradicted by context.

[0056] The recitation of ranges of values ​​herein merely serves as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise indicated herein, each separate value is incorporated herein as if it were individually set forth herein.

[0057] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples or exemplary language (e.g., "etc.") contained herein are intended merely to better illustrate the invention and do not limit the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0058] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing in this specification should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0059] The term "recombinant" in the context of the present invention means "produced by genetic engineering." Preferably, a "recombinant" such as a recombinant cell or nucleic acid, in the context of the present invention, is non-naturally occurring.

[0060] As used herein, the term "naturally occurring" means that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), that can be isolated from a natural source, and that has not been intentionally modified by man in the laboratory is naturally occurring.

[0061] As used herein, an "isolated" cell is a cell that is essentially free of other cell types. An isolated cell also refers to a cell that is separated from other cell types that it is normally associated with in its naturally occurring state. In some cases, a population of isolated cells refers to a homogenous population of cells. In other cases, the term simply refers to a cell that is separated from the cells that it is naturally associated with in its natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro. In one embodiment, the cell population described herein comprises more than 50% of the desired cells in the cell population, preferably more than 75% of the desired cells in the cell population, more preferably more than 85% or 90% of the desired cells in the cell population, and most preferably more than 95% of the desired cells in the cell population.

[0062] As used herein, a "purified" cell is one that is essentially free of undesirable components, such as cellular plasma components and / or contaminating proteins from the tissue source from which the cell is obtained. Purified cells also refer to cells that have been separated from undesirable components.

[0063] In one embodiment of the present invention, a population of cells containing bone marrow cells or their precursor cells refers to a population of cells that is substantially free of cells other than bone marrow cells or their precursor cells and / or undesirable components.

[0064] As used herein, the term "separated from" or "separate" refers to the characteristic that a population of a first material is removed from the vicinity of a population of a second material, where the population of the first material is not necessarily devoid of the second material, and the population of the second material is not necessarily devoid of the first material. However, a population of a first material that is "separated from" a population of a second material has a measurably lower content of the second material when compared to a mixture of the first and second materials that have not been separated.

[0065] Hematopoietic blood cells are derived from pluripotent bone marrow stem cells by proliferation and differentiation. Stem cells are characterized by their repertoire of cell surface receptors (e.g., CD34+) and are undifferentiated. Through repeated self-renewal and expansion by proliferation, followed by differentiation in response to cytokines and growth factors, these cells give rise to all of the mature functional blood cells (monocytes / macrophages, eosinophils, neutrophils, T lymphocytes, and B lymphocytes).

[0066] In blood cell differentiation, two lineage pathways are evident: myeloid (including monocytes / macrophages, eosinophils, and neutrophils) and lymphoid (including T and B lymphocytes). In general, myeloid cells have functional roles associated with rapid killing and elimination of pathogens (e.g. bacteria and parasites), whereas lymphoid cells are associated with pathogen memory and generation of antibody-mediated immune responses. Myeloid cells perform more transient functions and are therefore generated in large numbers, live for days / weeks rather than months or years, and are fully and terminally differentiated, functionally specialized cells that do not divide and remain in the GO / GI phase of the cell cycle. After performing their functions, myeloid cells die by apoptosis and are removed by phagocytosis. In contrast, and due to their specialized memory roles, T and B lymphocytes can survive longer and are capable of extensive proliferation and cell cycling.

[0067] Thus, the term "myeloid cells" refers to cells of myeloid origin and encompasses terminally differentiated, non-dividing (i.e., non-proliferating) cells derived from the myeloid lineage, including neutrophils or polymorphonuclear neutrophils (PMNs), eosinophils, and mononuclear phagocytes. The latter cells are known as monocytes when in the blood and as macrophages when migrated into tissues. Terminal differentiation is the normal end point of cell differentiation and is not usually reversible.

[0068] In a particularly preferred embodiment, the term "bone marrow cells" refers to monocytes. The term "monocytes" as used herein refers to white blood cells circulating in the bloodstream. Monocytes are found in circulating peripheral blood, where they account for 10-20% of the total mononuclear cell population. Monocytes are characterized by their CD14 phenotypic expression, and are therefore commonly referred to as CD14+ monocytes. They play an important role in host defense as circulating monocytes, and can differentiate into tissue macrophages and dendritic cells with strong antigen-presenting capacity. Monocytes differentiate into macrophages when they migrate into tissues. Thus, the term "monocytes" as used in the present invention also refers to macrophages.

[0069] The term "buffy coat" refers to the fraction of an anticoagulant-treated blood sample that contains most of the white blood cells and platelets following density gradient centrifugation of the blood.

[0070] Bone marrow progenitor cells include hematopoietic cells such as hematopoietic stem cells and bone marrow progenitor cells. In particular, bone marrow progenitor cells include cells expressing CD34+ (CD34+ cells). Cells expressing CD34 are usually found in the umbilical cord and bone marrow as hematopoietic cells. Bone marrow CD34+ cells include hematopoietic stem and progenitor cells and are known to differentiate into all of the various blood cell types. CD34+ cells can be isolated from blood samples using immunomagnetic or immunofluorescence techniques.

[0071] The terms "reduce", "inhibit" or "decrease" refer to the ability to cause an overall decrease in the level, preferably by 5% or more, 10% or more, 20% or more, more preferably by 50% or more, and most preferably by 75% or more. These terms include complete or essentially complete inhibition, i.e., reduction to zero or essentially zero.

[0072] The terms "elevation", "enhancement" or "promotion" refer to the ability to cause an overall increase in the level, preferably by 5% or more, 10% or more, 20% or more, 50% or more, 75% or more, 100% or more, 200% or more, or 500% or more. Such terms may relate to an increase, enhancement, or promotion from zero or an unmeasurable or undetectable level to above zero or a measurable or detectable level. Alternatively, such terms may mean that a particular level was present prior to the increase, enhancement, or promotion, and that the same level is increased following the increase, enhancement, or promotion.

[0073] According to the present invention, the expression "increasing the cellular level" in relation to a particular substance such as CFLAR relates to a measure that results in a higher degree or amount of the substance compared to normal circumstances, particularly in a cell, where the cellular levels are not artificially elevated / elevated.

[0074] The term "immune response" refers to the integrated body reaction to an antigen, and preferably refers to a cellular immune response, a humoral immune response, or both. According to the present invention, the phrase "immune response to" or "immune response against" in reference to an agent such as an antigen, cell or tissue, refers to an immune response, such as a cellular response, to the agent.

[0075] The terms "cellular immune response", "cellular response", "cell-mediated immunity" or similar terms are intended to include a cellular response to cells characterized by expression of an antigen and / or presentation of the antigen by class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes, which act as "helpers" or "killers". Helper T cells (CD4 + T cells (also called CD8 T cells) play a central role by regulating the immune response and act as killer cells (cytotoxic T cells, cytolytic T cells, CD8 + T cells or CTLs, kill cells such as reduced cells.

[0076] The term "humoral immune response" refers to the process in living organisms whereby antibodies are generated in response to agents and organisms that ultimately neutralize and / or eliminate them. The specificity of the antibody response is mediated by T and / or B cells through membrane-bound receptors that bind to a single specific antigen. Following binding of the appropriate antigen and receipt of various other activation signals, B lymphocytes divide, thereby generating clones of memory B cells and antibody-secreting plasma cells, each of which produces antibodies that recognize the same antigenic epitope as that recognized by its antigen receptor. Memory B lymphocytes remain dormant until subsequently activated by their specific antigen. These lymphocytes provide the cell-based memory and the resulting escalation of the antibody response upon re-exposure to the specific antigen.

[0077] The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to an epitope on an antigen. In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination of the above. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable and constant domains, respectively. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more highly conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are called HCDR1, HCDR2, and HCDR3, and the CDRs of VL are called LCDR1, LCDR2, and LCDR3. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody contains a heavy chain constant region (CH) and a light chain constant region (CL), and the CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged from amino terminus to carboxy terminus in the order CH1, CH2, CH3). The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. An antibody can be an intact immunoglobulin from a natural source or from a recombinant source, or an immunologically active portion of an intact immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. Antibodies can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single chain antibodies and humanized antibodies.

[0078] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, preferably antigen receptors, such as antibodies or B cell receptors (BCR). Immunoglobulins are characterized by structural domains with a characteristic immunoglobulin (Ig) fold, i.e. immunoglobulin domains. The term encompasses membrane-bound and soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface or membrane immunoglobulins, which are usually part of the BCR. Soluble immunoglobulins are generally called antibodies. Immunoglobulins usually comprise multiple chains, typically two identical heavy chains and two identical light chains linked via disulfide bonds. These chains are composed of immunoglobulin domains, e.g. V L (Variable light chain) domain, C L (constant light chain) domain, V H (variable heavy chain) domain, and C H (constant heavy chain) domains CH1, CH2, CH3, and CH4. There are five types of mammalian immunoglobulin heavy chains, namely α, δ, ε, γ, and μ, which account for the different classes of antibodies, namely IgA, IgD, IgE, IgG, and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxy termini. In mammals, there are two types of light chains, namely lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved among the different isotypes of immunoglobulins, while the variable region is highly diverse and accounts for antigen recognition.

[0079] An "antigen" according to the present invention covers any substance that will elicit an immune response and / or any substance that is the subject of an immune mechanism, such as an immune or cellular response. This also includes the situation where an antigen is processed into antigenic peptides, and the immune response or immune mechanism is directed to one or more antigenic peptides, especially when presented in the context of MHC molecules. In particular, "antigen" relates to any substance, preferably a peptide or protein, that reacts specifically with T lymphocytes (T cells). According to the present invention, the term "antigen" includes any molecule that comprises at least one epitope, such as an epitope of a T cell. Preferably, an antigen in the context of the present invention is a molecule that induces an immune response, optionally after processing, which is preferably specific against the antigen (including cells expressing the antigen). In an embodiment of the present invention, the antigen is preferably presented by cells in the context of MHC molecules, whereby an immune response against the antigen occurs.

[0080] The term "autoantigen" as used herein refers to an antigen that is native to a mammal and that may be involved in the pathology of an autoimmune disease.

[0081] The term "immunogenicity" relates to the relative effectiveness of an antigen to induce an immune response.

[0082] The term "immunostimulatory" is used herein to refer to an increase in the overall immune response.

[0083] The term "immunosuppression" is used herein to refer to the reduction of the overall immune response. In some cases, it is desirable to induce an antigen-specific immunosuppressive effect.

[0084] "Immune tolerance," "immunological tolerance," or simply "tolerance" describes a state of unresponsiveness of the immune system to substances or tissues capable of eliciting an immune response. This is in contrast to conventional immune-mediated elimination of foreign antigens. Although the mechanisms by which tolerance is established are different, the resulting effect is similar.

[0085] An "antigenic peptide" according to the present invention preferably relates to a part or fragment of an antigen capable of stimulating an immune response, preferably a cellular response, against an antigen or a cell characterized by antigen expression, preferably by antigen presentation. Preferably, the antigenic peptide is capable of stimulating a cellular response against a cell characterized by antigen presentation with class I MHC, preferably stimulating antigen-responsive cytotoxic T lymphocytes (CTLs). Preferably, the antigenic peptide is an MHC class I and / or class II presented peptide. Preferably, the antigenic peptide comprises an amino acid sequence that substantially corresponds to the amino acid sequence of a fragment of the antigen. Preferably, said fragment of the antigen is an MHC class I and / or class II presented peptide.

