US20100135974A1 - Redirected, genetically-engineered t regulatory cells and their use in suppression of autoimmune and inflammatory disease - Google Patents

Redirected, genetically-engineered t regulatory cells and their use in suppression of autoimmune and inflammatory disease Download PDF

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US20100135974A1
US20100135974A1 US12/525,270 US52527008A US2010135974A1 US 20100135974 A1 US20100135974 A1 US 20100135974A1 US 52527008 A US52527008 A US 52527008A US 2010135974 A1 US2010135974 A1 US 2010135974A1
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Zelig Eshhar
Eran Elinav
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Yeda Research and Development Co Ltd
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    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
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    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
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    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule

Definitions

  • the invention in the field of immunology and medicine relates to genetic modification of T regulatory cells with chimeric receptors with antibody-type specificity, and the use of such cells to suppress the action of T effector cells and treat any of a number of diseases and conditions in which such suppression is beneficial, primarily autoimmune and inflammatory diseases such as inflammatory bowel diseases (IBD), organ-specific autoimmune diseases, allograft rejection and Graft-vs. Host disease.
  • IBD inflammatory bowel diseases
  • organ-specific autoimmune diseases organ-specific autoimmune diseases
  • allograft rejection allograft rejection
  • Graft-vs. Host disease graft-vs.
  • Tregs Regulatory T-Cells
  • Treg cells One line of research that led to discovery of Treg cells was the observation that thymectomy of mice of certain susceptible strains on postnatal day 3 results in a spectrum of organ-specific autoimmune effects, which were preventable by “reconstitution” of these animals early in life with normal adult lymphocytes (Asano M et al., J Exp Med 1996; 184:387-96).
  • the effectors and suppressors of autoimmunity in this model of multi-organ autoimmunity were CD4+T-cells. It was subsequently shown that the regulatory CD4+Tregs that prevent disease coexpressed CD25.
  • CD4+CD25+ Tregs represent 5-10% of total peripheral CD4+ cells in mice and 3-6% of total peripheral blood CD4+T-cells in humans (Jonuleit H et al., J Exp Med 2001; 193:1285-94).
  • CD4+CD25+ Tregs have been studied for their function in autoimmune disease.
  • CD4+CD25+ Tregs suppressed disease induced by cloned autoantigen-specific T effector cells (Sufi-Payer, E et al., J. Immunol., 1998; 160:1212-18).
  • the CD4+CD25+ Treg cells appeared to be members of a unique lineage of regulatory T-cells.
  • Tregs express a transcription factor known as Foxp3 intracellularly.
  • Scurfin also forkhead box P3
  • Foxp3 was known to cause a rapidly fatal lymphoproliferative disease, similar to that seen in mice lacking cytolytic T lymphocyte-associated antigen 4 (CTLA-4).
  • CTL-4 cytolytic T lymphocyte-associated antigen 4
  • Foxp3 is believed to act through negative transcriptional regulation of cytokine genes, including IL2, IL4 and IFN- ⁇ (Kasprowicz D J et al., J Immunol. 2003; 171:1216-23, 2003), though many aspects of Foxp3 activity and regulation of its expression remain obscure.
  • mice which develop a severe systemic autoimmune disease including autoreactive T-cells and autoantibodies.
  • Injection of Foxp3-infected T-cells into these mice inhibited the ongoing development of autoimmune dermatitis and activation of cytotoxic CD8+T-cells.
  • This treatment also reduced serum concentrations of antinuclear antibodies, which was paralleled with reduced renal immunoglobulin depositions and increased kidney function.
  • Tregs are activated in an antigen-specific manner, but exert their suppressive function in an antigen-independent manner, mainly by producing and secreting suppressive cytokines such as IL-10 and TGF- ⁇ . Tregs can suppress “conventional” T-cells in vitro by direct cell contact.
  • CD25+CD4+ Treg cells play key roles in the maintenance of immunologic self-tolerance and negative control of a variety of physiological and pathological immune responses. Most of these cells are produced by the normal thymus as a functionally mature T cell subpopulation. Natural Tregs specifically express Foxp3, a transcription factor that plays a critical role in their development and function. Complete depletion of Foxp3-expressing natural Tregs (whether they are CD25+ or CD25 ⁇ ) activated even weak or rare self-reactive T effector cell clones, inducing severe and widespread autoimmune/inflammatory diseases.
  • Tregs are highly dependent on exogenously provided interleukin (IL)-2 for their survival in the periphery.
  • IL interleukin
  • a deficiency or functional alteration of other molecules may affect the development or function of Tregs, self-reactive T effector cells, or both, and consequently tip the balance between the two populations in the periphery toward autoimmunity.
  • Tregs suppress the activity of T effector cells that are a major cause of antigen-specific autoimmune inflammatory disorders.
  • Tregs induce anergy and promote suppression by a process that involves both cell-cell contact, and probably more importantly, by their secretion of TGF- ⁇ and IL-10.
  • the present invention is directed to one such novel approach to prevent or treat autoimmune disease and related immune/inflammatory conditions by imposing Treg-mediated control over T effector cells.
  • a chimeric receptor was made, for example, by fusing the variable portion of an antibody, such as an anti-tumor monoclonal antibody (mAb), to a lymphocyte intracellular triggering domain, so as to be expressed by the T-cell into which the gene has been transfected as the extracellular domain of that T-cell triggering molecule.
  • an antibody such as an anti-tumor monoclonal antibody (mAb)
  • mAb anti-tumor monoclonal antibody
  • T-cells and natural killer (NK) cells immune effector cells
  • T-bodies nicknamed “T-bodies”
  • the chimeric immune receptor confers redirected antigenic specificity coupled to direct, MHC-independent triggering of cellular activation in response to binding of pre-defined target antigen.
  • the heterodimeric CR configuration comprised the two T cell receptor (TCR) ⁇ and ⁇ chains in which each pair of TCR variable domains (V ⁇ and V ⁇ ) was replaced with a pair of V H and V L domain derived from a selected antibody.
  • TCR T cell receptor
  • V ⁇ and V ⁇ TCR variable domains
  • V H and V L domain V H and V L domain derived from a selected antibody.
  • T-cells could be activated to effector function such as cell killing or cytotoxic activity against an immunologically specific target in a manner that was MHC independent (and thereby non-restricted) (Gross et al., 1992, supra).
  • scFv single chain Fv
  • lymphocyte triggering moieties such as the TCR/CD3 complex-associated ⁇ chain, or the Fc receptor ⁇ chain (Eshhar Z et al., Proc Natl Acad Sci USA 1993; 90:720-4).
  • T cell signaling in a single continuous protein, was a modular structure with functional domains that are simple to manipulate, and could be readily expressed in human lymphocytes using retrovirus-based vectors (Eshhar Z et al., J Imm Meth 2001; 248:67-76).
  • the inventors have conceived expanded uses of such CR's for the treatment of undesired immune/inflammatory conditions such as autoimmune diseases (with a particular initial emphasis on inflammatory bowel disease, IBD) and graft rejection.
  • Inflammatory conditions are of particular importance in clinical medicine. These diseases, caused by actions of the immune system, involve inappropriate or excessive activation of certain T-cells, expression of regulatory cytokines and chemokines, loss of immune tolerance, and the like. Examples of autoimmune and/or chronic inflammatory diseases are multiple sclerosis, inflammatory bowel diseases (IBD), joint diseases such as rheumatoid arthritis, and systemic lupus erythematosus.
  • IBD inflammatory bowel diseases
  • joint diseases such as rheumatoid arthritis
  • systemic lupus erythematosus systemic lupus erythematosus.
  • intestine Crohn's Disease
  • skin psoriasis
  • myelinated nerves multiple sclerosis or MS
  • pancreatic islet or ⁇ cells insulin dependent diabetes mellitus (IDDM) or Type I Diabetes
  • salivary glands Sjogren's disease
  • skeletal muscle myasthenia gravis
  • thyroid Haashimoto's thyroiditis
  • Graves' Disease the anterior chamber of the eye
  • joint tissue rheumatoid arthritis
  • IBD Inflammatory bowel disease
  • autoimmune disease pathology is often obscure in humans where the diseases are largely sporadic, and symptoms may appear years after the first pathogenic T cell is activated. It has therefore been difficult to design effective therapies to block induction of disease. In contrast, there are common features in many of the later stages of these diseases. Inflammation at the disease site/target organ is typically present, caused by the release of inflammatory, also termed “proinflammatory,” cytokines (e.g. TNF- ⁇ and interferons) by T-cells and by other cells that contribute to the activation steps and effector pathways of immune/inflammatory processes. These cells include (among others) macrophages, dendritic cells and their precursors, B lymphocytes and plasma cells and NK cells (including NKT-cells). These reactions often involve destruction of “target” cells and tissue damage.
  • cytokines e.g. TNF- ⁇ and interferons
  • Th1 pathway dominates in most colitis models (and human Crohn's Disease).
  • TCR T cell receptor
  • TCR ⁇ KO mice TCR ⁇ KO mice
  • Such models are important for the development of therapeutic strategies to treat IBD.
  • the same group (Mizoguchi A et al., 2003 , Inflamm Bowel Dis. 9:246-259) noted that exaggerated immune responses to normal enteric microflora are involved in the initiation and perpetuation of chronic intestinal inflammation.
  • a major pathway involves development of “acquired” immune responses by the interactions of CD4+TCR ⁇ +T-cells with antigen-presenting cells (APC), particularly dendritic cells.
  • APC antigen-presenting cells
  • CD4+CD25+ Treg cells attenuated activated T cell responses.
  • a neutralizing anti-IL-12 monoclonal antibody mAb
  • mAb monoclonal antibody
  • Lamina limbal growth factor-12 and IFN ⁇ IL-12 and IFN ⁇
  • production of both cytokines declined dramatically and returned to pre-disease levels in the late phase.
  • the neutralizing anti-IL-12 reversed early, but not late, disease.
  • IL-4 and IL-13 production increased progressively from pre- to early to late disease. It was concluded that colitis developing in IL-10-deficient mice evolves into two distinct phases. IL-12 plays a pivotal role in early colitis, whereas other immune mechanisms, presumably mediated by IL-4 and IL-13, predominate in late disease to sustain chronic inflammation.
  • IL-10 affects the growth and differentiation of many hemopoietic cell types in vitro and is a particularly potent suppressor of macrophage and T cell functions. These observation were based in part from use of IL-10-deficient (knockout, KO) mutant mice by gene targeting (Kuhn R et al., Cell 1993; 75:263-74). In these mice, lymphocyte development and antibody responses are normal, but most animals are growth retarded, anemic and suffer from chronic enterocolitis. Alterations in the intestine include extensive mucosal hyperplasia, inflammation, and aberrant epithelial expression of major histocompatibility complex (MHC) class II molecules.
  • MHC major histocompatibility complex
  • IL-10 KO mutants kept under specific pathogen-free conditions, develop only localized inflammation (limited to the proximal colon). It was concluded that (1) bowel inflammation in these mutants originated from uncontrolled immune responses stimulated by enteric antigens and (2) IL-10 is an essential (negative) regulator in the intestinal tract.
  • peripheral regulatory CD4+CD25+ cells retain suppressive activity.
  • the number of these regulatory cells decreases in peripheral blood during active inflammation and only slightly increases in intestinal lesions (Maul J, et al., Gastroenterology 2005; 128(7):1868-78). This aberration suggests that Treg homing defects, as well as dysregulated in situ activation contribute to the pathogenesis of IBD
  • the present invention provides a novel approach, that of redirecting Treg cells, as a means to recruit Tregs to sites of inflammation, and activate them to suppress such immune/inflammatory reactions and protect against, alleviate and even cure such disease as IBD.