[0086] Peptides suitable for binding to MHC molecules, in particular class I MHC molecules, are preferably 7 to 20 amino acids in length, more preferably 7 to 12 amino acids in length, more preferably 8 to 11 amino acids in length, in particular 9 or 10 amino acids in length.

[0087] In one embodiment, the antigenic peptides are recognized by T cell receptors when presented in the context of MHC, such as the MHC of an antigen-presenting cell. When the antigenic peptides are recognized by the T cell receptor, they can, in the presence of an appropriate costimulatory signal, induce the expansion of a clone of T cells bearing a T cell receptor that specifically recognizes the antigenic peptide. Preferably, the antigenic peptides, especially when presented in the context of an MHC molecule, are capable of stimulating an immune response, preferably a cellular response, against the antigen from which they were derived, or against cells characterized by the expression of the antigen, preferably cells characterized by the presentation of the antigen.

[0088] The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, i.e. a part or fragment of a molecule that is recognized by the immune system, e.g. a part or fragment of a molecule that is recognized by T cells, especially when presented in the context of an MHC molecule. An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein and is preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, most preferably 10 to 25 amino acids long, e.g. an epitope is preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids long. In the context of the present invention, it is particularly preferred that the epitope is a T cell epitope.

[0089] The terms "epitope", "T cell epitope", "fragment of an antigen", "immunogenic peptide" and "antigenic peptide" are used interchangeably herein and relate to an incomplete representation of an antigen, preferably capable of eliciting an immune response against the antigen or a cell expressing or containing, preferably presenting, the antigen. Preferably, the term relates to an immunogenic part of an antigen. Preferably, it is the part of the antigen that is recognised (i.e. specifically bound) by the T cell receptor, especially when presented in the context of an MHC molecule. Certain preferred immunogenic parts bind to MHC class I or class II molecules.

[0090] The term "target" is intended to mean a cell or tissue that is the target of an immune response, such as a cellular immune response, for example an agent. Targets include cells that present an antigen or an antigen epitope, i.e. a peptide fragment derived from an antigen. In one embodiment, the target cell is a cell that expresses an antigen, preferably presenting said antigen with class I MHC.

[0091] The term "portion" refers to a fraction. With respect to a particular structure, such as an amino acid sequence or a protein, the term "portion" may refer to a contiguous or discontinuous fraction of said structure.

[0092] The terms "portion" and "fragment" are used interchangeably herein and refer to a contiguous element. For example, a portion of a structure, such as an amino acid sequence or a protein, refers to a contiguous element of said structure.

[0093] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of the antigen (e.g., degradation of a protein into peptides), and one or more of these fragments associate with (e.g., via binding to) an MHC molecule and are presented by a cell, preferably an antigen-presenting cell, to a specific T cell.

[0094] Antigen-presenting cells (APCs) are cells that display antigens in the context of the major histocompatibility complex (MHC) on their surface. T cells can recognize this system using their T cell receptor (TCR). Antigen-presenting cells process antigens and present them to T cells. Antigen-presenting cells include, but are not limited to, monocytes / macrophages, B cells, and dendritic cells (DCs).

[0095] Nonprofessional antigen-presenting cells do not constitutively express the MHC class II proteins required for interaction with naive T cells; these are expressed only upon stimulation of nonprofessional antigen-presenting cells with specific cytokines such as IFNγ.

[0096] Professional antigen-presenting cells are highly efficient at internalizing antigens by phagocytosis or by receptor-mediated endocytosis and then displaying fragments of antigens bound to class II MHC molecules on their membrane. T cells recognize and interact with antigen-class II MHC molecule complexes on the membrane of antigen-presenting cells. Further costimulatory signals are then generated by the antigen-presenting cells, leading to T cell activation. The expression of costimulatory molecules is a defining feature of professional antigen-presenting cells.

[0097] The main types of professional antigen-presenting cells are dendritic cells, which have the widest range of antigen presentation and are perhaps the most important antigen-presenting cells, macrophages, B cells, and certain activated epithelial cells.

[0098] Dendritic cells (DCs) are a population of leukocytes that present antigens captured in peripheral tissues to T cells via both the MHC class II and I antigen presentation pathways. Dendritic cells are well known to be potent inducers of immune responses, and activation of these cells is a critical step in the induction of immunity.

[0099] Dendritic cells are conveniently classified into "immature" and "mature" cells, which can be used as a simple method to distinguish between two well-characterized phenotypes, however, such classification should not be construed to exclude all possible intermediate stages of differentiation.

[0100] Immature dendritic cells are characterized as antigen-presenting cells with high antigen uptake and processing capacity, which correlates with high expression of Fcγ receptors and mannose receptors. The mature phenotype is typically characterized by lower expression of these markers and, conversely, high expression of cell surface molecules responsible for T cell activation, such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., CD40, CD80, CD86, and 4-1 BB).

[0101] Maturation of dendritic cells is considered a state of dendritic cell activation in which such antigen-presenting dendritic cells result in T cell priming, whereas presentation by immature dendritic cells results in tolerance. Dendritic cell maturation is primarily triggered by biomolecules with microbial characteristics detected by innate receptors (bacterial DNA, viral RNA, endotoxins, etc.), proinflammatory cytokines (TNF, IL-1, IFN), ligation of CD40 on the dendritic cell surface by CD40L, and substances released by cells undergoing stressful cell death. Dendritic cells can be obtained by culturing bone marrow cells in vitro with cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha.

[0102] The term "immunoreactive cells" or "effector cells", in the context of the present invention, relates to cells which exert an effector function during an immune response. "Immunoreactive cells" are preferably capable of binding to cells characterized by the expression and / or presentation of an antigen or an antigenic peptide derived from an antigen, and are capable of mediating an immune response. For example, such cells secrete cytokines and / or chemokines, kill microorganisms, secrete antibodies, recognize infected or cancerous cells, and optionally eliminate such cells. For example, immunoreactive cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present invention, "immunoreactive cells" are T cells, preferably CD4 + and / or CD8 + T cells.

[0103] Preferably, an "immunoreactive cell" recognizes an antigen or an antigenic peptide derived from an antigen with a certain degree of specificity, especially when presented in the context of an MHC molecule, such as on the surface of an antigen-presenting cell, or on a diseased cell, such as a tumor cell. Preferably, said recognition is capable of rendering the cell, which recognizes the antigen or an antigenic peptide derived from said antigen, responsive or reactive. The cell may be a helper T cell (CD4 + In the case of CD4+ T cells, such responsiveness or reactivity may include release of cytokines and / or CD8 + The activation of lymphocytes (CTL) and / or B cells may be included. If the cell is a CTL, such responsiveness or reactivity may include elimination, e.g., via apoptosis or perforin-mediated cytolysis, of cells presented in the context of MHC class I molecules, i.e., cells characterized by presentation of antigen with class I MHC. According to the present invention, CTL responsiveness may include sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of antigen-expressing target cells. CTL responsiveness may also be determined using artificial reporters that pinpoint CTL responsiveness. Such CTLs that recognize and are responsive or reactive to an antigen or antigenic peptides derived from an antigen are also referred to herein as "antigen-responsive CTLs." If the cell is a B cell, such responsiveness may include release of immunoglobulins.

[0104] The term "T cell" or "T lymphocyte" refers to cells derived from the thymus that participate in a variety of cell-mediated immune responses, including cytotoxic T cells (CTL, CD8 + T cells) and T helper cells (CD4 + T cells).

[0105] T cells belong to a group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells, by the presence of specific receptors on their cell surface called T cell receptors (TCRs). The thymus is the main organ responsible for the maturation of T cells. Several different subsets of T cells have been discovered, each with different functions.

[0106] T helper cells assist other white blood cells in immunological processes, including, among other functions, maturation of B cells into plasma cells and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 protein on their surface. Helper T cells become activated when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Upon activation, helper T cells divide rapidly and secrete small proteins called cytokines that regulate or support an active immune response.

[0107] Cytotoxic T cells destroy virally infected and tumor cells and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of almost every cell in the body.

[0108] Most T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor is generated from independent T cell receptor alpha and beta (TCRα and TCRβ) genes and contains two distinct peptide chains called α- and β-TCR chains. γδ T cells (gamma delta T cells) represent a small subset of T cells that have a different T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR is made up of one γ chain and one δ chain. This group of T cells is much less abundant than αβ T cells (2% of all T cells).

[0109] The structure of the T cell receptor is very similar to the immunoglobulin Fab fragment, the region defined as the combination of light and heavy chains of an antibody arm. Each chain of the TCR is a member of the immunoglobulin superfamily and has one N-terminal immunoglobulin (Ig)-variable (V) domain, one Ig constant (C) domain, a transmembrane / cell-spanning region, and a short cytoplasmic tail at the C-terminus. The variable domains of both the TCR α and β chains have three hypervariable or complementarity determining regions (CDRs), with the variable region of the β chain having an additional area of ​​hypervariability (HV4) that does not normally contact antigen and is therefore not considered a CDR. CDR3 is the main CDR responsible for recognition of processed antigens, although CDR1 of the α chain has also been shown to interact with the N-terminal part of antigenic peptides, and CDR1 of the β chain interacts with the C-terminal part of the peptide. CDR2 is thought to recognize MHC. CDR4 of the β chain is not thought to participate in antigen recognition, but has been shown to interact with superantigens. The constant domain of the TCR domain consists of a short connecting sequence in which cysteine ​​residues form disulfide bonds, forming the link between the two chains.

[0110] The term "peripheral blood mononuclear cells" or "PBMC" refers to peripheral blood cells with spherical nuclei. These cells consist of lymphocytes (T cells, B cells, NK cells) and monocytes, while red blood cells and platelets do not have nuclei and granulocytes (neutrophils, basophils, and eosinophils) have segmented nuclei. These cells can be extracted from whole blood using Ficoll and gradient centrifugation to separate the blood into a top layer of plasma, followed by a layer of PBMCs, and a bottom layer of polymorphonuclear cell fractions (e.g., neutrophils and eosinophils) and red blood cells.

[0111] The term "major histocompatibility system" and its abbreviation "MHC" refer to a complex of genes that includes MHC class I and MHC class II molecules and occurs in all vertebrates. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells in an immune response; MHC proteins or molecules bind peptides and present them for recognition by T cell receptors. Proteins encoded by MHC are expressed on the surface of cells and display both self antigens (peptide fragments derived from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms) to T cells.

[0112] The MHC region is divided into three subgroups: class I, class II, and class III. MHC class I proteins contain an α chain and β2 microglobulin (not part of the MHC encoded by chromosome 15). This protein presents antigen fragments to cytotoxic T cells. In most immune system cells, especially antigen-presenting cells, MHC class II proteins contain α and β chains, which present antigen fragments to T helper cells. The MHC class III region encodes other immune components, such as those encoding complement components and cytokines.

[0113] In humans, the genes within the MHC region that code for antigen-presenting proteins on the cell surface are called human leukocyte antigen (HLA) genes. However, the abbreviation MHC is often used to refer to the HLA gene products. The HLA genes include nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1.

[0114] In one preferred embodiment of all aspects of the invention, the MHC molecule is an HLA molecule.

[0115] The term "immune effector function" or "effector function" in the context of the present invention includes any function mediated by a component of the immune system, e.g., resulting in the killing of a cell. Preferably, an immune effector function in the context of the present invention is a T cell-mediated effector function. Such a function can be mediated by helper T cells (CD4 + In the case of T cells, recognition of an antigen or an antigenic peptide derived from an antigen in the context of an MHC class II molecule by the T cell receptor, release of cytokines and / or CD8 + Activation of lymphocytes (CTLs) and / or B cells includes, in the case of CTLs, recognition of antigens in the context of MHC class I molecules or antigenic peptides derived from antigens by the T cell receptor, elimination of cells presented in the context of MHC class I molecules, i.e., cells characterized by presenting antigen with class I MHC, e.g. via apoptosis or perforin-mediated cytolysis, production of cytokines such as IFN-γ and TNF-α, and specific cytolytic killing of antigen-expressing target cells.

[0116] A "nucleic acid" according to the invention is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferably RNA, most preferably in vitro transcribed RNA (IVT RNA) or synthetic RNA. A nucleic acid according to the invention includes genomic DNA, cDNA, mRNA, recombinantly produced molecules and chemically synthesized molecules. According to the invention, a nucleic acid can be present as a single-stranded or double-stranded, linear or covalently closed circular molecule. A nucleic acid can be isolated according to the invention. The term "isolated nucleic acid" means according to the invention that a nucleic acid has been (i) amplified in vitro, for example via polymerase chain reaction (PCR), (ii) produced by recombination by cloning, (iii) produced by excision and separation, for example by gel electrophoresis, or (iv) synthesized, for example by chemical synthesis. The nucleic acid can be used for introduction into a cell, i.e. for transfection of a cell, for example in the form of RNA, which can be prepared by in vitro transcription from a DNA template. The RNA can be further modified before application by stabilizing sequences, capping and polyadenylation.

[0117] The nucleic acids described herein may be included in vectors that can be used to deliver the nucleic acid to the interior of a cell. As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors, such as adenoviral or baculoviral vectors, retroviral or lentiviral vectors, transposon or artificial chromosome vectors, such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Such vectors include expression vectors as well as cloning vectors. Expression vectors include plasmids and viral vectors, and generally contain a desired coding sequence and appropriate DNA sequences required for expression of an operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are usually used to manipulate and amplify a particular desired DNA fragment and may lack functional sequences required for expression of the desired DNA fragment.

[0118] The tolerogenic or immunosuppressive cells described herein can be generated in vitro or in vivo by transfecting or transducing cells with a vector that results in increased expression of CFLAR. Cells can be transfected or transduced using any of a variety of methods well known to those of skill in the art.

[0119] Nucleic acid encoding CFLAR can be cloned into several kinds of vectors.However, the present invention is not limited to any particular vector.Rather, the present invention encompasses a wide variety of vectors that are readily available and well known in the art.In certain embodiments, the vector is selected from the group consisting of viral vectors, bacterial vectors, and mammalian cell vectors.Many such systems are widely available commercially.

[0120] The vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Preferably, the virus is a helper-dependent adenovirus (HD-Ad). In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.

[0121] Those skilled in the art of molecular biology will generally know how to use promoter, enhancer, and cell type combinations for protein expression. The promoters used can be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high levels of expression of the introduced nucleic acid segment encoding CFLAR. The promoters can be heterologous or endogenous. Constitutive promoter sequences used by the present invention include, but are not limited to, immediate early cytomegalovirus (CMV) promoter sequences, Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney virus promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and muscle creatine promoter. Furthermore, the present invention is not limited to the use of constitutive promoters. Inducible promoters are also considered part of the present invention. The use of an inducible promoter in the present invention provides a molecular switch that can turn on the expression of the polynucleotide sequence to which it is operably linked when such expression is desired, or turn off expression when such expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. Additionally, the present invention includes the use of tissue-specific promoters, which are active only in the desired tissue. Tissue-specific promoters are well known in the art and include, but are not limited to, the HER-2 promoter and PSA-related promoter sequences.

[0122] To assess the expression of CFLAR, the expression vector introduced into the cells can also contain a selection marker gene or a reporter gene, or both, which facilitates the identification and selection of expressing cells from a population of cells to be infected or transfected through a viral vector. In other embodiments, the selection marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selection marker and the reporter gene can be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selection markers are known in the art, including, for example, antibiotic resistance genes, such as neo. Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Reporter genes that code for proteins that are easy to assay are well known in the art. In general, reporter genes are genes that code for proteins that are not present or expressed by the recipient organism or tissue, and whose expression is manifested by some easily detectable property, such as enzymatic activity. The expression of the reporter gene is assayed at a suitable time after the nucleic acid is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene.

[0123] The vector can be easily introduced into the host cell by any method in the art. For example, an expression vector can be transferred into the host cell by physical, chemical or biological means. Physical methods for introducing nucleic acid into the host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0124] Biological methods for introducing nucleic acid of interest into host cells include the use of DNA and RNA vectors.Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells.Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, etc.

[0125] Chemical means for introducing nucleic acid into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system for use as a delivery vehicle in vitro and in vivo is liposome (i.e., artificial membrane vesicles). The preparation and use of such systems are well known in the art.

[0126] Regardless of the method used to introduce exogenous nucleic acid into a host cell or to otherwise increase cellular levels of CFLAR in the cell, various assays can be performed to determine the presence and / or amount of CFLAR or its encoding nucleic acid in a host cell. Such assays include, for example, Southern and Northern blotting, RT-PCR and PCR, and assays to detect the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot).

[0127] In the context of the present invention, the term "DNA" refers to a molecule that comprises deoxyribonucleotide residues, preferably composed entirely or substantially of deoxyribonucleotide residues. "Deoxyribonucleotide" refers to a nucleotide that lacks a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. The term "DNA" includes isolated DNA, e.g. partially or fully purified DNA, essentially pure DNA, synthetic DNA, and recombinantly produced DNA, including modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and / or modification of one or more nucleotides. Such modifications may include the addition of non-nucleotide material to the end(s) of the DNA or internally, e.g., to one or more nucleotides of the DNA. Nucleotides in a DNA molecule may also include non-standard nucleotides, e.g., non-natural nucleotides or chemically synthesized nucleotides. These modified DNAs may be referred to as analogs or analogs of naturally occurring DNA.

[0128] In the context of the present invention, the term "RNA" refers to a molecule that comprises ribonucleotide residues, preferably composed entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA, e.g. partially or fully purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or modification of one or more nucleotides. Such modifications may include the addition of non-nucleotide material to the end(s) of the RNA or internally, e.g. at one or more nucleotides of the RNA. Nucleotides in an RNA molecule may also include non-standard nucleotides, e.g. non-natural nucleotides or chemically synthesized nucleotides or deoxyribonucleotides. These modified RNAs may be referred to as analogs or analogs of naturally occurring RNA. According to the present invention, the term "RNA" includes, and preferably relates to, "mRNA", which means "messenger RNA", and relates to transcripts that may be generated using DNA as a template and that code for peptides or proteins. An mRNA typically comprises a 5' untranslated region (5'-UTR), a protein or peptide coding region and a 3' untranslated region (3'-UTR). An mRNA has a limited half-life in cells and in vitro. Preferably, an mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the present invention, an RNA is obtained by in vitro transcription or chemical synthesis. In vitro transcription methods are known to those skilled in the art. For example, various in vitro transcription kits are commercially available.

[0129] According to the present invention, the stability and translation efficiency of RNA can be modified as necessary.For example, the RNA can be stabilized and its translation can be increased by one or more modifications that have a stabilizing effect and / or increase the translation efficiency of RNA.To increase the expression of the RNA used according to the present invention, within the coding region, i.e. within the sequence that codes for the peptide or protein to be expressed, the RNA can be modified to increase the GC content, to increase the stability of the mRNA, to perform codon optimization, and thus to enhance translation in cells, preferably without changing the sequence of the peptide or protein to be expressed.

[0130] As used in the present invention, the term "modification" in the context of RNA includes any modification of RNA that does not naturally occur in said RNA.

[0131] In one embodiment of the invention, the RNA used according to the invention does not have uncapped 5'-triphosphates. Removal of such uncapped 5'-triphosphates can be achieved by treating the RNA with a phosphatase.

[0132] The RNA according to the present invention can have modified ribonucleotides to increase its stability and / or decrease its cytotoxicity.For example, in one embodiment, cytidine is partially or completely, preferably completely, replaced by 5-methylcytidine in the RNA used according to the present invention.Alternatively or additionally, in one embodiment, uridine is partially or completely, preferably completely, replaced by pseudouridine in the RNA used according to the present invention.

[0133] In one embodiment, the term "modification" relates to providing an RNA with a 5'-cap or a 5'-cap analog. The term "5'-cap" refers to the cap structure found at the 5' end of an mRNA molecule, which generally consists of a guanosine nucleotide attached to the mRNA via an unusual 5' to 5' triphosphate bond. In one embodiment, such a guanosine is methylated at position 7. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, preferably a 7-methylguanosine cap (m 7 In the context of the present invention, the term "5'-cap" includes 5'-cap analogs that resemble RNA cap structures and are preferably modified to have the ability to stabilize RNA and / or enhance translation of RNA upon attachment in vivo and / or in a cell.

[0134] The RNA may contain further modifications, for example, the extension or truncation of the naturally occurring poly(A) tail, or the alteration of the 5'- or 3'-untranslated region (UTR), such as the introduction of a UTR not associated with the coding region of said RNA, for example the replacement of an existing 3'-UTR with one or more, preferably two copies of a 3'-UTR obtained from a globin gene, such as alpha2-globin, alpha1-globin, beta-globin, preferably beta-globin, more preferably human beta-globin, or the insertion of such copies.

[0135] RNA with an unmasked polyA sequence is translated more efficiently than RNA with a masked polyA sequence. The term "poly(A) tail" or "polyA sequence" refers to a sequence of adenyl (A) residues typically located at the 3' end of an RNA molecule, and "unmasked polyA sequence" means that the polyA sequence at the 3' end of an RNA molecule ends with the A of the polyA sequence and is not followed by any nucleotides other than the A located at the 3' end, i.e. downstream, of the polyA sequence. Furthermore, a long polyA sequence of about 120 base pairs provides optimal transcript stability and translation efficiency of RNA.

[0136] Thus, to increase / enhance the stability and / or expression of the RNA used according to the invention, the RNA can be modified to be present with a polyA sequence, preferably having a length of 10 to 500, more preferably 30 to 300, even more preferably 65 to 200, especially 100 to 150 adenosine residues. In a particularly preferred embodiment, the polyA sequence has a length of about 120 adenosine residues. To further increase / enhance the stability and / or expression of the RNA used according to the invention, the polyA sequence can be left unmasked.

[0137] The term "stability" of an RNA relates to the "half-life" of the RNA. "Half-life" relates to the period required to eliminate half of the activity, amount, or number of a molecule. In the context of the present invention, the half-life of an RNA indicates the stability of said RNA. The half-life of an RNA may affect the "duration of expression" of the RNA. It can be expected that an RNA with a long half-life will be expressed for a long period of time.

[0138] Needless to say, according to the present invention, if it is desired to decrease the stability and / or translation efficiency of the RNA, the RNA can be modified to interfere with the function of the above-mentioned elements that increase the stability and / or translation efficiency of the RNA.

[0139] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a nucleic acid to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have a defined sequence of nucleotides or a defined sequence of amino acids. Thus, a nucleic acid encodes a protein when expression (translation and optionally transcription) of the nucleic acid produces the protein in a cell or other biological system.