  • the present invention is based on the inventors' conception that CR-mediated redirection and activation of Treg cells at sites of inflammation results in suppression of inflammatory conditions, commonly part of organ-specific autoimmune disease and exemplified herein as inflammation in the colon in experimental IBD.
  • the inventors have further conceived of using these cells to overcome rejection of mismatched cells and tissues by T effector cells that arise in transplant recipients or to inhibit the pathogenic action of transplanted immunocompetent cells in the case of GVH disease.
  • the invention relies on the inventors' innovative T-body approach that has thus far proven useful for immunotherapy of cancer (and is currently in phase I/II clinical trials).
  • the invention provides a new approach to the exploitation of Treg cells for amelioration of pathologic and undesired immune responses, particularly immunotherapy of autoimmune and inflammatory conditions, including various organ-predominant autoimmune diseases, and other pathologic or undesirable immune responses such as graft rejection and graft vs. host disease.
  • Treg cells are endowed with CRs that are specific for a selected target antigen or ligand.
  • Such modification causes activation of redirected Tregs at sites of inflammation to suppress the proinflammatory effector-type immune responses.
  • a targeted site such as the inflamed colon, where they will suppress disease-mediating T effector cells.
  • the Tregs may, where possible, be administered directly at or to such site, where they will become activated and suppress disease-mediating T effector cells.
  • Treg cells that have been genetically engineered to express a CR, preferably a tripartite chimeric receptor (TpCR) that is made of a single chain extracellular recognition unit, a transmembrane region, and an intracellular signaling region.
  • a CR preferably a tripartite chimeric receptor (TpCR) that is made of a single chain extracellular recognition unit, a transmembrane region, and an intracellular signaling region.
  • TpCR tripartite chimeric receptor
  • the extracellular recognition region is specific for a selected target antigen or ligand and may preferably be a single chain antibody variable (scFv) region or another ligand that is capable of binding to the target antigen or ligand.
  • the extracellular recognition region preferably does not comprise an MHC protein extracellular domain.
  • the redirected Tregs of the present invention are sometimes referred to herein as “T-bodies” despite the fact that the extracellular recognition region is not necessarily an antibody domain. Thus, this term is not intended to be limited to Tregs with antibody-like specificity, but also includes Tregs with ligand-receptor-like specificity or otherwise.
  • a flexible spacer region may be present between the extracellular recognition region and the transmembrane region.
  • Such flexible spacer is preferably an immunoglobulin (Ig)-like hinge, such as any hinge region derived from the Ig superfamily.
  • the intracellular region includes a combination of T-cell signaling polypeptide moieties, fused in tandem, which combination of moieties, upon binding of the extracellular recognition region to the selected target antigen or ligand, triggers activation of the Treg cells to cause suppression of T-cell mediated immunity.
  • the T-cell signaling moieties preferably include one or more cytoplasmic domains of a costimulatory molecule (e.g., CD28) and a cytoplasmic T-cell stimulatory domain, e.g., of FcR ⁇ or a CD3- ⁇ chain.
  • the redirected Treg cells become specifically activated, upon binding of the extracellular recognition region of the CR to its target antigen or ligand, in a manner that is preferably (1) not restricted by, or dependent upon, the binding of the target antigen or ligand to an MHC, nor is it otherwise dependent in any way on the MHC (HL-A) haplotype of the recipient and (2) independent of engagement of costimulatory ligand(s) on a target cell.
  • a preferred target disease of this invention is an IBD such as ulcerative colitis, in which the present methods, as used successfully in an animal model, will permit Treg cells to reach bowel lesions in IBD patients and become efficiently activated at the inflammation site.
  • the present invention results in site-specific Treg accumulation, ultimately resulting in CR-mediated, antigen-specific activation that results in the production of suppressive cytokines which in turn suppress effector autoimmune T-cells in an antigen-nonspecific manner, leading to alleviation of symptoms and thereby treating the disease.
  • the present therapeutic approach has several unique advantages. In contrast to other immunotherapeutic models, it involves T-bodies redirected with a CR, and preferably a TpCR, that combines antibody/antigen or ligand/receptor recognition with stimulatory and costimulatory motifs. Thus, T-bodies can be fully activated in a way that is not restricted by the MHC and is independent of a requirement for costimulation.
  • the second advantage of the present invention stems from the fact that, although Treg activation is antigen-dependent, the suppressive action of these cells is antigen-, TCR-, and MHC-independent.
  • a chimeric receptor that is specific for one or more tissue-associated antigens rather than requiring specificity for an unknown number of yet undefined autoimmune disease-specific antigens.
  • Expression of such chimeric receptors in Tregs redirects these cells and their activation to the appropriate target tissue (in a preferred embodiment, the colon) so that they are activated in an antigen-specific manner, where their potent suppressive effects take place without a need for further recognition of disease-associated-antigens (the “bystander effect”).
  • Tregs By using specifically-activated Tregs, many fewer cells are required to treat autoimmune inflammatory conditions; such as IBD, or allograft-associated reactions in patients than would have been possible prior to this invention when much higher numbers of non-specific Tregs would have been needed.
  • the present inventors have constructed strains of transgenic (Tg) mice whose T-cells and natural killer (NK) cells express an antigen-specific TpCR.
  • Tg transgenic mice whose T-cells and natural killer (NK) cells express an antigen-specific TpCR.
  • TNP trinitrophenyl
  • TNP specific Tregs isolated from these Tg mice suppressed TNP-specific effector T-cells in vitro and in vivo and were able to suppress trinitrobenzenesulfonic acid (TNBS)-induced colitis in mice.
  • TNBS trinitrobenzenesulfonic acid
  • TNP-specific Tregs suppressed oxazolone-induced colitis in mice in which a low dose of TNP was introduced into the colon together with the oxazolone challenge.
  • the TNP-specific Tregs had no effect on the oxazolone-induced colitis in the absence of TNP introduction. This establishes the “bystander” effect of the present invention, i.e., that the target antigen need not be the pathogenic antigen, as long as the redirected Tregs are activated in the vicinity of the pathogenic or undesired immune response.
  • One distinct advantage of the present invention is that it provides cells and methods that permit antigen-specific activation and antigen-nonspecific action of Treg cells used to suppress effector T cell responses (and treat consequent pathologies) in a way that does not require identity between the ligand (e.g., the antigen) recognized by the TpCR (e.g., by its target recognition portion) and the ligand/antigen that plays a pathogenic role in the disease process.
  • the antigen that is pathogenic does not have to be recognized by the T effector cells being suppressed, and, indeed, may be unrelated to the disease or condition being treated.
  • the invention exploits the “bystander” effect.
  • the redirected Tregs of the present invention can be triggered or activated at that location to release of suppressive cytokines (e.g., IL-10 and TGF- ⁇ ), that will result in suppression of any “bystander” effector T-cells, and by this mechanism, quell an ongoing inflammatory/autoimmune response.
  • suppressive cytokines e.g., IL-10 and TGF- ⁇
  • redirected human Tregs constitute an effective cell-based therapeutic modality for IBD or ulcerative colitis and, more broadly, for any T effector cell-mediated disease or condition.
  • the present invention includes methods to induce these cells using cytokines (e.g. TGF- ⁇ ) or by expression of transgenes (e.g. encoding the Foxp3 transcription factor) that will, together with the TpCR, allow antigen-specific Treg expansion.
  • cytokines e.g. TGF- ⁇
  • transgenes e.g. encoding the Foxp3 transcription factor
  • human Treg cells derived from either the subject with the autoimmune/inflammatory disease or condition to be treated, or from an HLA-matched healthy donor (or a universal cell that is not recognized by the recipient's immune system), are endowed with antigen/ligand-specificity, by transduction with the antigen/ligand-specific TpCR as disclosed herein.
  • the cells being endowed with antigen-specificity are the entire T-cell population and the nucleic acid construct including the sequence encoding the TpCR further includes a Foxp3 transgene that is present as an independently transcribed cistron.
  • a Foxp3 transgene is separately transfected into the T-cell population, to turn the T-cells into Treg cells.
  • Examples provided below include studies using murine colonoscopy, in vivo imaging and immunofluorescence, and provide the basis for a novel cell-based therapeutic modality for IBD, and, by extension, for other pathologic and undesired immune responses mediated by antigen specific T effector cells.
  • the extracellular recognition region is an antibody-derived scFv domain that is specific for a selected antigen.
  • the extracellular recognition region is a member of a ligand-receptor pair, which is specific for the other member of that pair.
  • the extracellular recognition region is linked to the transmembrane region through a flexible spacer, which, more preferably, is a hinge from a molecule of the immunoglobulin family.
  • the intracellular signaling region preferably includes a signaling moiety from a chain of an antigen-specific T-cell receptor, which more preferably is one having a polypeptide region comprising an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • antigen-specific T-cell receptors are chains of the TCR/CD3 complex, a TCR ⁇ , ⁇ , ⁇ or ⁇ chain, and the ⁇ chain of an Ig Fc receptor (FcR ⁇ ).
  • the chain of an antigen-specific T-cell receptor is preferably the CD3/ ⁇ chain or an FcR ⁇ subunit.
  • the intracellular signaling region further preferably includes a signaling moiety of a costimulatory-receptor protein of a T-cell.
  • the costimulatory-receptor protein is preferably selected from CD28, OX40, CD40L (gp39), 4-1BB and PD-1 (or preferably the human form or homolog of these costimulatory molecules). Most preferred among these is CD28 or 4-1BB.
  • the intracellular signaling region includes more than one of the costimulatory-receptor protein signaling moieties.
  • the combination of T-cell signaling polypeptide moieties in the intracellular signaling region may include both CD28 and 4-1BB.
  • the extracellular hinge and transmembrane regions of CD28 are used as the extracellular hinge and transmembrane regions of the chimeric receptor.
  • the intracellular signaling region may also include a signaling moiety from a cytokine receptor of a T-cell, such as the IL-2 receptor or the TGF- ⁇ receptor.
  • a cytokine receptor of a T-cell such as the IL-2 receptor or the TGF- ⁇ receptor.
  • the latter may help to induce the T-cell containing the chimeric receptor to adopt the characteristics of a Treg cell.
  • the intracellular region may also include a signal-transducing enzyme that (a) is an enzyme in the signal transduction pathway of an antigen-specific T-cell receptor or (b) is an enzyme with corresponding specificity and activity as the enzyme of (a), derived from a non-T-cell lymphocyte.
  • a signal-transducing enzyme that (a) is an enzyme in the signal transduction pathway of an antigen-specific T-cell receptor or (b) is an enzyme with corresponding specificity and activity as the enzyme of (a), derived from a non-T-cell lymphocyte.
  • Such enzyme is preferably a kinase, such as the Syk kinase.
  • the chimeric nucleic acid encoding the CR may also include a nucleotide sequence that encodes Foxp3 arranged such that Foxp3 is expressed by the Treg cell independently of the chimeric receptor.
  • the chimeric nucleic acid may be bicistronic such that the Foxp3 transgene is present as an independently transcribed cistron.
  • the target antigen or ligand is preferably one that is present or expressed at a site or target tissue of an immune or inflammatory response mediated by effector T-cells.
  • the autoimmune or inflammatory response may comprise an autoimmune response or disease, an allograft or xenograft response or rejection, or graft vs. host (GVH) disease.
  • the target antigen or ligand may be an autoantigen or an antigen that is cross-reactive with an autoantigen, i.e., is also bound by an antibody that is specific to the autoantigen.