[0140] The term "expression" is used according to the present invention in its most general sense and includes the production of RNA and / or peptides or polypeptides, e.g. by transcription and / or translation. With respect to RNA, the terms "expression" or "translation" particularly relate to the production of peptides or polypeptides. This term also includes partial expression of a nucleic acid. Furthermore, expression can be transient or stable.

[0141] In the context of the present invention, the term "transcription" refers to the process whereby the genetic code in a DNA sequence is transcribed into RNA. The RNA can then be translated into a protein. According to the present invention, the term "transcription" includes "in vitro transcription", which refers to the process whereby RNA, in particular mRNA, is synthesized in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, a cloning vector is applied for the generation of the transcript. These cloning vectors are usually called transcription vectors and are included in the term "vector" according to the present invention. According to the present invention, the RNA used in the present invention is preferably in vitro transcribed RNA (IVT-RNA), obtained by in vitro transcription of a suitable DNA template. The promoter for controlling the transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are T7, T3 and SP6 RNA polymerase. Preferably, the in vitro transcription according to the present invention is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription is obtained by cloning a nucleic acid, in particular a cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA is obtained by reverse transcription of RNA.

[0142] The term "translation" according to the present invention relates to the process in the ribosomes of a cell in which a chain of messenger RNA directs the assembly of a sequence of amino acids to form a peptide or polypeptide.

[0143] According to the present invention, expression control sequences or regulatory sequences operably linked to a nucleic acid can be homologous or heterologous to the nucleic acid. A coding sequence and a regulatory sequence are "operably" linked together when they are covalently linked together such that transcription or translation of the coding sequence is under the control or influence of the regulatory sequence. If the coding sequence is to be translated into a functional protein, the functional linkage of the regulatory sequence with the coding sequence allows transcription of the coding sequence upon induction of the regulatory sequence without shifting the reading frame within the coding sequence or losing the ability of the coding sequence to be translated into the desired protein or peptide.

[0144] The term "expression control sequence" or "regulatory sequence" includes, according to the invention, promoters, ribosome binding sequences and other control elements that control the transcription of a nucleic acid or the translation of the resulting RNA. In certain embodiments of the invention, the regulatory sequence can be controlled. The exact structure of the regulatory sequence may vary depending on the species or cell type, but usually includes 5'-non-transcribed and 5'- and 3'-non-translated sequences involved in the initiation of transcription or translation, such as TATA-boxes, capping-sequences, CAAT-sequences, etc. In particular, 5'-non-transcribed regulatory sequences include promoter regions that include promoter sequences for transcriptional control of functionally linked genes. Regulatory sequences may also include enhancer sequences or upstream activator sequences.

[0145] According to the invention, expression of said peptides or proteins is preferably brought about by a nucleic acid, such as an RNA, encoding a peptide or protein that is taken up or introduced, i.e. transfected or transduced, into a cell, which may be present in vitro or in a subject. The cell may express these encoded peptides or proteins intracellularly (e.g. in the cytoplasm and / or nucleus), may secrete the encoded peptides or proteins, or may express them on its surface.

[0146] In accordance with the present invention, the terms "nucleic acid expression" and "nucleic acid encoding" or similar terms are used interchangeably herein and refer to a particular peptide or polypeptide, meaning that a nucleic acid, when present in an appropriate environment, preferably inside a cell, can be expressed to produce said peptide or polypeptide.

[0147] According to the present invention, any means can be used to engineer cells to increase the cellular level of CFLAR.In one embodiment, cells are engineered to increase the cellular level of CFLAR by transfecting with the nucleic acid encoding CFLAR polypeptide.

[0148] The terms "transfer", "introduce", "transfect" or "transduce" are used interchangeably herein and relate to the introduction of a nucleic acid, in particular an exogenous or heterologous nucleic acid, for example the introduction of RNA, into a cell. According to the invention, the cell can be in vitro or in vivo, for example the cell can form part of an organ, tissue and / or organism. According to the invention, transfection can be transient or stable. For some applications of transfection, it is sufficient that the transfected genetic material is expressed transiently. Since the nucleic acid introduced during the transfection process is not usually integrated into the nuclear genome, the exogenous nucleic acid will be diluted or degraded by mitosis. Cell lines that allow episomal amplification of the nucleic acid greatly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, stable transfection must occur. RNA can be transfected into the cell to transiently express its encoded protein.

[0149] The term "cellular FLICE [Fas-associated death domain (FADD)-like IL-1β-converting enzyme]-inhibitory protein," "cellular FLICE-inhibitory protein," "CFLAR," or "c-FLIP" is a key anti-apoptotic regulator and resistance factor that suppresses tumor necrosis factor-α (TNF-α), Fas-L, and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, as well as apoptosis triggered by chemotherapeutic agents in malignant cells. CFLAR is a long (CFLAR L ), short (CFLAR S ), and CFLAR R It is expressed in human cells as a splice variant. CFLAR binds to FADD and / or caspase-8 or -10 and TRAIL receptor 5 (DR5) in a ligand-dependent and ligand-independent manner to form the apoptosis inhibitory complex (AIC). Such interactions then prevent the formation of the death-inducing signaling complex (DISC) and subsequent activation of the caspase cascade. CFLAR L and CFLAR S is also known to have a multifunctional role in activating and / or upregulating various signaling pathways and multiple cytoprotective and pro-survival signaling proteins, including Akt, ERK, and NF-kB. According to the present invention, the term "CFLAR" includes any variants, particularly mutants, splice variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those that occur naturally, and is intended to include any variants, particularly mutants, splice variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those that occur naturally, and is intended to include any variants, particularly mutants, splice variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those that occur naturally, and is intended to include any variants, particularly mutants, splice variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those that occur naturally, and are ... L , CFLAR S , CFLARR and v-CFLAR.

[0150] The term "CFLAR L " preferably relates to a protein comprising an amino acid sequence according to SEQ ID NO: 1 of the sequence listing or a variant of said amino acid sequence. In one embodiment, the amino acid sequence according to SEQ ID NO: 1 of the sequence listing is encoded by a nucleic acid sequence according to SEQ ID NO: 2 of the sequence listing.

[0151] The term "CFLAR S" preferably relates to a protein comprising an amino acid sequence according to SEQ ID NO: 3 of the sequence listing or a variant of said amino acid sequence. In one embodiment, the amino acid sequence according to SEQ ID NO: 3 of the sequence listing is encoded by a nucleic acid sequence according to SEQ ID NO: 4 of the sequence listing.

[0152] The term "CFLAR R " preferably relates to a protein comprising an amino acid sequence according to SEQ ID NO: 5 of the sequence listing or a variant of said amino acid sequence. In one embodiment, the amino acid sequence according to SEQ ID NO: 5 of the sequence listing is encoded by a nucleic acid sequence according to SEQ ID NO: 6 of the sequence listing.

[0153] The term "v-CFLAR" preferably relates to a protein comprising an amino acid sequence according to SEQ ID NO: 7 of the sequence listing or a variant of said amino acid sequence. In one embodiment, the amino acid sequence according to SEQ ID NO: 7 of the sequence listing is encoded by a nucleic acid sequence according to SEQ ID NO: 8 of the sequence listing.

[0154] According to the present invention, the term "peptide" refers to a substance which comprises two or more, preferably three or more, preferably four or more, preferably six or more, preferably eight or more, preferably ten or more, preferably thirteen or more, preferably sixteen or more, preferably twenty-one or more, preferably up to 8, 10, 20, 30, 40 or 50, in particular 100, amino acids covalently joined by peptide bonds.

[0155] The term "protein" refers to large peptides, i.e., polypeptides, preferably peptides having more than 100 amino acid residues, although in general, the terms "peptide," "polypeptide," and "protein" are synonymous and are used interchangeably herein.

[0156] The present invention also includes "variants" of the peptides, proteins, or amino acid sequences described herein.

[0157] For the purposes of the present invention, "variants" of an amino acid sequence include amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants.

[0158] Amino acid insertion variants include the insertion of a single or two or more amino acids into a particular amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific sites in the amino acid sequence, although random insertions with appropriate screening of the resulting products are also possible.

[0159] Amino acid addition variants include amino and / or carboxy terminal fusions of one or more amino acids, for example 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.

[0160] Amino acid deletion mutants are characterized by the removal of one or more amino acids from the sequence, for example the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion may be in any part of the protein. Amino acid deletion mutants that contain deletions at the N-terminus and / or C-terminus of the protein are also called N-terminal and / or C-terminal truncation mutants.

[0161] Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another in its place. It is preferred that modifications are made at positions in the amino acid sequence that are not conserved between homologous proteins or peptides and / or that amino acids are replaced by others with similar properties. Preferably, the amino acid changes in the protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include the substitution of one of a family of amino acids related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.

[0162] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably provided over at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably contiguous amino acids. The degree of similarity or identity is preferably provided over a segment of at least 80, at least 100, at least 120, at least 150, at least 180, at least 200, or at least 250 amino acids. In preferred embodiments, the degree of similarity or identity is provided over the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using the best sequence alignment, e.g., using Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0163] "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences.

[0164] The term "percentage identity" is intended to express the percentage of identical amino acid residues between the two sequences compared, obtained after the best alignment, this percentage being purely statistical, the differences between the two sequences being distributed randomly and over their entire length. Sequence comparison between two amino acid sequences is generally performed by comparing these sequences after optimal alignment, said comparison being performed segment by segment or "window of comparison" in order to identify and compare local regions of sequence similarity. In addition to being generated manually, optimal alignments of sequences for comparison can be generated using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, using the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, using the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or using computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0165] The percentage identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions being compared, and multiplying the result by 100 to obtain the percentage identity between the two sequences.

[0166] Homologous amino acid sequences according to the invention exhibit an amino acid residue identity of at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98 or at least 99%.

[0167] The amino acid sequence variants described herein can be readily prepared by one of skill in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or proteins with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily prepared with the aid of known peptide synthesis techniques, for example, solid phase synthesis and similar methods.

[0168] The present invention includes derivatives of the peptides or proteins described herein, encompassed by the terms "peptide" and "protein". According to the present invention, "derivatives" of proteins and peptides are modified forms of proteins and peptides. Such modifications include any chemical modification, including single or multiple substitutions, deletions and / or additions of proteins or peptides, such as sugars (carbohydrates), lipids and / or any molecules associated with the protein or peptide. In one embodiment, a "derivative" of a protein or peptide includes modified analogs resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage or binding to an antibody or another cellular ligand. The term "derivative" also covers all functional chemical equivalents of said proteins and peptides. Preferably, the modified peptides have increased stability and / or increased immunogenicity.

[0169] According to the present invention, a variant or derivative of a peptide or protein preferably has the functional properties of the peptide or protein from which it is derived. Preferably, a variant or derivative of a CFLAR protein has the same or similar properties as the CFLAR protein from which it is derived.

[0170] The term "derived from" means according to the invention that a particular entity, in particular a particular sequence, is present in the object, in particular an organism or molecule, from which it originates. In the case of amino acid sequences, in particular a particular sequence region, "derived from" means in particular that the relevant amino acid sequence is derived from the amino acid sequence in which it is present.