  • the autoantigen may be a pathogenic antigen in the pathophysiology of the autoimmune disease.
  • the antigen is not necessarily an autoantigen, but can be, for example, an antigen that is part of the bacterial flora, such as LPS derived from the bacteria native to the colon.
  • the autoimmune disease or graft response and the antigen/ligand or antigens/ligands against which the Treg is specific is preferably selected from the following group:
  • Treg cell that is able to act and suppress IBD or ulcerative colitis, and may be specific for an antigen associated with IBD such as carcinoembryonic antigen (CEA) or an antigen of intestinal bacterial flora such as bacterial lipopolysaccharide (LPS) or a component thereof, preferably a Lipid A component.
  • CEA carcinoembryonic antigen
  • LPS bacterial lipopolysaccharide
  • the Treg may be specific for an activation antigen expressed on T effector cells such as CD69 or CD107a.
  • the Treg may be specific for an antigen expressed on a dendritic cell, macrophage/monocyte, granulocyte or eosinophil present at the inflammation site.
  • the above Treg cell is specific for an antigen that is introduced exogenously to a subject to the site or target tissue of the immune or inflammatory response, either before, concomitantly with, or after administration of the Treg cell.
  • the above Treg cell preferably is one that expresses CD4 or CD8, along with CD25 on its surface and expresses the Foxp3 transcription factor intracellularly.
  • the Foxp3 transcription factor may be expressed in the cell endogenously (i.e., from the cells' own Foxp3 gene); this expression is enhanced by exposure of cells to TGF- ⁇ or another cytokine that induces Foxp3 expression and induces a Treg phenotype in T-cells.
  • Foxp3 is expressed from a nucleic acid that has been introduced into the cell exogenously (i.e., transduced) as a recombinant nucleic acid expression construct encoding Foxp3 and regulating its expression.
  • the above Treg may be obtained from a mammalian subject prior to introduction of the chimeric nucleic acid and prior to stimulation that induces Foxp3 expression or prior to transducing the exogenous Foxp3-encoding construct.
  • the chimeric nucleic acid encoding the chimeric receptor and the nucleic acid construct encoding Foxp3 may be co-transduced into the cell.
  • co-transduction is achieved using a bicistronic vector that includes, in a single vector, a sequence of (i) the chimeric nucleic acid encoding the chimeric receptor and (ii) the nucleic acid construct encoding Foxp3, under the control of a common (or separate) promoter and regulatory sequences.
  • Treg cells may be enriched or purified from a mixed population of lymphocytes or T-cells on the basis of the Treg cells' expression of CD4 (or CD8) and CD25 and/or Foxp3.
  • the cell may be subjected to the following treatment:
  • an immunoregulatory pharmaceutical composition for suppressing a T effector cell-mediated immune/inflammatory response or treating a T effector cell-mediated immune/inflammatory disease or condition, comprising a redirected Treg as described above and a pharmaceutically and immunologically acceptable carrier, excipient or diluent.
  • This invention is also directed to a method for producing the above redirected Treg that expresses a chimeric receptor as described.
  • This method preferably comprises:
  • this method comprises:
  • This invention further is directed to a method of suppressing undesired activity of T effector cells in mediating an immune or inflammatory response, comprising delivering to a population of T effector cells to be suppressed (or to a site where such T effector cells are present) an amount/number of redirected Tregs as above, effective to suppress activity of the T effector cells.
  • the redirected Treg cells are delivered by injection or infusion to a subject in whom the T effector cell activity is to be suppressed, preferably by a route selected from intravenous, intramuscular, subcutaneous, intraperitoneal, intra-articular, intrathecal, intraluminal, intracerebroventricularly, rectal, and topical.
  • the Treg cells are delivered regionally or locally to a site of inflammation.
  • the above method is intended for use in situations wherein the T effector cells mediate an autoimmune inflammatory response or disorder, rejection of a transplant or GVH disease.
  • the method for treating or ameliorating symptoms of a disease or condition in a subject that is mediated by undesired activity of T effector cells comprises administering to the subject in need thereof an effective amount/number of Treg cells as described above, or a pharmaceutical composition described above, wherein the target recognition domain of the redirected Treg cells is specific for an antigen/ligand present in the subject in the vicinity of the T effector cells so that, upon recognizing and binding the antigen, these redirected Treg cells are activated to secrete suppressive cytokines that suppress the T effector cells in an antigen-nonspecific manner.
  • the Treg cell activation occurs in a manner that is not restricted by the MHC and does not require costimulation by a ligand for the costimulatory signaling protein.
  • the invention is directed to a method for suppressing a T effector cell-mediated immune/inflammatory process in a subject in need thereof, comprising administering to the subject an effective amount/number of redirected Treg cells that express on their surface an antigen-specific chimeric receptor that includes portions that activate Treg cells upon contact with the antigen for which the receptor is specific, the antigen being one that is present in the vicinity of the immune/inflammatory activity.
  • the disease or condition to be treated or ameliorated is preferably: (a) IBD; (b) rheumatoid arthritis; (c) Type I diabetes mellitus or autoimmune insulitis; (d) multiple sclerosis; (e) thyroiditis; (f) gastritis; (g) uveitis or uveoretinitis; (h) orchitis; (i)) oophoritis; (j) psoriasis; (k) prostatitis; (l) encephalomyelitis; (m) vitiligo; (n) rejection of a mismatched cell, tissue or organ graft; or (o) GVH disease.
  • the present method is used to inhibit the rejection of transplanted cells, tissue, or an organ (alto- or xeno-) that is, for example, mismatched for a major and/or one or more minor histocompatibility antigens.
  • the recipient In the case of GVH disease, the recipient generally has received a transplant of allogeneic, semi-allogeneic or non-MHC-mismatched bone marrow cells or enriched or isolated hematopoietic stem cells that are responsible for mediating pathogenic effects.
  • the present invention is further directed to the novel chimeric DNA that can be used to produce the redirected T-cells described above, as well as to the chimeric receptor protein encoded thereby.
  • Such chimeric DNA comprises:
  • a first DNA segment encoding an extracellular recognition region specific for a selected target antigen or ligand, which does not comprise an MHC protein extracellular domain, the selected target antigen or ligand being one that is present or expressed at a site or target tissue of a pathogenic or undesired immune response mediated by effector T-cells;
  • a third DNA segment encoding an intracellular signaling region comprising a combination of T-cell signaling polypeptide moieties, which combination of moieties, upon transfection of the chimeric DNA into a regulatory T lymphocyte (Treg cell) and binding of the extracellular recognition region to the selected target antigen or ligand thereof, triggers activation of the Treg cells to cause suppression of T-cell mediated immunity, which chimeric DNA, upon transfection into a Treg cell, expresses the extracellular recognition region, the transmembrane region and the intracellular signaling region in one single, continuous chain on the surface of the transfected cell such that the transfected Treg is triggered to activate and cause suppression of T-cell mediated immunity when the expressed extracellular recognition region binds to its selected target antigen or ligand.
  • FIG. 1 Schematic diagram of TNP-specific TPCR structure.
  • A Schematic presentation of the TNP-specific chimeric receptors.
  • the TNP-specific CR encompasses a scFv derived from the anti-TNP mAb, Sp6. In the tripartite configuration, the scFv is joined in tandem to a short portion of CD28 (lacking the ligand-binding site) of the extracellular and including the transmembrane, and cytoplasmic domains fused to the FcR ⁇ ITAM domain.
  • B Chimeric receptor transgene constructs. Constructs used to generate the transgenic mice were placed under the control of the human CD2 promoter/enhancer that directs expression only in T and NK cells.
  • CYT indicates cytoplasmic domain; H, hinge domain; L, immunoglobulin leader; LCR, locus control region; P, promoter; pL, plasmid sequence; TM, transmembrane domain; VH and VL, immunoglobulin heavy and light-chain variable domains, respectively; ACD28, truncated CD28 containing part of the extracellular and the transmembrane domain, and lacking the cytoplasmic signaling moiety.
  • FIG. 2 Flow cytometry results of Foxp3 staining of TNP-specific Tregs.
  • Splenocytes isolated from WT and TNP-Tg mice were stained for intracellular Foxp3 and for TNP-specific chimeric receptor using antiidiotypic antibody to the Sp6 scFv.
  • Representative flow cytometry analyses are shown for an individual mouse out of five tested mice. Percentages indicate double-stained cells.
  • FIG. 3 Graph showing the ratio of CD4+CD25+ cells to CD4+ cells in splenocytes of wildtype and Tg mice.
  • the groups are: wildtype mice, mice Tg for a chimeric receptor specific for an “irrelevant” antigen, ErbB2 (also referred to as ErbB2-Tg mice), TNP-Tg mice that have been transfected with a vector lacking the transgenic costimulatory CD28 domain (also referred to as TNP ⁇ CD28-Tg mice), and TNP-Tg.
  • TNP-Tg Tregs fully express the TNP-specific TpCR
  • FIG. 4 Graph (left) showing Foxp3+/CD4 cell ratio in wildtype, ErbB2-Tg, TNP ⁇ CD28-Tg, and TNP-Tg mice. Flow cytograms (right) showing splenic Foxp3 expression. Results compare wildtype, ErbB2-Tg, TNP-Tg and TNP ⁇ CD28-Tg mice.
  • FIG. 5 Flow cytograms showing Foxp3 staining in sorted wildtype, ErbB2-Tg, TNP-Tg and TNP- ⁇ CD28-Tg CD4+CD25+ effector T-cells.
  • FIG. 6 Graph (left) showing ratio of Foxp3+ to CD25+/Foxp3+ cells. Flow cytogram (right) showing co-staining of Foxp3 and CD25. Results compare wildtype, ErbB2-Tg, TNP-Tg and TNP- ⁇ CD28-Tg splenocytes.
  • FIG. 7 Graph showing percentage of Foxp3+ splenocytes in the total CD3+T-cell population following induction of TNBS colitis.
  • Splenic lymphocytes were isolated from WT and TNP-Tg mice prior to or 48 hours following induction of TNP colitis, and double-stained with anti-Foxp3 and anti-CD3 antibodies.
  • FIG. 8 Graph showing percentage of Foxp3+ lymphocytes extracted from colonic lamina intestinal following induction of TNBS colitis. Lymphocytes were isolated from WT and TNP-Tg mice prior to and 48 hours following induction of TNP colitis, and double-stained as in FIG. 7 . The percentage of Foxp3+ lymphocytes in the CD3+ population is presented as the average Foxp3/CD3 ratio ⁇ s.d. of each five-mouse group. Data shown are averages of two independent experiments performed. Differences in ratios between na ⁇ ve and colitis-induced TNP-Tg mice were significant (P ⁇ 0.05).
  • FIG. 9 Series of 6 graphs showing stimulation of proliferation of redirected Tregs (left) and T effector cells (right) by an antigen-nonspecific stimulus (anti CD3 and anti CD28 mAbs) and antigen-specific (TNP) stimulus.
  • FIG. 10 Graph showing polyclonal activation with Concanavalin A (Con A) of co-cultures of Tregs and T effectors cells (and control cultures of individual cell populations)
  • FIG. 11 Graphs showing Specific activation of TNP-Tg Tregs and their suppression of effector T-cells requires TNP and CD28-co-stimulation.
  • specific (TNP) activation of Tregs is shown.
  • WT or TNP-Tg Tregs (5 ⁇ 10 4 ) were cocultured with WT or TNP-Tg Teff (5 ⁇ 10 4 ) in the presence of irradiated, T-cell depleted, TNPylated splenic APC (1.5 ⁇ 10 5 ). Teff proliferation was measured after 48 hours by 3 H-Thymidine incorporation.
  • Right panel TNP-loaded APCs as stimulus.
  • FIG. 12 Graph showing dose-response of TNP-specific stimulation of Treg cell+T effector cell cocultures.
  • APCs were TNP-modified stimulator P815 mastocytoma cells, which do not express B7 (P815-TNP) or TNP-modified P815 cells into which the B7 gene was stably transfected (B7-TNP).
  • FIG. 13 Graph showing Specific activation of TNP-Tg Tregs and their suppression of effector T-cells requires TNP and CD28-co-stimulation.
  • FIG. 14 Photograph of colon of wildtype mice (left) and TNP-Tg mice (right) four days after induction of high-dose TNBS colitis by intrarectal instillation of TNBS at day 0.
  • FIG. 15 Mortality curve of wild-type (WT), TNP-Tg, ErbB2-Tg and TNP- ⁇ CD28-Tg mice following induction of TNBS colitis by intrarectal instillation of TNBS at day 0.
  • FIG. 16 Photomicrograph of stained tissue (H&E, 40 ⁇ ) of the colons from FIG. 14 .
  • OXA a hapten/antigen that is distinct from TNP
  • FIG. 18 Flow cytograms of Foxp3 staining of wildtype, ErbB2-Tg, TNP- ⁇ CD28-Tg and TNP-Tg T effector cells after a week of culture in the presence of the following “stimuli” (across top): anti-CD3, TGF- ⁇ , anti-CD3+TGF- ⁇ , TNP, or TNP+TGF- ⁇ .
  • FIG. 19 Graphs showing mortality or survival rate of wildtype mice subjected to induction of moderate (left panel) and severe (right panel) TNBS colitis following adoptive transfer of the following Treg populations: WT, ErbB2-Tg, TNP-Tg and TNP- ⁇ CD28-Tg.
  • FIG. 20 Graph showing Wallach Colitis Severity Score of wildtype mice subjected to induction of TNBS colitis after adoptive transfer of cells from WT, ErbB2-Tg or TNP-Tg donors.
  • Tregs (1 ⁇ 10 5 ) from TNP-Tg, ErbB2-Tg or WT mice were adoptively transferred to the recipient mice. Each experiment was repeated three times. The data shown represent the average of a representative experiment.
  • FIG. 21 Photograph of excised colons of wildtype mice in which TNBS colitis was induced, following adoptive transfer of wildtype, ErbB2-Tg and TNP-Tg mice in the experiment described in FIG. 20 .
  • FIG. 22 Photomicrograph of stained colon tissue sections (H&E, 40 ⁇ ) from wildtype mice with TNBS colitis after adoptive transfer of Tregs from the following donors: (A) wildtype (B) ErbB2-Tg and (C) TNP-Tg. Panel D shows normal control colon.
  • FIG. 23 Localization of Tregs in the colon. Flow cytograms of fluorescent staining Tregs labeled with the intracellular dye carboxyfluorescein diacetate succinimidyl ester (CFSE) in the colonic lamina intestinal of na ⁇ ve mice or mice with TNBS-colitis. Labeled Tregs were intraperitoneally injected 24 hours following induction of TNBS colitis. Lymphocytes from lamina intestinal were obtained 16 hrs after adoptive transfer of 10 6 wildtype or TNP-Tg Tregs to indicated recipients. CFSE-labeled Tregs were 9-fold more abundant in diseased colons. Data shown represent the percentages of CFSE-positive cells in the corresponding gates of one representative mouse of each four-mouse group. Each experiment was repeated twice.
  • CFSE carboxyfluorescein diacetate succinimidyl ester
  • FIG. 25 Localization of Tregs in the colon. In-situ fluorescent microendoscopic (Cell Vizio) evaluation of CFSE-labeled Tregs accumulating at the colonic pre-luminal mucosal layer.
  • the experimental design is identical to the one described in FIG. 23 .
  • the figure shows representative frames taken 48 hours following adoptive transfer. Each group consisted of four mice, and each experiment was repeated twice.
  • FIG. 26 Intrarectal administration of TNBS results in TNP-Tg Treg-mediated protective effect from oxazolone colitis.
  • FIG. 27 shows 8 schematic drawings of T cells which are transduced with a retroviral vector that carried one of 8 CR constructs that include the detectable fluorescent protein, eGFP.
  • eGFP detectable fluorescent protein
  • Those depicted in the lower half of the Figure are bicistronic constructs that encode a fusion of GFP and the transcription factor Foxp3.
  • Light and fluorescence microscopy were used to follow expression of the GFP in the cytoplasm of nucleus of the transduced cells.
  • the constructs are labeled as follows (where “TPCR” refers to “tripartite chimeric receptor” even though, some of these CR's were “more” than tripartite).
  • FIG. 28 An annotated nucleotide sequence (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) of a TNP-specific tripartite CR as used herein.
  • the annotations include the origin of the regions (scFv, here the “Sp6” mAb), the “CD28” region, and the FcR ⁇ regions (indicated as “GAMMA”), as well as restriction sites, leader sequence, etc.
  • the mature protein begins at amino acid residue 23.
  • FIG. 29 An annotated nucleotide sequence (SEQ ID NO:3) of a pBullet plasmid that includes a CR-encoding construct that comprises a nucleotide sequence encoding the scFv of mAb HB 9081 (i.e., produced by a hybridoma given ATCC Accession No. HB9081) fused to C28/FcR ⁇ .
  • This mAb and, hence, the scFv is specific for LPS.
  • Annotations show various restriction enzyme recognition sites, the leader sequence, and plasmid sequences.
  • FIG. 30A-30B is an annotated amino acids sequence of Human CD14 (SEQ ID NO:4). See GenBank Accession No. P08571. A signal sequence and an LPS-binding motif (residues 100-119) are noted. This protein serves as an LPS receptor on cells.
  • FIG. 30B is an annotated amino acids sequence of Human MD-2 protein (SEQ ID NO:5). See GenBank Accession No. NP — 056179. A signal sequence and an LPS-binding motif (residues 120-132) are noted. This LPS-binding protein interacts with TLR-4 as a co-receptor.
  • FIG. 31A-31B is an annotated nucleotide sequence (SEQ ID NO:6) showing the nucleotide sequence of a Chimeric Receptor comprising CD14 motif-CD28-FcR ⁇ .
  • FIG. 31B is an annotated nucleotide sequence (SEQ ID NO:7) showing the nucleotide sequence of a chimeric, bicistronic receptor: CD14 motif-CD28-FcR ⁇ -IRES-GFP-Foxp3. Also shown is the amino acid sequence (single letter code) of the CD14 motif (residues 110-119 of SEQ ID NO:4).
  • Annotations show various restriction sites, beginnings and ends of protein regions, IRES region, etc.
  • FIG. 32A-32B is an annotated nucleotide sequence (SEQ ID NO:8) showing the nucleotide sequence of a Chimeric Receptor comprising MD2 motif-CD28-FcR ⁇ .
  • FIG. 32B is an annotated nucleotide sequence (SEQ ID NO:9) showing the nucleotide sequence of a chimeric, bicistronic receptor: MD2 motif-CD28-FcR ⁇ -IRES-GFP-Foxp3. Also shown is the amino acid sequence of the MD2 motif (residues 120-132 of SEQ ID NO:4. Annotations show various restriction sites, beginnings and ends of protein regions, IRES region, etc.
  • FIGS. 33A and 33B are an annotated nucleotide sequence (SEQ ID NO:10) showing the nucleotide sequence of a Chimeric Receptor comprising MD2 motif-CD14 motif-CD28-FcR ⁇ .
  • FIG. 33B is an annotated nucleotide sequence (SEQ ID NO:11) showing the nucleotide sequence of a chimeric, bicistronic receptor: MD2 motif-CD14 motif-CD28-FcR ⁇ -IRES-GFP-Foxp3.
  • nucleotides 106-148 of SEQ ID NO:11 double underlined
  • SEQ ID NO:12 also double underlined
  • Annotations show various restriction sites, beginnings and ends of protein regions, IRES region, etc.
  • FIG. 34 is an annotated nucleotide sequence (SEQ ID NO:13) showing the nucleotide sequence of a Chimeric Receptor comprising MD2-CD28-FcR ⁇ (SEQ ID NO:13). Also shown is the amino acid sequence of the full length MD2 protein (SEQ ID NO:4). The LPS-binding region of this amino acid sequence is underscored. Annotations show various restriction sites, beginnings and ends of protein regions, etc.
  • FIG. 35 is an annotated nucleotide sequence (SEQ ID NO:14) showing the nucleotide sequence of a chimeric, bicistronic receptor: MD2-CD28-FcR ⁇ -IRES-GFP-Foxp3. Also shown is the amino acid sequence of the full length MD2 protein (SEQ ID NO:4). The LPS-binding region of this amino acid sequence is underscored. Annotations show various restriction sites, beginnings and ends of protein coding regions, IRES, vector sequence, etc.
  • Treg cells are a specialized subpopulation of T cells that act in a “regulatory” way to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens. Tregs have sometimes been referred to suppressor T-cells. Treg cells are characterized by expression of the forkhead family transcription factor Foxp3 (forkhead box p3). They may also express CD4 or CD8 surface proteins. They usually also express CD25.
  • Foxp3 forkhead box p3
  • Tregs include natural Tregs and induced or adaptive Tregs and Tregs that have been created using recombinant DNA technology.
  • Naturally-occurring Treg cells (CD4+CD25+Foxp3+) arise like all other T cells in the thymus.
  • adaptive Treg cells also known as Trl cells or Th3 cells
  • Antigen-specific activation of human effector T-cells leads to inducible expression of Foxp3 in a subgroup of the activated effector cells, and this subgroup can develop a regulatory (Treg) phenotype.
  • Treg regulatory phenotype.
  • One way to induce Tregs is by prolonged exposure of T effector cells to TGF- ⁇ .
  • T-cells may also be converted to Treg cells by transfection or transduction of the Foxp3 gene into a mixed population of T-cells.
  • Redirected Treg is intended to be a comprehensive term for Tregs carrying a chimeric receptor (CR) as described and claimed herein which confers on the cells the ability to bind to and be activated by a target antigen or ligand that is different from that to which a Treg population may have been previously specific (as controlled by its endogenous antigen-specific TCR).
  • Redirected Tregs are “MHC-independent” and “non-MHC restricted” in the process of their activation and in their actions as they do not require association of a peptide derived from their target antigen or ligand with MHC in order to recognize it.
  • Treg that recognizes a specific epitope of an MHC molecule per se, e.g., functioning as a transplantation antigen.
  • these redirected Tregs are still non-MHC restricted.
  • selected target antigen or ligand means a molecule to which the extracellular recognition region of the redirected Treg is intended to bind so as to activate that Treg. If the selected target is an antigen, then an antibody can be raised against it and the binding regions of such an antibody used to construct the extracellular recognition region of the redirected Treg. If the target molecule is a member of a receptor/ligand pair (defined below), then the other member of that pair can be used as part of the extracellular recognition region of the redirected Treg.
  • the intended target tissue or site where the Treg is to be employed is first identified, and then, an antigen or ligand that is present on or near this intended target tissue or site is identified.
  • An antibody or ligand/receptor that binds thereto is then identified, or, if necessary, created or constructed for use on the redirected Treg.
  • ligand as used herein, and particularly as part of the term “target antigen or ligand” or the term “receptor/ligand pair” refers to a molecule that is able to bind to and form a complex with another biomolecule to serve a biological purpose. Often the binding partner of a ligand is called a receptor so that the two binding partners are termed a “receptor/ligand pair.”