[0171] The term "cell" or "host cell" is preferably an intact cell, i.e. a cell with an intact membrane that has not released its normal intracellular components such as enzymes, organelles, or genetic material. An intact cell is preferably a viable cell, i.e. a living cell capable of carrying out its normal metabolic functions. Preferably, the term relates to any cell that can be transformed or transfected with an exogenous nucleic acid according to the present invention. The term "cell" according to the present invention includes prokaryotic cells (e.g. E. coli) or eukaryotic cells (e.g. dendritic cells, B cells, CHO cells, COS cells, K562 cells, HEK293 cells, HELA cells, yeast cells, and insect cells). Exogenous nucleic acids can be found in the cell (i) intact and freely divergent, (ii) incorporated in a recombinant vector, or (iii) integrated into the genomic or mitochondrial DNA of the host cell. Mammalian cells, such as cells of human, mouse, hamster, pig, goat, and primate origin, are particularly preferred. Cells can be derived from a number of tissue types and include primary cells and cell lines.

[0172] Cells containing the nucleic acid, eg, cells transfected with the nucleic acid, preferably express the peptide or protein encoded by the nucleic acid.

[0173] The term "expansion" refers to the process of doubling of a particular entity. In one embodiment of the invention, the term is used in the context of an immunological response in which lymphocytes are stimulated by an antigen, proliferate, and the particular lymphocytes that recognize said antigen are amplified. Preferably, clonal expansion leads to lymphocyte differentiation.

[0174] The tolerogenic or immunosuppressive cells described herein (either generated in vitro for administration to a subject or generated in situ in a subject, e.g., by administration of a nucleic acid encoding CFLAR) are useful in methods of suppressing an immune response in a mammal for the treatment or prevention of autoimmune or inflammatory diseases, or graft rejection, including organ, tissue, cell, and / or protein transplants. Without wishing to be bound by any particular theory, the tolerogenic or immunosuppressive cells administered to a patient or formed in situ in a patient inhibit the activation and proliferation of the patient's T cells.

[0175] Tolerogenic or immunosuppressive cells are useful in avoiding or reducing side effects that arise as a result of a patient's immune response mounted against a protein administered to the patient (optionally through expression of a nucleic acid administered to the patient), thus reducing the efficacy and safety of the protein. Protein therapeutics include, but are not limited to, monoclonal antibodies, enzymes, cytokines, and toxins.

[0176] Additionally, the tolerogenic or immunosuppressive cells are useful for reducing and / or eliminating immune responses to the graft in the recipient by administering or generating to the recipient an amount of the tolerogenic or immunosuppressive cells effective to reduce or inhibit host graft rejection. The graft may include a biocompatible lattice or a donor tissue, organ, cell, or molecule to be transplanted. Examples of grafts include, but are not limited to, skin cells or tissue, bone marrow, and solid organs such as the heart, pancreas, kidney, lung, and liver. In some cases, the graft is a nucleic acid or a protein. Based on the disclosure provided herein, bone marrow cells or their progenitors can be obtained from any source, such as the donor of the transplant, the recipient of the transplant, or from an otherwise unrelated source (a completely different individual or species). The tolerogenic or immunosuppressive cells are preferably administered to or generated in the recipient prior to or simultaneously with the transplant to reduce and / or eliminate host graft rejection. In yet another embodiment, tolerogenic or immunosuppressive cells can be administered to or generated in the transplant recipient after administration of the graft.

[0177] The present invention also includes the use of tolerogenic or immunosuppressive cells as a therapy to suppress graft-versus-host disease after transplantation. In this embodiment, the invention encompasses a method of contacting a donor graft with tolerogenic or immunosuppressive cells prior to transplantation of the graft into a recipient. The tolerogenic or immunosuppressive cells serve to ameliorate, inhibit or reduce adverse reactions of the donor graft to the recipient. The tolerogenic or immunosuppressive cells can be obtained from any source, for example, the transplant donor, transplant recipient or other unrelated source (completely different individual or species), for use in eliminating or reducing unwanted immune responses of the graft to the transplant recipient. Thus, the tolerogenic or immunosuppressive cells can be autologous, allogeneic or xenogeneic to the transplant donor, transplant recipient or other unrelated source. In one embodiment of the invention, a transplant recipient suffering from or at risk of suffering from graft-versus-host disease is treated by administering tolerogenic or immunosuppressive cells to the recipient or generating tolerogenic or immunosuppressive cells in the recipient to reduce, inhibit or eliminate the severity of graft-versus-host disease. In this embodiment of the invention, preferably, the tolerogenic or immunosuppressive cells can be obtained from the recipient prior to transplantation and expanded and / or stored in culture to provide a sufficient reserve of tolerogenic or immunosuppressive cells to treat the ongoing graft-versus-host reaction. However, as described elsewhere herein, the tolerogenic or immunosuppressive cells can be obtained from any source, for example, the transplant donor, the transplant recipient or an otherwise unrelated source (an entirely different individual or species).

[0178] Based on the disclosure herein, it is envisioned that the tolerogenic or immunosuppressive cells described herein can be used in combination with current modes of treatment, such as the use of immunosuppressive therapy for the treatment of host rejection of donor tissue or graft-versus-host disease. The advantage of using tolerogenic or immunosuppressive cells in combination with immunosuppressants in transplantation is that the amount of immunosuppressive therapy used and / or the frequency of administration of immunosuppressive therapy can be reduced by using tolerogenic or immunosuppressive cells to improve the severity of the immune response in the transplant recipient. The advantage of reducing the use of immunosuppressive therapy is the mitigation of systemic immunosuppression and unwanted side effects associated with immunosuppressive therapy.

[0179] The invention described herein specifically encompasses a method of preventing or treating graft rejection and / or graft-versus-host disease by administering to or producing in a patient a prophylactically or therapeutically effective amount of a tolerogenic or immunosuppressive cell described herein for the prevention, treatment or amelioration of host graft rejection and / or graft-versus-host disease. Based on the present disclosure, a therapeutically effective amount of a tolerogenic or immunosuppressive cell is an amount that inhibits activated T cells or reduces the number of activated T cells when compared to the number of activated T cells in the absence of administration of the tolerogenic or immunosuppressive cell. In the context of host graft rejection, an effective amount of a tolerogenic or immunosuppressive cell is an amount that inhibits activated T cells in the recipient or reduces the number of activated T cells in the recipient when compared to the number of activated T cells in the recipient prior to administration of the tolerogenic or immunosuppressive cell. In the context of graft-versus-host disease, an effective amount of a tolerogenic or immunosuppressive cell is an amount that inhibits activated T cells present in the graft or reduces the number of such activated T cells.

[0180] An effective amount of tolerogenic or immunosuppressive cells can be determined by comparing the number of activated T cells in the recipient or graft prior to administration of the tolerogenic or immunosuppressive cells with the number of activated T cells present in the recipient or graft following administration of the tolerogenic or immunosuppressive cells. A decrease, or the absence of an increase, in the number of activated T cells in the graft recipient or in the graft itself associated with administration of the tolerogenic or immunosuppressive cells indicates that the number of tolerogenic or immunosuppressive cells administered is a therapeutically effective amount of tolerogenic or immunosuppressive cells.

[0181] Instead of administering tolerogenic or immunosuppressive cells to a patient, the present invention can also be used to express CFLAR in mammalian cells to generate tolerogenic or immunosuppressive cells in the mammal. This aspect of the present invention relates to a type of gene therapy in which a therapeutic protein is administered to a mammal by introducing a nucleic acid encoding it into the mammalian cells. In all cases in which a nucleic acid is transfected into a cell, the nucleic acid sequence encoding CFLAR is operably linked to the necessary regulatory sequences to achieve expression of the nucleic acid sequence in the cell. The nucleic acid construct is preferably provided as an expression vector that includes a coding sequence for CFLAR operably linked to the necessary regulatory sequences such that the coding sequence is expressed by the cell when the expression vector is transfected into the cell. The coding sequence is operably linked to the necessary regulatory elements for expression of the sequence in the cell. The nucleic acid encoding CFLAR can be a cDNA, genomic DNA, synthetic DNA or a hybrid thereof, or an RNA molecule such as mRNA.

[0182] The nucleic acid construct comprises a nucleotide sequence encoding CFLAR operably linked to a regulatory element, and can remain in the cell as a functional cytoplasmic molecule, a functional episomal molecule, or can be integrated into the chromosomal DNA of the cell. Exogenous genetic material can be introduced into the cell, where it remains as separate genetic material in the form of a plasmid. Alternatively, linear DNA that can be integrated into a chromosome can be introduced into the cell. When introducing DNA into the cell, a reagent that promotes the integration of DNA into a chromosome can be added. A DNA sequence useful for promoting integration can be included in the DNA molecule. Alternatively, RNA can be introduced into the cell.

[0183] The basic approach of using the tolerogenic or immunosuppressive cells of the present invention to induce tolerance has a wide application as an adjunct to therapies that utilize potentially immunogenic molecules for therapeutic purposes. For example, an increasing number of therapeutic methods utilize proteinaceous molecules, such as antibodies or fusion proteins, for the treatment of clinical disorders. A limitation of the therapeutic use of such molecules is that they may elicit an immune response against the therapeutic molecule in the treated subject (e.g., the efficacy of factor VIII in human subjects is hindered by the induction of an immune response against factor VIII in human subjects). The present invention is an improvement over general protein therapy in the context of inducing tolerance to the administered molecule.

[0184] Administration of the tolerogenic or immunosuppressive cells of the invention to inhibit T cell responses can be applied in these therapeutic situations, allowing long-term use of therapeutic molecules in subjects without eliciting an immune response.

[0185] According to the present invention, the term "disease" or "disorder" refers to any pathological condition.

[0186] The term "inflammatory disease" refers to any disease characterized by or associated with high levels of inflammation in tissues, particularly connective tissues, or degeneration of such tissues. Chronic inflammatory diseases are medical conditions characterized by persistent inflammation. Examples of (chronic) inflammatory diseases include celiac disease, vasculitis, lupus, chronic obstructive pulmonary disease (COPD), irritable bowel disease, atherosclerosis, arthritis, ankylosing spondylitis, Crohn's disease, colitis, chronic active hepatitis, dermatitis, and psoriasis.

[0187] The term "autoimmune disease" or "autoimmune disorder" refers to any disease / disorder in which the body generates an immunogenic (i.e. immune system) response against some component of its own tissues. In other words, the immune system loses its ability to recognize some tissue or system within the body as self, and targets and attacks it as if it were foreign. Autoimmune diseases can be classified into those in which one organ is primarily affected (e.g. hemolytic anemia and anti-immune thyroiditis) and those in which the autoimmune disease process spans many tissues (e.g. systemic lupus erythematosus). For example, multiple sclerosis is thought to result from T cells attacking the sheath surrounding nerve fibers in the brain and spinal cord. This causes lack of coordination, weakness, and blurred vision. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, and type I diabetes mellitus, among others.

[0188] "Graft" refers to a biocompatible lattice that is transplanted, or to a donor tissue, organ, or cells. Examples of grafts include, but are not limited to, skin cells or tissue, bone marrow, and solid organs such as the heart, pancreas, kidney, lung, and liver. Graft can also refer to any substance that is administered to a host. For example, graft can refer to a nucleic acid or a protein.

[0189] The term "graft rejection" refers to the rejection of a graft, such as a transplanted tissue or organ, by the recipient's immune system, which can ultimately destroy the graft.

[0190] The term "graft-versus-host disease" or "GvHD" as used herein is defined as a condition in mammals, including humans, that occurs when allogeneic, immunologically active cells, such as those in the bone marrow or spleen, are introduced, transplanted or grafted into a host, resulting in a severe, potentially fatal, reaction due to immune incompatibility between the host and the graft in which the immunocompetent cells of the graft immunologically attack the host.

[0191] "Treat" means administering to a subject an agent or composition as described herein to prevent or eliminate disease, including halting or slowing the progression of disease in a subject; inhibiting or slowing the onset of new disease in a subject; reducing the frequency or severity of symptoms and / or recurrences in a subject currently suffering from or previously suffering from a disease; and / or prolonging or increasing the survival of the subject. In particular, the term "treatment of a disease" includes curing, shortening the duration, ameliorating, preventing, slowing or inhibiting the progression or worsening of, or preventing or delaying the onset of a disease or a symptom thereof.

[0192] "At risk" refers to a subject, i.e., a patient, who is identified as having a higher than normal likelihood of developing the disease compared to the general population. In addition, subjects who have had the disease in the past or present are at high risk of developing the disease, as they may continue to develop the disease. Furthermore, subjects who have received or will receive a transplant are at high risk of developing GvHD.

[0193] In the context of the present invention, the terms "protect", "prevent", "prophylactic", "prophylactic" or "protective" relate to the prevention or treatment or both of the occurrence and / or spread of a disease in a subject, and in particular to minimizing the likelihood that a subject will develop a disease or to delay the onset of a disease. For example, a person at risk of a disease is a candidate for a therapy to prevent the disease.

[0194] Prophylactic administration of an agent or composition described herein preferably protects the recipient from developing the disease. Therapeutic administration of an agent or composition described herein can result in inhibition of disease progression / growth. This includes slowing down disease progression / growth, particularly halting disease progression, and preferably leading to elimination of the disease.

[0195] The agents and compositions provided herein may be used alone or in combination with common therapeutic regimens such as surgery, radiation, chemotherapy and / or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated).

[0196] The term "in vivo" relates to the context of a subject.

[0197] The terms "subject", "individual", "organism" or "patient" are used interchangeably and relate to vertebrates, preferably mammals. For example, in the context of the present invention, mammals are humans, non-human primates, domestic animals (dogs, cats, sheep, cows, goats, pigs, horses, etc.), laboratory animals (mice, rats, rabbits, guinea pigs, etc.), as well as domestic animals such as zoo animals. The term "animal" as used herein also includes humans. The term "subject" can also include patients, i.e. animals, preferably humans, having a disease, preferably a disease described herein.

[0198] The term "autologous" is used to describe something that originates from the same subject. For example, an "autologous graft" refers to a transplant of a tissue or organ that originates from the same subject. Such procedures are advantageous because they overcome immune disorders that would otherwise result in rejection.

[0199] The term "allogeneic" is used to describe something that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical.

[0200] The term "syngeneic" is used to describe individuals or tissues that have the same genotype, ie, derived from identical twins or the same inbred strain of animals, or tissues thereof.

[0201] The term "xenogeneic" is used to describe something that is made up of multiple dissimilar elements. As an example, the transfer of bone marrow from one individual to a different individual constitutes a xenogeneic transplant. A xenogeneic gene is a gene that is derived from a source other than the subject.

[0202] The agents and compositions described herein can be administered via any common route, including injection or infusion. Administration can be performed, for example, orally, intravenously, intraperitoneally, intramuscularly, subcutaneously, or transdermally. In particular, the cells described herein can be administered systemically, i.e., parenterally, by intravenous injection, or can be targeted to a specific tissue or organ, such as bone marrow. Additionally, the cells can be administered via subcutaneous implantation of the cells, or by injection of the cells into connective tissue, for example, into the muscle.

[0203] The agents and compositions described herein are administered in an effective amount. "Effective amount" refers to an amount that achieves a desired reaction or a desired effect, such as a detectable level of immunosuppression or resistance, either alone or together with further doses. In the case of the treatment of a particular disease or a particular condition, the desired reaction is preferably related to the inhibition of the disease process. This includes slowing down the progression of the disease, in particular stopping or reversing the progression of the disease. The desired reaction in the treatment of a disease or condition may also be the delay or prevention of the onset of said disease or said condition.

[0204] The effective amount of the agents or compositions described herein will depend on the condition to be treated, the severity of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, duration of treatment, type of concomitant treatment (if any), specific route of administration, and similar factors. Thus, the dosage of the agents and compositions described herein may depend on such various parameters. If the patient's response is inadequate at the initial dose, a higher dose (or a higher dose of the effect achieved by a different, more localized route of administration) may be used.

[0205] The pharmaceutical compositions described herein are preferably sterile and contain an effective amount of the therapeutically active material to produce the desired reaction or desired effect.

[0206] The pharmaceutical compositions described herein are usually administered in pharma- ceutically compatible amounts and in pharma- ceutically compatible preparations. The term "pharma-ceutically compatible" refers to non-toxic substances that do not interact with the action of the active ingredients of the pharmaceutical composition. Such preparations may usually include salts, buffer substances, preservatives, carriers, and other therapeutically active compounds, if appropriate. When used in medicines, the salts must be pharma- ceutically compatible. However, salts that are not pharma- ceutically compatible can also be used to prepare pharma- ceutically compatible salts and are included in the present invention. Such pharmacologically and pharma- ceutically compatible salts include, but are not limited to, the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Pharmaceutically compatible salts can also be prepared as alkaline earth metal salts or alkali metal salts, such as sodium salts, potassium salts, or calcium salts.

[0207] The pharmaceutical compositions described herein may include a pharma- ceutical compatible carrier. The term "carrier" refers to a natural or synthetic organic or inorganic component with which the active ingredient is mixed to facilitate application. According to the present invention, the term "pharma-ceutical compatible carrier" includes one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a patient. The components of the pharmaceutical compositions described herein are typically such that there is no interaction that would substantially impair the desired pharmaceutical efficacy.

[0208] The pharmaceutical compositions described herein may contain suitable buffer substances, such as, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt.

[0209] The pharmaceutical compositions may also contain, where appropriate, suitable preservatives, such as benzalkonium chloride, chlorobutanol, parabens and thimerosal.

[0210] The pharmaceutical compositions are usually provided in a uniform dosage form and can be prepared in a known manner. The pharmaceutical compositions described herein can be in the form of, for example, capsules, tablets, lozenges, solutions, suspensions, syrups, or elixirs, or in the form of an emulsion.

[0211] Compositions suitable for parenteral administration usually comprise a sterile aqueous or non-aqueous preparation of active compound, which is preferably isotonic with the blood of the recipient. Examples of compatible carriers and solvents are Ringer's solution and isotonic sodium chloride solution. In addition, sterile non-volatile oils are usually used as a solution or suspension medium.

[0212] The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. EXAMPLES

[0213] The techniques and methods used herein are either described herein or carried out in a known manner, e.g., as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. All methods, including the use of kits and reagents, are carried out according to manufacturer's information unless otherwise specified.

[0214] Example 1: Specific CFLAR L and CFLAR S Transfection of human stem cells with lentivirus encoding (CD34 + cells) and monocytes (CD14 + Transduction of IL-16 cells promotes an immunosuppressive phenotype The inventors of CFLAR L (Figure 1A) or CFLAR STwo plasmid constructs were generated containing the sequences of (Figure 1B). Both constructs contained a reporter gene: green fluorescent protein (GFP) with tracking and marker functions. For lentiviral recombinant protein production, a calcium chloride precipitation method was used. Briefly, plasmid vectors encoding important proteins for virus construction were purchased from Takara (Takara Clontech, CA, USA). Calcium chloride CaCl2 (2 mM) was added to the pGp vector, pE-eco vector, and CFLAR vector. L or CFLAR S The transfection mixture was then added to a solution containing a vector encoding , or luciferase (control) (25 μg). Then, buffer HBS 2X (274 mM NaCl2, 10 mM KCl, 1.5 mM Na2HPO4, 12 mM dextrose, 42 mM HEPES, pH 7.1) was added under stirring. The transfection mixture was then added dropwise to a culture plate of 293T cells, and the medium containing the viral particles was collected after 48 hours, centrifuged, filtered through a filter with 0.45 μm pores to remove cells and debris, and concentrated by ultracentrifugation at 50,000 g for 140 minutes at room temperature. The infection was performed by spin-inoculation: the cells (human stem cells or monocytes) were placed in a multi-well plate swinging bucket rotor and centrifuged; the plates were spun at 2500 rpm for 4 hours at 30 °C. After this step, we set up an immunosuppression assay. Human stem cells (bone marrow CD34 + cells, 1 million, cat. 2M-101C, Lonza, Switzerland) were infected with CD34 +Cells were differentiated in vitro for four days in the presence of G-CSF (40 ng / ml; Miltenyi Biotec Srl, Italy) and GM-CSF (40 ng / ml, Miltenyi Biotec Srl, Italy) to promote the acquisition of MDSC-associated immunosuppressive activity. After four days, cells were harvested and co-cultured in the presence of activated cell-tracking labeled PBMCs (Figure 2A, left panel). Briefly, PBMCs were harvested, washed in IMDM medium (Lonza, Switzerland) and supplemented with 10% FBS (EuroClone, Italy), 100 U / ml penicillin / streptomycin (Lonza, Switzerland), β-mercaptoethanol (Sigma-Aldrich, Italy) and 10 mM HEPES (Lonza, Switzerland). PBMCs were diluted in PBS for 10 min at 20°C for 1 h. 7 Cells were resuspended to a final concentration of 1000 cells / ml and stained with Cell Trace Violet stock solution (Invitrogen Molecular Probes, UK) at a final working concentration of 2.5 μM, followed by incubation for 5 min at 37°C and protected from light. The dye readily diffused into the cells and was cleaved by intracellular esterases to give a highly fluorescent compound. This compound covalently bound to intracellular amines, resulting in a stable and well-retained fluorescent stain. After each division the dye was evenly distributed to the daughter cells, resulting in a halving of cell fluorescence. In the flow cytometry histogram, discrete peaks appeared towards the left, representing each cell division or generation. Cells were then washed and resuspended in culture medium. Labeled "target" PBMCs were stimulated with coated anti-CD3 (clone OKT-3; eBioscience, CA, USA) at 1 μg / ml and soluble anti-CD28 (clone CD28.2; eBioscience, CA, USA) at 5 μg / ml for four days to generate "effector" in vitro differentiated CD34+ / CD4+ cells at different effector-to-target ratios in 384-flat-bottom well plates (BD Biosciences, NJ, USA). +Cells were co-cultured with 100% erythrocytes. Cell cultures were incubated at 37°C with 5% CO2 in arginine- and glutamine-free RPMI (Biochrom AG, Germany) supplemented with 2 mM L-glutamine, 150 μM arginine, 10% FBS (Sigma-Aldrich, MO, USA), 10 U / ml penicillin and streptomycin (Lonza, Switzerland), and 0.1 mM HEPES (Lonza, Switzerland). At the end of the culture, cells were stained with PE-Cy7 conjugated anti-CD3 (UCHT1, eBioscience, CA, USA), all samples were acquired with a FACS-CantoII (BD Biosciences, NJ, USA), and cell tracking signals of gated lymphocytes were analyzed by FlowJo software (Treestar Inc.). The extent of cell proliferation was quantified by analyzing the number of proliferating cells and the number of in vitro differentiated CD34 + The immunosuppressive capacity of MDSCs was assumed to be 100% without CFLAR cells. We demonstrated that upregulation of CFLAR enhanced the immunosuppressive capacity of differentiated MDSCs in vitro, suggesting a novel biological role for this protein. Indeed, CFLAR-expressing CD34 + Cells were luciferase-expressing CD34 + The cells showed significantly increased suppressive activity compared to the control cells (Figure 2A, right panel). To further validate these data, we used purified buffy coat-derived CD14 cells, which do not normally exhibit any immunosuppressive function. + Monocytes were immunomagnetically infected with CFLAR or luciferase expressing lentivirus (cat.130-050-201; Miltenyi Biotec Srl, Italy) and co-cultured with anti-human CD3 / CD28 activated cell tracker labeled PBMCs (Figure 2B, left panel). +The cells acquired a stronger suppressive ability compared to luciferase-infected monocytes (Figure 2B, right panel). Using flow cytometry with the APC-AnnexinV Apoptosis Detection Kit (cat. 640930, Biolegend, CA, USA) using 7-AAD, CD14 cells after in vitro co-culture were significantly suppressed. + 7-AAD - Annexin-V - This potent immunosuppressive function was due to the increased expression of CFLAR-expressing CD14 cells compared with controls, as the percentages of cells were comparable (data not shown). + It did not correlate with better cell viability. All samples were acquired by FACS-CantoII (BD Biosciences, NJ, USA) and analyzed by FlowJo software (Treestar Inc.).