  • receptor when used in the sense of a “receptor/ligand pair,” has a broader meaning than, for example, a typical definition of a “receptor” as a protein in or on a cell that binds to a specific ligand.
  • IL-2 can be a ligand because it binds to and forms a complex with another biomolecule, i.e., an IL-2 receptor (IL-2R), to serve a biological purpose.
  • IL-2R IL-2 receptor
  • IL-2R is also a “ligand” because it is a molecule that binds to and forms a complex with another biomolecule, i.e., IL-2, to serve a biological purpose.
  • IL-2R may be considered the receptor, and vice versa.
  • a “chimeric receptor,” as used in the present specification and claims, is a recombinant polypeptide that includes an extracellular recognition region that is derived from one molecule and at least one intracellular signaling moiety that is derived from a different molecule. In that sense it is chimeric.
  • a “chimeric nucleic acid” is a recombinant polynucleotide that includes a sequence that encodes a chimeric receptor.
  • recombinant or “recombinantly” when applied to a polynucleotide, polypeptide or cell means that the molecule or cell is made using genetic engineering techniques and would not exist but for the hand of man.
  • T-cell signaling polypeptide moiety means that portion of a molecule endogenous to a T-cell that mediates signaling. It may be a portion of a T-cell receptor molecule that mediates signaling, or a downstream signal-transducing enzyme or a portion thereof that mediates signaling, i.e., that has enzymatic activity.
  • antibody-derived scFv domain means a single-chain antibody in which the V L of a specific antibody is linked to the V H thereof by a flexible linker or spacer.
  • an MHC protein extracellular domain refers to the disclosure of Meal D J et al. ( Proc Nall Acad Sci USA 2005; 102:11817-22), discussed below, and Jodi M D et al., Nat. Biotechnol., 2002, 20:1215-1220. These publications describe Treg cells redirected against T-cells in a murine system. They used the class II MHC Is ⁇ and Is ⁇ chains as extracellular regions of two separate chimeric receptors for use in a redirected Treg. The term MHC protein extracellular domain is defined so as to encompass what was used in the Meal et al. and Jodi et al.
  • flexible spacer means any flexible peptide moiety that will facilitate the functionality of the extracellular recognition region. When this region is not rigidly attached to the transmembrane region, but is allowed some degree of flexibility with respect to the cell membrane, the ability of the recognition region to recognize and bind to its target antigen or ligand is facilitated. Small neutral amino acids, such as glycine and serine, confer such flexibility. Examples are Gly 4 Ser and Gly 4 Ser 3 .
  • immunoglobulin superfamily means the large group of cell surface and soluble proteins that are involved in the recognition, binding, or adhesion processes of cells. Molecules are categorized as members of this superfamily based on shared structural features with immunoglobulins (also known as antibodies); they all possess a domain known as an immunoglobulin domain or fold. Members of the Igs include various cell surface antigen receptors, co-receptors and co-stimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules and certain cytokine receptors. They are commonly, though not exclusively, associated with roles in the immune system.
  • hinge when referring to a region of a molecule of the Igs means the region between the C H 1 and C H 2 domains consisting of a small number of amino acids.
  • the hinge is flexible and allows the binding region to move freely relative to the rest of the molecule.
  • disulfide bridges which link the two dimers, creating the tetramer structural unit. Examples of such immunoglobulin hinge sequences may be found in U.S. Pat. No. 6,165,476, which is incorporated herein by reference.
  • antigen-specific receptor of a T-cell refers to a receptor that is found on a T-cell that is antigen-specific, i.e., naturally has an extracellular region that binds specifically to a particular antigen in preference to another.
  • antigen-specific receptors of a T-cell are the TCR ⁇ , ⁇ , ⁇ or ⁇ chains, the TCR ⁇ dimer and TCR dimer.
  • TCR/CD3 complex is sometimes called the “TCR complex.”
  • CD3 is a protein complex composed of four chains in mammals (CD3 ⁇ , CD3 ⁇ and two CD38 chains), that associate with molecules known as the T cell receptor (TCR; see above) and with the ⁇ -chain and ⁇ -chain (as homo- or heterodimers) to generate an activation signal in T lymphocytes.
  • TCR T cell receptor
  • the intracellular tails of these CD3 molecules contain a single conserved motif known as an “immunoreceptor tyrosine-based activation motif” or ITAM for short, which is essential for the signaling capacity of the TCR.
  • ITAM immunoimmunoreceptor tyrosine-based activation motif
  • T-cell costimulatory-receptor protein means a receptor of the T-cell that provides a costimulatory signal. During the activation of T cells, costimulation is often crucial to the generation of an effective immune response. T cells require two signals to become fully activated. A first, antigen-specific, signal is provided through the T cell receptor/CD3 complex. A second signal, the costimulatory signal, is antigen-nonspecific and is provided by costimulatory molecules expressed on the T cell membrane. Examples of T-cell costimulatory-receptor proteins are CD28, OX40, CD40L, 4-1BB and PD-1.
  • the present invention is based on the conception that regulatory T-cells (Treg) that have been modified to possess antibody-type antigen specificity, can be harnessed to suppress T effector cells function in vivo.
  • Treg regulatory T-cells
  • the action of these Treg cells is mediated in an antigen-nonspecific manner, primarily by release of suppressive cytokines in the vicinity where the Tregs are activated or stimulated by an antigen recognized by their TpCR. Once activated, Tregs can suppress bystander T cell responses.
  • preferred target diseases or conditions for this invention are autoimmune diseases, more preferably, organ-specific, T cell-mediated autoimmune diseases.
  • Other examples of undesired immune responsiveness to be targeted herein are graft rejection of solid tissue and organ grafts as well as grafts of suspended cells (e.g. bone marrow (BM) transplants or hemopoietic stem cell (HSV) transplant).
  • BM bone marrow
  • HSV hemopoietic stem cell
  • Another target disease is graft-vs-host disease (GVH) that is a common consequence of a mismatched BM or HSV transplant.
  • An additional condition targeted by this invention is transplant rejection (e.g., of a mismatched kidney) where the recipient's immune effector cells reject the graft.
  • the present invention provides methods for producing Tregs, as well as providing the Tregs produced by those methods, that are based on induction of Foxp3 in T-cells in a process of driving cells along the pathway to Treg status.
  • DNA encoding Foxp3 is transduced or transfected into T-cells using any suitable expression vector as a delivery vehicle in a process to drive these cells to become Tregs. Further support for this conception is found in reports that prevention of Foxp3 expression in vivo results in animals with a propensity for development of autoimmune and lymphoproliferative disorders (Sakaguchi S, et al., J Immunol 1995; 155:1151-64; Hori S et al., Science 2003; 299:1057-61; Khattri R, et al., supra; Fontenot J D et al., Nat Immunol. 2003; 4:330-6.).
  • the starting population can be total PBL, T-cells that have been enriched or isolated from PBL, or CD4+T-cells that have been enriched or isolated from such T-cells (either expressing CD25 or not). These cells can be redirected by transducing the TpCR prior to, concomitantly with, or after transducing DNA encoding Foxp3 DNA, preferably in the form of a Foxp3 expression vector.
  • Walker M R. et al., 2005 , Proc Natl Acad Sci USA. 102:4103-8 have shown that antigen-specific human CD4+CD25+ Treg cells can be generated de novo from CD4+CD25 ⁇ cells. The advantage of the present invention over the approach described by Walker et al.
  • the antigen-specificity and Treg activation requirements are independent of the MHC. This important improvement makes isolation and activation of antigen-specific Tregs simpler and allows for therapeutic methods (described below) in which the antigen can be conveniently administered together with the transferred Treg cells to a desired site, such as an inflammatory site, exemplified by the colon in IBD.
  • Tregs are thymus-derived, express high levels of Foxp3 and suppress activation of effector lymphocytes. Antigen-specific activation of human effector T-cells leads to inducible expression of Foxp3 in a subgroup of the activated effector cells, which subgroup can develop a regulatory (Treg) phenotype. These induced regulatory T-cells can suppress (independently of cell contact) freshly isolated effector cells (Walker M R., et al., 2005, supra; Walker M R., et al., 2003 , J Clin Invest. 112:1437-43).
  • Tregs In mice, both in vitro and in vivo induction of Tregs is achieved by prolonged exposure of effector cells to TGF- ⁇ (Wan Y R, et al., 2005 , Proc Natl Acad Sci USA. 102:5126-31; Mantini M C, et al., 2004 , J Immunol. 72:5149-53; Mantini et al., 2006, supra).
  • This small, peripherally generated population of inducible Tregs are believed to play a central role in regulating and containing ongoing immune responses just as the lack of Treg induction is associated with a propensity for autoimmunity.
  • approaches that specifically redirect regulatory T-cells to suppress the activity of pathological T-cells are beneficial in inflammatory conditions by facilitating localization of Tregs to inflammatory sites and their specific activation by inflammation-associated antigens.
  • Tregs When specifically activated in inflammatory lesions, such Tregs are expected to attenuate inflammatory disease by suppressing pathogenic effector T lymphocytes in an antigen-nonspecific, MHC-unrestricted, manner.
  • the MHC-independent activation and action of Treg cells according to the present invention is an important advantage. Such action is contrasted with the report of Meal D J et al. ( Proc Natl Acad Sci USA 2005; 102:11817-22) of a study of experimental allergic encephalomyelitis (EAE) which described CD4+25+ Treg cells redirected against myelin basic protein (MBP) epitope 89-101-reactive T cells by a CR that included the MBP epitope linked to MHC class II protein.
  • EAE experimental allergic encephalomyelitis
  • MBP myelin basic protein
  • the Treg activity is antigen-specifically focused against the autoreactive T-cells.
  • Such a model requires some degree of MHC-dependency as a single CR can only have domains of a single MHC and thus can only be used for patients with that HLA characteristic.
  • the Treg cells of the present invention act to suppress pathogenic T effector cells in an MHC-independent manner, making them more advantageous for treating autoimmune/inflammatory conditions because they can target common target antigens shared among many individuals.
  • Tregs acted by MHC- and antigen-restricted engagement.
  • these Tregs which express the ligand that is recognized by the TCR of the autoreactive T-cells, are stimulated by such interaction and suppress the effector cells.
  • HLA MHC
  • HLA-DR MHC-II
  • T-body was designed by one of the present inventors and his colleagues as a novel modality for specific redirection and activation of effector T lymphocytes towards pre-defined targets, mostly those associated with neoplastic processes (e.g., Pin thus JHU et al., J Clin Invest 2004; 114:1774-81) and infectious diseases (Bitton N, et al., Curr Top Microbiol Immunol 2001; 260:271-300).
  • the T-body approach was intended to overcome the relative inaccessibility of antibodies to certain sites (such as solid tumors) and the general ineffectiveness of tumor-infiltrating lymphocytes to combat solid tumors by combining into one effector cell population the properties of the humoral and cellular arms of the immune system (Gross G et al., 1989; supra Eshhar Z, et al., Br J Cancer Suppl, 1990; 10:27-9).
  • the preferred T-body chimeric receptors comprise a ligand binding portion, preferably (1) a single chain antibody variable region (scFv) directed against a disease-associated antigen, linked to (2) an optional extracellular spacer and a transmembrane region and (3) one or more intracytoplasmic moieties of T cell costimulatory and stimulator/signaling molecules.