[0215] Example 2: Enforced CFLAR expression on monocytes induces an MDSC-like molecular program that controls immunosuppressive functions To improve the identification of CFLAR-activated molecular pathways underlying the immunosuppressive functional program in myeloid cells, we used CFLAR- and luciferase-transfected CD14 cells isolated from four healthy donors. +Cellular transcriptome analysis was performed. Briefly, total RNA was isolated using TRIzol reagent (Life technologies, CA, USA) and RNA integrity was assessed using Agilent-2100-Bioanalyzer (Agilent Technologies, CA, USA). RNA was further purified using RNeasy MinElute Cleanup kit (Qiagen, Venlo, Netherlands), and cDNA was synthesized and amplified from total purified RNA by Ovation Pico WTA System V2 (NuGEN, CA, USA). All samples were hybridized to Affymetrix U133 PLUS 2.0 arrays and scanned with an Affymetrix GCS 3000 7G scanner. Samples and genes are clustered using Pearson correlation coefficient and average as distance metric and association, respectively. As shown in Figure 3, surveillance clustering (q-value < 0.05, fold change > 2) generated a list of 750 upregulated genes (Figure 4) and 1,130 downregulated genes (data not shown) in CFLAR-infected monocytes compared to controls. Notably, these differentially expressed genes were significantly enriched in categories involved in inflammatory pathways, Notch-related pathways, and IL-10-related pathways. Furthermore, forced expression of CFLAR activates the upregulation of MDSC-associated immunosuppressive genes, such as SOCS2, FAS, CCR7, CCL5, NF-kB, STAT3, CD38, CD274, CD273, IL4R, IL6, IL10, CFS3, PTGS2, and IDO1. Several of the putative CFLAR-associated targets were tested and validated (Figure 5). In particular, we found that CFLAR-transduced monocytes upregulated the CFLAR-associated immunosuppressive genes, which are involved in the upregulation of MDSCs. L and CFLAR SReal Time PCR demonstrated that both CFLAR and luciferase-infected monocytes up-regulated expression of IFN-γ and IDO1, as well as IDO2. To this end, we isolated total RNA from both CFLAR-infected and luciferase-infected monocytes with TRIzol reagent (Life technologies, CA, USA) and RNA integrity was assessed using an Agilent-2100-Bioanalyzer (Agilent Technologies, CA, USA). RNA was further purified using the RNeasy MinElute Cleanup Kit (Qiagen, Venlo, Netherlands) and cDNA was synthesized and amplified from the total purified RNA with an Ovation Pico WTA System V2 (NuGEN, CA, USA). Real Time PCR was set up with custom primers designed by ABI Biosystems, Primer Express, using all samples. Post-hoc qRT-PCR analysis to quantify relative gene expression was performed using the comparative Ct method (2 -ΔΔCt As shown in Figure 5A, CFLAR-transduced monocytes expressed significantly higher levels of the putative target genes. Furthermore, the inventors found that CFLAR-transduced CD14 + Cells were luciferase-transduced CD14 +The secretion of significantly higher levels of IL-10 from the cells was verified by an ELISA (Enzyme Linked ImmunoSorbent Assay) assay (R&D System, MN, USA). Briefly, this assay consists of transferring the medium into specific 96-well plates pre-incubated with purified antibodies specific for IL-10. In some wells, a calibration curve was performed using a 1:2 scalar dilution of the cytokine in question instead of the sample. After 2 hours of incubation at 37°C, the plates were marked with a biotinylated anti-IL-10 antibody for 1 hour, followed by a 30 minute incubation at room temperature with streptavidin conjugated to peroxidise. The reaction of peroxidise with its substrate (TMB-3,3',5,5'-tetramethylbenzidine) allows the quantification of the cytokine of interest by reading at 450 nm. Finally, we demonstrated by flow cytometry that forced CFLAR-expression on monocytes promoted the upregulation of specific surface markers: briefly, CD14 24 h after infection, as described above. +Cells were stained with mouse anti-human CD273-PE (cat. 557926, BD Biosciences, NJ, USA), mouse anti-human CD274-APC (cat. 228489, BD Biosciences, NJ, USA), mouse anti-human CD80-APC (cat. 305220, Biolegend CA, USA), anti-human CD163-PE (cat. 556018, BD Biosciences, NJ, USA), anti-human CD44-PE (cat. 103024, Biolegend, NJ, USA), anti-human CD38-APC (cat. 554262, BD Biosciences, NJ, USA), and anti-human CD124-APC (cat. FAB23OP, R&D The cells were resuspended in 100 μl of FACS buffer (0.9% NaCl solution containing 2% BSA and 0.02% NaN3 (Sigma-Aldrich)) containing 10% ethanol (Sigma-Aldrich, MN, USA). All samples were acquired using a FACS-CantoII (BD Biosciences, NJ, USA) and analyzed using FlowJo software (Treestar Inc.).

[0216] Example 3: Adoptive transfer of CFLAR-expressing monocytes controls the progression of GvHD in immunodeficient mice engrafted with human PBMCs Based on these observations, the present inventors have demonstrated that CFLAR-infected CD14 controls the development of graft-versus-host disease (GvHD). + We decided to evaluate the immunosuppressive function of the cells (described above) in vivo using a xenogeneic mouse model. To this end, we used sublethally irradiated ( 137 Cs source irradiation), immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Sug / JicTac (NOG) (purchased from Taconic, NY, USA) mice were treated with 10 6 Human PBMCs were injected intravenously (iv). Circulating human CD3 + When the lymphocyte frequency reaches a percentage of ~5% of the total cells, 10 6Treatment of mice was initiated by iv transfer of 100 CFLAR-expressing or luciferase-expressing monocytes. Briefly, 100 μl of blood was isolated by retro-orbital bleeding; red blood cells were eliminated with ACK (ammonium-chloride-potassium) lysine buffer, and cells were stained with rat anti-mouse CD45-APC (clone A20, Biolegend, NJ, USA), anti-human CD45-PerPC-Cy5.5 (clone 2D1, Biolegend, NJ, USA), and anti-human CD3-PE-Cy7 (clone UHCT1, Biolegend, NJ, USA). All samples were acquired by FACSCanto II (BD Biosciences, NJ, USA) and analyzed using FlowJo software (Treestar Inc.). Treatment was repeated four times, 21, 28, 42, and 49 days after the initial challenge, and mice were monitored for immunological, clinical, and histopathological GvHD scores (Figure 6A). CFLAR infection CD14 + Injection of the cells efficiently reduced the incidence of GvHD and consequently improved the long-term survival of transplanted mice compared with GvHD-affected mice treated with luciferase-expressing monocytes or untreated (Figure 6B). In addition, tissue-infiltrating human CD3 +Histological analysis and quantification of T cells showed less aggressive GvHD pathology in all analyzed organs of mice treated with CFLAR-expressing monocytes (Figure 6C). Briefly, tissues were removed at necropsy, fixed in 10% buffered formalin, and embedded in paraffin. Histopathological evaluation was performed on standard hematoxylin and eosin sections. Pathological scores were assessed blindly by two pathologists using previously published baseline guides (Cooke KR, Kobzik L, Martin TR, Brewer J, Delmonte J, Crawford JM, Ferrara JLM: An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: The roles of minor H antigens and endotoxin. Blood 8:3230, 199). Finally, paraffin sections of spleen, liver, lung, skin, colon, kidney and stomach were used for immunohistochemistry: sections were stained with anti-human CD3 (clone ab5690, Abcam, United Kingdom) antibody to quantify human tissue-infiltrating lymphocytes.

[0217] To check the ability of CFLAR-expressing monocytes to control the in vivo expansion of T cells in GvHD-affected mice, we investigated the uptake of human CD3 +The presence of human T cell frequency was confirmed (Figure 7A). After only two treatments, GvHD-affected mice treated with CFLAR-expressing monocytes showed a lower human T cell frequency compared to luciferase-treated mice (Figure 7B). In particular, GvHD-affected mice were sacrificed 35 days after PBMC transfer, and human T cell frequency was assessed by flow cytometry in blood (Figure 7Bi), spleen (Figure 7Bii), and bone marrow (Figure 7Biii). For blood analysis, 100 μl of blood was isolated by retro-orbital bleeding; red blood cells were eliminated with ACK (ammonium-chloride-potassium) lysine buffer, and cells were stained with PE-Cy7-conjugated anti-CD3 (UCHT1, eBioscience, CA, USA) antibody. For spleen analysis, fresh splenocytes (10 6Each mouse (100 μl / sample) was resuspended in 100 μl FACS buffer containing anti-human FcγR blocking solution (cat.130-046-702, Miltenyi Biotec Srl, Italy) for 10 min at room temperature to reduce non-specific staining. After washing, cells were resuspended in FACS buffer and labeled with anti-human PE-Cy7 conjugated anti-CD3 (UCHT1, eBioscience, CA, USA) antibody. Finally, for bone marrow cell analysis, tibias and femurs were removed from GvHD-affected mice using sterile technique and BM was drained. Red blood cells were eliminated with ACK (ammonium-chloride-potassium) lysine buffer and cells were stained with anti-human PE-Cy7 conjugated anti-CD3 (UCHT1, eBioscience, CA, USA) antibody. All samples were acquired by FACSCanto II (BD Biosciences, NJ, USA) and analyzed using FlowJo software (Treestar Inc.). In addition, the activation state of human T cells was evaluated. Briefly, splenocytes from treated GvHD-affected mice were stimulated or not (resting state) with phorbol 12-myristate 13-acetate (PMA, 50 ng / ml) and ionomycin (1 μg / ml) in vitro for 24 h. Splenocytes were washed, stained with surface antibodies (anti-human Pacific Blue-conjugated anti-CD8 (cat. 34417, Biolegend, CA, USA) antibodies), fixed, permeabilized with Perm / Wash Kit (cat. 554723, BD Biosciences, NJ, USA), and incubated with PE-labeled hIFN-γ antibodies (cat. 506506, Biolegend, CA, USA) (Figure 7B iiii). All samples were acquired by FACSCanto II (BD Biosciences, NJ, USA) and analyzed with FlowJo software (Treestar Inc.). We found that T cells isolated from CFLAR-treated mice expressed statistically lower frequencies of human CD8+IFNγ compared to luciferase-treated mice. +We demonstrated that CFLAR-monocytes express monocytes, which indicates that CFLAR-monocytes can mediate an exhausted or non-activated state. Finally, we assessed that injection of CFLAR-expressing monocytes in GvHD-affected mice induces the expansion of circulating T regulatory cells (Tregs) when compared to luciferase-expressing monocytes (Figure 7C).

[0218] Example 4: Rosa26.vFLIP;LysMcre Mouse Model To better understand the molecular pathways induced by CFLAR, we utilized a transgenic mouse model expressing a viral form of CFLAR: Rosa26.vFLIP knock-in mice. Kaposi's sarcoma-associated herpesvirus encodes the FADD-like interferon-converting enzyme or caspase-8 (FLICE) inhibitor protein (vFLIP) in its genome. Rosa26.vFLIP knock-in mice were crossed with mice expressing Cre recombinase under the control of the endogenous Lyz2 promoter (LysM-Cre), resulting in mice with vFLIP expression restricted to the myeloid lineage. Bone marrow and splenocytes were obtained from both Rosa26.vFLIP;LysMcre and Rosa26.vFLIP mice and analyzed by fluorescence-activated cell sorting (FACS) to identify CD11B + Ly6G - Ly6C high and CD11B + Ly6G + Ly6C low The immunosuppressive activity of these cells was then assessed by plating freshly isolated bone marrow cells in 96-well plates at a final concentration of 24% of the total cells in culture in the presence of splenocytes from transgenic 37B7 mice (a mouse model presenting all CD8+ T cells containing an engineered T cell receptor (TCR) specific for the melanoma-associated antigen TRP2, labeled with 1 μM CFSE+TRP-2). 180-188The peptides were diluted 1:10 with CD45.1+ splenocytes in the presence of peptide (1 μg / ml). After 3 days of co-culture, we performed FACS analysis with a FACS-Canto (BD Biosciences, NJ, USA) and plotted CD8+ / CD9+ / CD10+ cells to assess inhibition of proliferation. + Gating on T cells, the percentage of CFSE-diluted cells was assessed using FlowJo software (Treestar Inc.). The percentage of proliferating cells was then used to calculate the percent inhibition of proliferation using the formula: 1-(% proliferation with myeloid cells / % proliferation without myeloid cells) x 100 (Figure 8).

[0219] Example 5: c-FLAR induces an immunosuppressive program independent of its anti-apoptotic function Starting from buffy coats of healthy donors (HD), we identified CD14 + Monocytes were purified and infected with c-FLAR- or luciferase-encoding lentiviruses and demonstrated that the immunomodulatory properties of c-FLAR did not correlate with improved survival of c-FLAR-expressing monocytes compared to controls, both when monocytes were cultured alone and when co-cultured with CD3 / CD28-activated PBMCs. Indeed, survival (7-AAD) at different time points was significantly improved. - Annexin-V - )CD14 + The percentage of cells was not significantly different between c-FLAR- or luciferase-monocytes (Fig. 9a), indicating that the immunomodulatory activity may be separate from the anti-apoptotic properties of c-FLAR, at least in mature monocytes. In addition, to distinguish between the induction of pro-survival and immunosuppressive functions, we generated lentiviral expression vectors of other anti-apoptotic genes (i.e., Bcl-2 and Bcl-xL) and tested the immunosuppressive properties after their forced expression in human monocytes. As shown in Fig. 9b, only forced expression of c-FLAR can significantly suppress T cell activation and proliferation in vitro.

[0220] Example 6: Enforced c-FLAR expression in monocytes drives an MDSC-associated molecular program. Using gene ontology (GO) enrichment analysis, differentially expressed genes were significantly enriched in categories related to inflammation, cytokine networks and genes upregulated in response to interferon proteins, as well as the IL-10 pathway and NF-κB-related pathways as a whole (Fig. 10a,b).

[0221] Example 7: c-FLAR activates the canonical NF-κB pathway in myeloid cells. Forced expression of c-FLAR triggers the canonical NF-κB pathway. Gene expression data suggested unexpected transcriptional and signaling activities of c-FLAR, possibly linked to the NF-κB pathway. To clarify the molecular program under the control of c-FLAR, we transiently transfected THP1 monocytic cells with in vitro transcribed (IVT) c-FLAR-encoding RNA. The rationale was to induce sustained and virus-dependent production of the target protein; moreover, IVT-RNA does not activate the production of innate immune receptors and type I interferons. Forced introduction of synthetic c-FLAR RNA into THP1 cells triggered an immunosuppressive program consistent with data previously shown for lentiviral expression vectors (Figure 11a). Transfection of c-FLAR RNA significantly promoted the nuclear translocation of NF-κB subunits p65 and p50, mediators of the canonical NF-κB activation pathway, whereas no difference was detected in the translocation of the p52 subunit (Fig. 11b). To test whether NF-κB signaling is required for the immune regulatory program downstream of FLAR, we silenced the kinases IKKα (encoded by Chuk) and IKKβ (encoded by Ikbkb), both essential for canonical NF-κB activation. We used v-FLAR-expressing Ly6C1 cells isolated from Tg mice. +We demonstrated that the immunosuppressive activity of the cells was significantly reduced after silencing of IKKα or IKKβ expression ( FIG. 11 c). Collectively, our data indicate that c-FLAR activates a signaling pathway that controls an immunosuppressive program in monocytes through NF-κB activation.

Claims

1. A population of cells, including bone marrow cells or their precursor cells, engineered to elevate cellular levels of cellular FLICE [Fas-associated death domain (FADD)-like IL-1β-converting enzyme]-inhibitory protein (CFLAR).

2. The population of cells of claim 1 , wherein increasing the cellular level of CFLAR increases the immunosuppressive activity of the cells.

3. The population of cells of claim 1 or 2, wherein the bone marrow cells or their progenitor cells are engineered to increase cellular expression of CFLAR.

4. 4. The population of cells according to any one of claims 1 to 3, wherein bone marrow cells or their progenitor cells are transfected with a nucleic acid encoding CFLAR.

5. The population of cells of claim 4 , wherein the transfection is stable or transient transfection.

6. 6. The population of cells according to claim 4 or 5, wherein the bone marrow cells or their progenitor cells are transfected using a viral vector, such as a lentiviral vector, as a carrier.

7. The population of cells of claim 4 or 5, wherein the nucleic acid is RNA.

8. CFLAR is CFLAR Ｌ and / or CFLAR Ｓ 8. The population of cells according to any one of claims 1 to 7,

9. 9. The population of cells according to any one of claims 1 to 8, wherein the bone marrow cells or their progenitor cells are isolated and / or purified cells, in particular cells isolated and / or purified by magnetic cell separation.

10. 10. The population of cells according to any one of claims 1 to 9, wherein the bone marrow cells or their precursor cells are monocytes.

11. 11. The population of cells according to any one of claims 1 to 10, wherein the bone marrow cells or their progenitor cells are CD14+ monocytes.

12. 12. The population of cells according to any one of claims 1 to 11, wherein the bone marrow cells or their progenitor cells are CD14+ monocytes isolated from buffy coat.

13. 10. The population of cells according to any one of claims 1 to 9, wherein the bone marrow cells or their progenitor cells are stem cells.

14. 14. The population of cells according to any one of claims 1 to 9 and 13, wherein the bone marrow cells or progenitor cells thereof are bone marrow CD34+ cells.

15. 15. The population of cells of any one of claims 1 to 9, 13 and 14, wherein the bone marrow cells or progenitor cells thereof are bone marrow CD34+ cells isolated from bone marrow mononuclear cells (MNC).

16. 16. The population of cells of any one of claims 1 to 15, wherein the cells have been treated to promote acquisition of immunosuppressive activity.

17. 17. The population of cells of any one of claims 1 to 16, wherein the treatment to promote acquisition of immunosuppressive activity comprises in vitro differentiation in the presence of G-CSF and GM-CSF.

18. 20. A population of cells according to any one of claims 1 to 17 for therapy.

19. The population of cells of claim 18 , wherein the treatment requires immunosuppression.

20. 20. A population of cells according to any one of claims 1 to 17 for treating a patient in need of immunosuppression.

21. 21. The population of cells of claim 20 which is autologous to the patient.

22. 18. A pharmaceutical composition comprising a population of cells according to any one of claims 1 to 17.

23. 23. A pharmaceutical composition according to claim 22 for therapy.

24. 24. The pharmaceutical composition of claim 23, wherein the therapy requires immunosuppression.

25. 23. The pharmaceutical composition of claim 22 for treating a patient in need of immunosuppression.

26. 26. The pharmaceutical composition of claim 25, wherein the population of cells is autologous to the patient.

27. 20. Use of a population of cells according to any one of claims 1 to 17 for the manufacture of a medicament for therapy.

28. 28. The use according to claim 27, wherein the therapy requires immunosuppression.

29. 20. Use of a population of cells according to any one of claims 1 to 17 for the manufacture of a medicament for treating a patient in need of immunosuppression.

30. 30. The use of claim 29, wherein the population of cells is autologous to the patient.

31. 20. A method for treating a patient in need of immunosuppression comprising administering a population of cells according to any one of claims 1 to 17.

32. 32. The method of claim 31 , wherein the population of cells is autologous to the patient.

33. A method for treating a patient in need of immunosuppression comprising increasing cellular levels of CFLAR in bone marrow cells or progenitor cells of the patient.

34. 34. The method of claim 33, wherein increasing the cellular level of CFLAR increases the immunosuppressive activity of the cell.

35. 35. The method of claim 33 or 34, wherein increasing the cellular level of CFLAR in the patient's bone marrow cells or progenitor cells comprises administering a nucleic acid encoding CFLAR.

36. 36. The method of claim 35, wherein the nucleic acid encoding CFLAR transfects a bone marrow cell or a progenitor cell thereof.

37. 37. The method of claim 36, wherein the transfection is stable or transient transfection.

38. 38. The method of claim 37, wherein the bone marrow cells or their progenitor cells are transfected using a viral vector, such as a lentiviral vector, as a carrier.

39. 38. The method of any one of claims 35 to 37, wherein the nucleic acid is RNA.

40. CFLAR is CFLAR Ｌ and / or CFLAR Ｓ 40. The method of any one of claims 33 to 39, wherein

41. 41. The method of any one of claims 33 to 40, wherein the bone marrow cells or progenitor cells thereof are monocytes.

42. 42. The method of any one of claims 33 to 41, wherein the bone marrow cells or progenitor cells thereof are CD14+ monocytes.

43. 41. The method of any one of claims 33 to 40, wherein the bone marrow cells or progenitor cells thereof are stem cells.

44. 44. The method of any one of claims 33 to 40 and 43, wherein the bone marrow cells or progenitor cells thereof are bone marrow CD34+ cells.

45. Nucleic acids encoding CFLAR for therapeutic purposes.

46. 46. ​​The nucleic acid of claim 45, wherein the treatment requires immunosuppression.

47. A nucleic acid encoding CFLAR for treating a patient in need of immunosuppression.

48. A pharmaceutical composition comprising a nucleic acid encoding CFLAR.

49. 49. A pharmaceutical composition according to claim 48 for use in therapy.

50. 50. The pharmaceutical composition of claim 49, wherein the therapy requires immunosuppression.

51. 49. The pharmaceutical composition of claim 48 for treating a patient in need of immunosuppression.

52. Use of a nucleic acid encoding CFLAR for the manufacture of a medicament for therapy.

53. 53. The use of claim 52, wherein the therapy requires immunosuppression.

54. Use of a nucleic acid encoding CFLAR for the manufacture of a medicament for treating a patient in need of immunosuppression.

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