  • a ligand binding portion preferably (1) a single chain antibody variable region (scFv) directed against a disease-associated antigen, linked to (2) an optional extracellular spacer and a transmembrane region and (3) one or more intracytoplasmic moieties of T cell costimulatory and stimulator/signaling molecules.
  • scFv single chain antibody variable region
  • Such CRs as initially developed enable non-MHC restricted, specific antibody-type recognition, homing and penetration of neoplastic tissues.
  • antigen-specific activation of chimeric-receptor bearing T effector cells enabled T cell-mediated destruction of tumor cells either by direct cyto
  • the scFv domain is the preferred recognition unit of the present invention, in other embodiments, it may be substituted by another structure that serves as a targeting ligand (or ligand binding partner) that will facilitate bringing the Treg cells expressing the CR to a selected site or a selected antigen.
  • a targeting ligand or ligand binding partner
  • Capon and colleagues have disclosed a number of CRs of this sort, such as one where a ligand binding partner polypeptide is fused at its C-terminus to the N-terminus of an immunoglobulin constant region. See, for example, Roberts M R. et al., Blood 1994; 84:2878-89; Ashkenazi A et al., Int Rev Immunol.
  • the CR polypeptide of the present invention is characterized broadly as comprising (1) an extracellular portion or domain capable of binding to a ligand (such as a target antigen) in a non-MHC restricted manner, (2) an optional extracellular spacer and a transmembrane domain and (3) a cytoplasmic region (one or more domains) capable of activating an intracellular signaling pathway.
  • a ligand such as a target antigen
  • T cell CRs comprise a first binding domain, a preferred example of which is an extracellular scFv fragment derived from a monoclonal antibody (mAb) specific for a selected antigen.
  • the foregoing domain is fused to a spacer domain (preferably a hinge domain of the Ig family that provides spacing and flexibility), a transmembrane domain, a costimulatory region, for example parts of a CD28 molecule, and a further intracellular signaling moiety for T-cells.
  • spacer domain preferably a hinge domain of the Ig family that provides spacing and flexibility
  • a transmembrane domain preferably a hinge domain of the Ig family that provides spacing and flexibility
  • a costimulatory region for example parts of a CD28 molecule
  • a further intracellular signaling moiety for T-cells include a TCR/CD3 complex-associated ⁇ chain or ⁇ chain, or an ITAM-containing cytoplasmic region such as the ⁇ chain of an Ig Fc receptor (F
  • An ITAM is an “immunoreceptor tyrosine-based activation motif; for reviews, see Humphrey M B et al., Immunol Rev. 2005 December; 208:50-65; Pitcher L A et al., Trends Immunol. 2003; 24:554-60; Isakov N, Receptors Channels. 1998; 5:243-53; Daeron M, Annu Rev Immunol. 1997; 15:203-34; Isakov N, J Leukoc Biol. 1997, 61:6-16; Cambier J C, J Immunol. 1995; 155:3281-5; Flaswinkel H et al., Semin Immunol.
  • TCR ⁇ , ⁇ , ⁇ or ⁇ receptor molecules in the CR for this purpose.
  • the signaling moiety of a cytokine receptor may also be present in the chimeric receptor chain for use in the present invention.
  • adding the signaling portion of the IL-2 receptor will cause the Treg cell to further act as if it had been subjected to external IL-2 upon binding of the extracellular targeting domain to the selected target antigen or ligand.
  • adding the signaling moiety of the TGF ⁇ receptor will induce a T effector cell to become a Treg cell and thus this may also be a useful addition to the chimeric receptor chain of the present invention.
  • CRs expressed on T-cells are known to be functional and, upon exposure to antigen, promote cytokine production (and, when expressed on appropriate effector cell type in the prior art, promoted lysis of antigen-bearing target cells (Stancovski I, et al., J Immunol 1993; 151:6577-82)).
  • An early configuration of a scFv-based CR comprised an extra-cellular recognition domain and an intracellular signaling moiety. Full activation of such T-bodies through the CR required either pre-stimulation of the T-body or activation of a costimulatory pathway by exposure to CD08/CD86 (B7)-bearing antigen presenting cells (APCs).
  • TpCR tripartite chimeric receptor
  • Another useful intracellular signaling domain for the present invention is all or part of the cytoplasmic domain of a phosphotyrosine kinase (e.g., a molecule of the Syk family) which is fused to the CR.
  • a phosphotyrosine kinase e.g., a molecule of the Syk family
  • Use of such a signaling moiety bypasses membrane-proximal signaling events that are often defective in T-cells of subjects with acute or chronic inflammation or cancer.
  • Retroviral-mediated expression of CRs in T-cells in general requires T cell activation which activation is commonly achieved by combined use of anti-CD3 and anti-CD28 antibodies. Such pre-activation was sufficient to prime the T-cells to respond to a signal mediated through the CR upon interaction with the antigen for which the CR is specific—both in vitro and in vivo (e.g., Schwartz R H; Annu Rev Immunol 2003; 21:305-34).
  • a costimulatory signal is advantageous for optimal and sustained T cell function and antigen-driven re-activation, even by targets that often lack ligands for costimulatory molecules.
  • the first (recognition) domain preferably an scFv domain
  • a costimulatory signaling molecule preferably CD28
  • an intracellular activation region such as from the CD3 ⁇ chain or the FcR ⁇ chain.
  • an extracellular recognition site preferably an antibody-based recognition site such as an scFv
  • a CD28 intracellular domain “in series” is linked to a CD28 intracellular domain “in series” and further linked to the intracellular signaling region of the TCR complex ⁇ chain.
  • Such a construct was 20-fold more potent in stimulating IL-2 production upon exposure to solid phase antigen (compared with transfectants expressing CR's lacking the CD28 domain (Finney H M et al., J Immunol 1998; 161:2791-7)). Intracellularly, this domain in the CR binds the p85 subunit of phosphatidylinositol 3′-kinase.
  • One of the present inventors designed a novel tripartite CR composed of a scFv recognition moiety fused to the non-ligand binding part of the extracellular domain (ECD) of CD28, the entire transmembrane and intracellular domains of CD28, and the intracellular stimulatory domain of FcR ⁇ (“scFv-CD28- ⁇ ”) (Eshhar et al., 2001, supra).
  • ECD extracellular domain
  • scFv-CD28- ⁇ the intracellular stimulatory domain of FcR ⁇
  • T lymphocytes from unprimed, na ⁇ ve mice that are Tg for the scFv-CD28- ⁇ TpCR manifested potent responses (proliferation, IL-2 secretion, and rescue from apoptosis) upon stimulation solely by the cognate antigen in immobilized form (Friedmann-Morvinski D et al., supra).
  • CD28 molecules other than, or in addition to, CD28 are exploited to provide costimulatory signals when included in the present CR configuration.
  • Preferred examples of these are the members of the “inducible co-stimulator” (ICOS) family, including OX40 (CD134), CD40 ligand (CD40L, CD154), PD-1 (“programmed death receptor-1), and 4-1BB (CD137).
  • ICOS inducible co-stimulator
  • CD134 CD40
  • CD40L CD40 ligand
  • CD154 CD154
  • PD-1 programmed death receptor-1
  • 4-1BB 4-1BB
  • Each of these ligand/receptor pairs possess distinct functions that differ according to the nature of the stimulus and the “antigenic history” of the T-cells on which they are expressed.
  • CD28 signaling is accompanied by induction of ICOS, which, in turn, co-stimulates CD4+T cell activation.
  • OX40 (studied in the context of tumor-specific adoptive immunotherapy) improved survival and anti-metastatic actions of T effector cells by CD4+T helper cell-dependent mechanism (Weinberg A D, Trends Immunol 2002; 23:102-9).
  • Activation of OX40 promotes expression of anti-apoptotic proteins Bcl-XL and Bcl-2 and, accordingly, enhances the survival and hence the number of antigen-specific CD4+ T-cells, resulting in strong antigen-specific CD4+T cell memory.
  • 4-1BB CD137 costimulatory receptor
  • 4-1BBL Engagement of 4-1BB (CD137) costimulatory receptor with its ligand, 4-1BBL, increased TCR-induced proliferation, survival, and cytokine production in both CD4+ and CD8+T-cells (Cheuk A T et al., Cancer Gene Ther 2004; 11:215-26). Cell survival was associated with increased expression of the anti-apoptotic genes bcl-XL and bfl-1.
  • the interacting ligand/receptor pair 4-1BB/4-1BBL acts to amplify existing costimulatory signals, particularly those emanating from CD28 (Guinn B A et al., J Immunol 1999; 162:5003-10).
  • Human CD4+T-cells express PD-1 and its ligands, PD-L1 and PD-L2, upon activation.
  • Antibodies to the receptor can be agonists or antagonists of the apoptotic pathway.
  • PD-1 engagement can promote ICOS- or CD28-mediated costimulation. (e.g., Bennett F et al., J Immunol. 2003; 170:711-8.
  • CD28 costimulatory domains of CD28, ICOS, OX40 (CD134), and 4-1BB (CD137) in CRs
  • CD4+ and CD8+T-cells The activity of costimulatory domains of CD28, ICOS, OX40 (CD134), and 4-1BB (CD137) in CRs is also known in human CD4+ and CD8+T-cells (Finney H M et al., J Immunol 2004; 172:104-13). In that study, the tripartite genes were electroporated into cells to avoid pre-activation of the cells. When CR-bearing T-cells were stimulated by their specific antigen (CD33), cytokine release and cytotoxic activity were dramatically enhanced compared to cells in which the CRs lacked costimulatory signaling structures.
  • CD33 specific antigen
  • the present invention includes the use of an intracellular domain or part of any of these costimulatory sequences in the CR, it is not certain that signaling evoked by these molecules has practical advantages over use of the CD28 costimulatory sequences alone. So, even though the performance of CD28 appears thus far to be quite satisfactory both in vitro and in vivo, the present invention includes within its scope the use of additional or alternative costimulatory systems to CD28 for generating Treg cells that perform optimally in suppressing T effector cells and treating autoimmune/inflammatory and other conditions as described herein. 4-1BB has been used successfully as an alternative to CD28 in T-bodies. See Zhang et al., J. Immunol., 2007; 179:4910-4918.
  • T-cells Use of transferred T-cells in vivo in adoptive therapy requires that transferred cells survive, overcome the host's homeostatic control mechanisms that may serve to hinder the acceptance of these cells, and migrate to (home to, or traffic to) and accumulate or localize at, the desired target site(s).
  • the immune system utilizes internal stimuli to regulate the total size of lymphocyte pools.
  • the total number of peripheral T lymphocytes remains fairly constant, despite production of new cells, turnover of existing cells, and clonal expansion of antigen-specific cells during an immune response (Jameson S C. Nat Rev Immunol 2002; 2:547-56.).
  • These “internal stimuli,” include cytokines and self-peptide-MHC ligands for the TCR. At least two general mechanisms are believed to be responsible for homeostatic effects of bystander T-cells in limiting proliferation: (1) inhibition by physical T cell-T cell interactions; and/or (2) competition for limited “resources” (e.g., IL-7 and access to APCs with suitable self-MHC ligands).
  • cytokines include those that signal through receptors containing a common ⁇ chain, termed collectively “ ⁇ C cytokines.” These include IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Homeostatic control of na ⁇ ve T cell expansion (examined in vitro) is supported by IL-4, IL-7, IL15 and IL21 through the CD28 transmembrane region, whereas only IL-7 appears to be required in vivo (Jameson, supra).
  • Lymphodepletion or “lymphoablation” is preferably performed to condition a recipient of the transduced Tregs of the present invention. Any method known in the art may be used, for example, irradiation, treatment with certain antimetabolites such as fludarabine, etc. Such treatments have been used in conjunction with adoptive T cell therapy in other contexts. Lymphodepletion in vivo performed as a precursor to adoptive cell transfer is known to boost antitumor immunotherapeutic activity in mice and in humans (as studied particularly with autologous, tumor-reactive T effector cells; Klebanoff C A et al., Trends Immunol 2005; 26:111-7).
  • treatment with homeostatic cytokines may be used to maintain the Treg populations in the recipient.
  • the present inventors' group found that activation of T-cells in general and T-bodies in particular (such as that required during the ex vivo manipulations to express the CR with certain vectors) down regulated expression of the chemokine receptor CXCR4, thereby impairing T cell homing in response to the chemokine SDF-1, for example.
  • SDF-1 is a chemoattractant for T-cells that express the CXCR4 (Bleul C C et al., J Exp Med 1996; 184:1101-9; Beider K et al., Blood 2003; 102:1951-8).
  • Treg cells must either home/migrate to the desired target sites or be administered to such sites.
  • a key factor for success of adoptively transferred T cell therapy (which thus far has been examined most thoroughly with T effector cells in cancer) is maintenance of the transduced T-cells' (effector) function.
  • it is desired to maintain the function of Tregs that have been administered to perform a suppressive function.
  • it may be preferred that the Tregs act in shorter “bursts” to curtail a more acute (rather than a chronic) T effector response.
  • lymphocytes found in tumor patients include CD4+CD25+ Treg cells that suppress T effector cells (Wang H Y et al., Immunity 2004; 20:107-18; Curiel T J, et al., Nat Med 2004; 10:942-9), such “endogenous” suppressive activity must be overcome to optimize the action of redirected T effector cells.
  • the objective is the converse: redirected Treg cells are administered to a subject in need thereof to quell or otherwise inhibit immune/inflammatory responses that characterize autoimmune conditions, transplant rejection, etc.
  • Tregs has a much lower risk of (3).
  • direct administration of Tregs to sites of inflammation should overcome the limitation of (1)-(3).
  • Adjustment of dose regimens (number of cells, frequency of administration) using routine clinical considerations are expected to limit the impact of the above limiting factors.
  • an effective amount of redirected Treg cells are administered to a subject.
  • Preferred carriers for the Treg cells are phosphate buffer, preferably 0.01-0.1M, more preferably 0.05M, or 0.8% saline. Acceptable diluents or carriers for various routes of administration are well-known.
  • Typical dosages are between about 10 6 and about 10 11 Treg cells per injection or infusion, more preferably, about 10 7 to about 10 10 cells. If an antigen is to be administered with the cells (or separately, but to a site where it is intended to activate the cells), a dose of about 0.01 to 100 mg/kg/body preferably, 0.1 to 50 mg/kg/body wt is preferred.
  • An effective amount of Treg cells is that needed to induce a measurable change, generally a decrease, in the severity of any measurable symptom of the disease, preferably more than one symptom, and most preferably, would result in cessation of symptoms and cure of the disease or condition.
  • the above decrease may be at least about 10%, more preferably at least about 20%, more preferably at least about 30%, even more preferably, at least about 40%, and more preferably, at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99%. It is within the skill of the clinical arts to determine when such therapeutic goals have been achieved, and to adjust the dose or frequency of administration accordingly, or to cease further treatment.
  • the Treg cells of the invention may be given once, or on multiple occasions, via a single or multiple routes.
  • the cells may be administered daily, or preferably on alternate days, preferably weekly or biweekly. Administration can range over an interval of several days to weeks, or even months or years.
  • the frequency and duration of administration can be determined empirically, or based on the clinical history and experience of the subject.
  • the cellular compositions of the present invention can be administered by any of a number of means and routes known in the art. Administration is preferably parenteral. Preferred routes include, intravenous, intramuscular, subcutaneous, intraperitoneal, intra-articular, intracerebroventricular, intraluminal (preferably into the lumen of the ileum or colon), rectal or the topical route. Also included is the “intrathecal” route, which is intended to encompass injection, infusion or instillation directly into a cavity or space (thecum) surrounding an organ or body region in which an undesired immune/inflammatory response is occurring. Such spaces include the pleural space, peritoneum, subarachnoid space or dural space, or pericardial space.
  • intracranial spaces The generic term for administration into a sheath encasing an organ is termed “intrathecal (see, for example, definition in Dorland's Medical Dictionary 29 th Edition, WB Saunders (2000) and Stedman's Medical Dictionary, 27 th Edition, Lippincott, Williams & Wilkins (2000)) as meaning “within a sheath.” As used herein, this term is intended to be broader than a more commonly used definition which is limited to intracranial spaces.
  • compositions, methods, and products of this invention are applicable to human and veterinary uses.
  • the preferred subject is a human.
  • mice that express the TNP-specific TpCR, that were recently produced by the present inventors and their colleagues (Friedmann-Morvinski D, 2005) are described herein. These mice are the source of TNP-specific T effector and T regulatory cells and are used as experimental animals in which the induction of colitis is evaluated using the ‘classical’ reactive hapten, TNBS. As a control for these CR-bearing cells, cells from erbB-2-specific TpCR Tg mice that were produced in the present inventors' laboratory are used as they express a CR specific for an irrelevant antigen.
  • mice All mature T-cells and NK cells in these Tg mice express the scFv-CD28-FcR ⁇ construct.
  • Naive Tg T-cells can be fully activated by plastic-immobilized TNP without the need for pre-sensitization. (Friedmann-Morvinski D, et al., supra). Results in the Examples herein show that splenic CD4+CD25+ Tregs isolated from such mice specifically suppress proliferation and cytokine secretion by TNP-specific effector T-cells. Moreover, these Tregs are responsible for the delayed development and attenuation of TNBS-induced colitis in these animals.
  • the level of Tregs in the periphery of the TNP-specific TpCR-expressing strains is higher than in wild-type (WT) mice and that the Tregs do not require pre-activation to exhibit their suppressive activity in vivo. This is believed to result from the cross-reactivity of the SP6 mAb, from which the scFv of the TpCR was derived.
  • retroviral vectors Genetic modification of human peripheral T-cells is achieved in one embodiment using retroviral vectors (Eshhar Z, et al., 2001, supra).
  • the pBullet vector is used, into which the CR-encoding cDNA (Weijtens M E, et al., 1998) is introduced.
  • a bicistronic expression construct is used in which the TpCR and eGFP cDNA are expressed under control of the LTR. This serves to generate a packaging cell based on PG13 that is being used to pseudotype the retroviral vector with the gibbon ape leukemia virus (GALV).
  • GALV gibbon ape leukemia virus
  • lymphocytes are activated in culture with plate-bound anti-CD3 and anti-CD28 mAbs (or using commercial microbeads coated with these antibodies; e.g. from Invitrogen, Miltenyi Biotec, Inc.,) and are transferred to plates coated with RetronectinTM (fibronectin fragment CH-296) plates together with fresh supernatants taken from the packaging cells.
  • RetronectinTM fibronectin fragment CH-296
  • Useful additional reagents are anti-idiotypic antibodies against idiotopes of the scFv of the TpCR. These enable direct labeling and visualization of TpCR on cell membranes. Such antibodies against the SP6 scFv (exemplified below) have been made and used by the present inventors.
  • SEQ ID NO:1 An annotated nucleotide sequence (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) of the TNP-specific TpCR used herein is shown in FIG. 27 .
  • the mature protein begins at amino acid residue 23 of SEQ ID NO:2.
  • a preferred sequence that excludes the scFv above, and that can be linked to any other appropriate ligand binding region, preferably a different scFv specific for another antigen, is that defined by the above sequences beginning at the CD28 region.
  • a preferred nucleotide coding sequence is nucleotides 2203-2523 of SEQ ID NO:1 and amino acids 260-367 of SEQ ID NO:2. Additional nucleotides comprising a 5′ restriction site, and amino acids “inadvertently” encoded thereby, may also be included in a preferred sequence.
  • Additional coding sequence added at the 3′ end of 2203-2523 of SEQ ID NO:1, or additional amino acids encoded thereby and added to at the C-terminus of 260-367 of SEQ ID NO:2, may be present, provided that they permit the encoded sequence, as expressed on the redirected Tregs, to function as a TpCR in ways described herein.
  • Those skilled in the art of cloning and recombinant DNA technology will understand how to modify theses sequences to achieve the desired objective without undue experimentation.
  • Expression vectors comprising the foregoing sequences are also used in the present invention, in the production of redirected, TpCR-expressing Tregs.
  • Redirected T-cells are “converted” to Tregs by causing them to express both Foxp3 transcription factor and the antigen-specific TpCR.
  • Such manipulation permits production of large numbers of Tregs for evaluation and therapeutic use.
  • Successful co-transduction or co-expression is tested by including a Foxp3-GFP fusion gene in the same construct as a TpCR to express both in the same cells.
  • This approach is particularly useful when the starting cell populations are human PBL in which Tregs constitute only about 3-5% of CD4+T-cells. This avoids the complications of another approach, also within the scope of the invention, in which large scale Treg propagation is required for effective transduction with retroviral vectors. Moreover it will simplify the isolation of the Tregs and assessment of their fate in vivo.
  • messenger RNA (mRNA) for Foxp3 is cloned from purified Tregs using PCR.
  • Foxp3 cDNA is cloned into an eGFP Clontech plasmid to create a Foxp3-GFP fusion protein.
  • the fusion protein is cloned into the pBullet vector containing TpCR inserted after an IRES to create a bicistronic expression vector.
  • Both a Foxp3-GFP single gene retroviral vector and a bicistronic TpCR—IRES ⁇ Foxp3-GFP double gene retroviral vector are transduced into isolated CD4+CD25 ⁇ human peripheral blood T-cells following their activation with anti-CD3 and anti-CD28 antibodies.
  • the resulting cells are tested for expression of the three genes by FACS using (1) antiidiotypic antibodies specific for the scFv idiotype, or anti-hinge region antibodies and (2) intracellular GFP and Foxp3 by staining fixed cells with primary antibodies specific for Foxp3 (Alexis Biochemicals, Lausanne, Switzerland).
  • sequential expression protocols are used (first TpCR and then Foxp3-GFP genes) or co-expression protocols.
  • first TpCR and then Foxp3-GFP genes are expressed, relatively large number of Tregs can be obtained and separated using cell sorter (FACSaria fluorescence-activated cell sorting (Becton Dickinson, Mountain View, Calif.), sorting for GFP and TpCR co-expressing cells.
  • the Foxp3 construct may be in the form of a bicistronic vector that includes DNA encoding a reporter molecule such as a fluorescent protein.
  • reporter molecules are well-known in the art and include fluorescent, chemiluminescent or chromogenic proteins, for example Green fluorescent protein (GFP) or enhanced yellow fluorescent protein (EYFP) or a fluorescent homologue thereof, firefly luciferase protein (encoded by the Luc gene) the enzymes chloramphenicol acetyl-transferase (CAT), or bacterial LacZ, ( ⁇ -galactosidase) or the thymidine kinase gene (encoded by the HSV1 TK gene.
  • GFP Green fluorescent protein
  • EYFP enhanced yellow fluorescent protein
  • CAT chloramphenicol acetyl-transferase
  • CAT chloramphenicol acetyl-transferase
  • CAT chloramphenicol acetyl-transferase
  • GFP and EYFP are detected by fluorimetry or fluorescence histochemistry; enzymes are detected by use of a chromogenic substrate that is converted into a colored product which can be used in histochemical colorimetric detection of enzymatic activity. Luciferase is measured by activation of luciferin which emits light at a known wavelength. Reporter molecules may be detected in vivo by non-invasive detection techniques such as fluorescence optical imaging (FOI), bioluminescence optical imaging (BOI), cooled charged coupled device (CCD) camera optical imaging (CCOI) and positron emission tomography (PET).
  • FCI fluorescence optical imaging
  • BOI bioluminescence optical imaging
  • CCD cooled charged coupled device
  • COI positron emission tomography
  • Any method for introducing DNA into a cell and expressing it may be used in the present invention, including, but not limited to vectors such as retroviral or lentiviral vectors, electroporation, lipofection, and the like.
  • Tregs can be determined using co-culture tests as described in the Examples. If APCs are to be used in such tests, a preferred source is irradiated monocytes. The antigen is loaded into irradiated human APCs which will present it to T effectors and Tregs. In the case of antigens such as CEA, human colon carcinoma cells stably transfected with the CEA epitope may be used. In such coculture tests, one may detect specific activation of TpCR-bearing Tregs through the TpCR.
  • Treg activation is assessed by examining these cells' action on T effector cell (1) proliferation and (2) cytokine secretion profile, focusing on IL2, IL4, IL10, IFN- ⁇ and TGF- ⁇ (using commercial ELISA kits, e.g., Ready-Set Go ELISA kit, Ebioscience CA). It is preferred to assay TGF- ⁇ and/or IL-10 as an indication of the cells' Treg phenotype.
  • the present invention redirects Tregs to sites of colonic inflammation, by introducing into such cells CRs with antibody-type specificity.
  • the redirected Tregs are activated to suppress IBD-associated immune response.
  • Tregs endowed with predefined specificity migrate and home to inflamed sites in the colon where they undergo activation and, as a result, suppress T effector cells that mediate the disease processes.
  • the present redirected Tregs represent a novel form of the ‘T-bodies’ discussed above and are employed as a novel therapeutic modality in IBD.
  • These T-bodies are T-cells that have been genetically engineered to express TpCR in which an antibody variable region is the recognition unit linked to T-cell costimulatory and stimulatory domains that enable specific activation of these T-cells but in a manner that is MHC independent and not MHC-restricted. Based on previous studies using tumor models described above, these redirected Tregs are tested in murine models of IBD models.
  • the colon-associated antigen(s) to which the T-bodies are redirected and targeted are not necessarily the pathogenic autoantigens recognized by the autoaggressive T effector cells.
  • this invention can exploit the phenomenon of “bystander” reactivity—where the presence of the relevant antigens at the sites of the inflammatory reactions serve to attract and “hold” or localize the redirected Tregs, permitting them to be activated and to exert their suppressive effects in a paracrine manner—acting on target effector cells in the vicinity irrespective of differences in the T effector cells' and Treg cells' antigen specificity.
  • CEA and LPS-Colonic Antigens as Targets for Redirected Human Tregs in IBD
  • hapten-specific IBD model that is based on specificity to the hapten TNP to study the suppressive effects of Tregs.
  • other antigens that are expressed in intestinal or colonic tissue either normally or in the relevant disease state are preferred targets.
  • The include carcinoembryonic antigen, CEA, and bacterial floral antigens such as lipopolysaccharide, LPS.
  • Treg activation is indeed antigen-specific and thus depends on TCRs, or in the present Tregs, on antibody-based specificity, associated with costimulation together with the activation/mediated by the intracellular signaling moieties of the present constructs.
  • Tregs are activated, their suppressive action is antigen-independent, and is carried out by secretion of suppressive cytokines (e.g., TGF- ⁇ and IL-10) even after the activating antigen has been eliminated.
  • suppressive cytokines e.g., TGF- ⁇ and IL-10
  • CEA is significantly over-expressed in diseased colon tissue in patients with active ulcerative colitis compared to normal individuals and to patients with quiescent IBD (Smithson J E et al., J Pathol. 1996; 180:146-51; Pavelic Z P et al., Anticancer Res. 1991; 11:1671-5). This enhanced tissue expression of CEA was independent of dysplastic changes and is a result of the mucosal reaction to the inflammatory process itself.
  • CEA is a preferred candidate for Treg TpCR targeting in active ulcerative colitis.
  • a second candidate antigen (or “non-antigen” ligand) to which Tregs may be redirected is endotoxin or LPS, derived from the outer membrane of Gram-negative bacteria resident in the colon.
  • the antibody-like part (scFv) the CR's extracellular recognition region may be derived from an anti-LPS antibody, such as the mAb produced by the hybridoma with ATCC Accession No. HB9081.
  • the nucleotide sequence of an scFv made from this mAb is shown as an annotation in FIG. 29 as part of the full sequence of a plasmid (pBullet) comprising this scFv—SEQ ID NO:3.
  • a Treg expressing a TpCR that displays this scFv extracellularly will, at a site where LPS is present such as inflamed colon tissue (whether the gut lumen, the lamina basement or even regional lymph nodes and other gut-associate lymphatic tissue) bind the LPS and be activated to cause suppression of any T effectors cells in the vicinity in an antigen-nonspecific and MHC-independent manner.
  • LPS inflamed colon tissue
  • CD14 (SEQ ID NO:4) is a class of LPS receptor that is a GPI-anchored 356 aa glycoprotein. It contains a 19aa signal peptide, an extracellular domain which contain 11 leucine-rich repeat (LRR) domains, 4 N-glycosylation sites and an unknown number of O-glycosylation sites. At least 2 soluble forms of CD14 have been described, one retains GPI and is released from the cell surface which results in an approximately 48 kDa molecule and the other is released prior to the addition of the GPI anchor resulting in a higher molecular weight (>48 kDa).
  • LRR leucine-rich repeat
  • CD14 While LPS interacts with CD14, CD14 is not capable of initiating a transmembrane activation signal because it is a glycosylphosphatidylinositol (GPI)-anchored protein. Thus, LPS must interact with a transmembrane receptor(s) that is responsible for signal transduction. LPS is recognized by the toll-like receptor TLR4 and MD-2 (SEQ ID NO:5; human), a molecule associated with the extracellular domain of TLR4. CD14 greatly enhances the formation of LPS-TLR4-MD-2 complexes, apparently by LPS loading onto TLR4-MD-2 but not in the interaction itself between LPS and TLR4-MD-2. (Akashi S, et al., J. Exp. Med. 198:1035-42 (2003)).
  • Interaction of LPS with MD-2 in a TLR4-MD-2 complex triggers an intracellular signal transduction cascade that leads to the production and release of proinflammatory cytokines, particularly TNF- ⁇ (Dauphinee S M et al., 2006 , Lab. Invest. 86, 9-22).
  • proinflammatory cytokines particularly TNF- ⁇
  • Patients with IBD show increased colon and serum levels of endotoxin, LBP, CD14, and MD-2 (Pastor Rojo O, et al., 2006 , Inflamm Bowel Dis., December 19 (epub); Amati L et al., Curr Pharm Des. 2003; 9:1937-45; Cario E et al., J Immunol. 2006; 176:4258-66).
  • This change correlates with disease activity, and proinflammatory cytokine levels return to normal after treatment.
  • a motif of human MD-2 for example, from amino acids 119-132 (14 residues) of SEQ ID NO: ______ can substitute for MD-2 in MD-2-TLR4 complex binding to the lipid A moiety of LPS, which (Mancek M et al., Biochem Biophys Res Comm 2002; 292: 880-5; Kobayashi M et al., J Immunol. 2006; 176:6211-8).
  • the extracellular recognition region comprises, in place of an antibody-like structure (e.g., an scFv), a receptor that binds to a ligand that is not acting as an “antigen.”
  • a preferred ligand in the present invention is LPS.
  • the extracellular recognition region may comprise any of the following receptor structures:
  • any of these constructs when displayed on a Treg surface, will allow the redirected Treg to bind to, and be activated by LPS molecules, for example, at colon inflammatory sites, and thereby exert their suppressive activities in that vicinity.
  • LPS molecules for example, at colon inflammatory sites
  • this is an example of receptor-ligand binding/recognition that is not “antibody-like” but nevertheless permits the TpCR to act in accordance with this invention and activate Tregs in an antigen-nonspecific (and MHC-independent) manner.
  • the present invention includes an embodiment in which redirected Tregs bearing a TpCR are designed to be specific for an antigen, referred to herein as “AgX,” that may have no inherent relationship with the tissue being targeted or the disease being treated.
  • the Tregs specific for AgX are activated specifically in a selected site by administering them together with AgX to that site.
  • the site is one where T effector cells are situated and active, where the ongoing inflammation is to be suppressed.
  • the AgX-specific antibody-like receptor of the Tregs will recognize AgX without a need for antigen presentation, MHC, etc., and the linked signaling moieties on the TpCR will serve to activate the Tregs to release inhibitory cytokines at that site. This process will lead to nonspecific suppression of the ongoing T effector cell and inflammatory activity.
  • autoimmune diseases which involve undesired effector T-cells activity as an underlying cause or as a consequence of the pathophysiology.
  • diseases include, but are not limited to, IBD, rheumatoid arthritis, Type I diabetes, multiple sclerosis, autoimmune thyroiditis, autoimmune uveoretinitis, autoimmune orchitis, autoimmune insulitis, autoimmune oophoritis, psoriasis, autoimmune polymyositis and the like. See, for example, Theofilopoulos, A., In: Stites, D P et al., eds., Basic and Clinical Immunology , Lange Medical Publications, Los Altos, Calif., 1988)).
  • CD4+CD25+ Tregs were purified from splenic lymphocytes or peripheral blood mononuclear cell populations using several methods.
  • One method utilized magnetic bead separation (MACS). Spleens are mashed gently into HBSS/5% FCS to prepare single cell suspensions.
  • MCS magnetic bead separation
  • CD4+T-cells were purified by negative selection by incubation with biotin-conjugated CD4 MACS beads (Miltenyi Biotec, Inc., Auburn, Calif.). Further purification of CD4+CD25+ cells was conducted by incubation with phycoerythrin (PE)-conjugated anti-CD25 antibodies or anti-CD45RB high , followed by incubation with anti-PE microbeads (Miltenyi Biotec, Inc., Auburn, Calif.). Magnetic separation was conducted using magnetic columns according to manufacturer's instructions. For highly-purified (>99%) Treg and effector T lymphocyte subpopulation, high-speed cell sorting is be applied, using BD FACSaria (®) cell-sorting system (BD Bioscience)
  • Lamina intestinal lymphocytes from colon were isolated as previously described (Han X et al., Gastroenterology. 2005; 129:185-203). Briefly, colonic mucosa was dissected, followed by incubation with Ca 2+ —Mg 2+ -free Hanks' balanced salt (HBSS) solution containing 1 mM dithiothreitol (Sigma-Aldrich, St. Louis, Mo.) for 30 min to remove mucus, and then serially incubated twice times in medium containing 0.75 mM EDTA (Sigma-Aldrich) for 60 min at each incubation.
  • HBSS Hanks' balanced salt
  • the supernatants from these incubations containing epithelium and intraepithelial lymphocyte population are discarded, and the residual fragments pooled and treated with 2 mg/mL collagenase A (Worthington Biomedical, Freehold, N.J.) and 0.01% DNase (Worthington) in humidified air at 37° C. for 2 hours.
  • the cells are then be pelleted twice through a 40% isotonic Percoll solution, after which they are purified further by Ficoll-Hypaque density gradient centrifugation (40%/75%).
  • Tregs are thymus derived, express high levels of Foxp3 forkhead transcription factor and suppress activation of effector lymphocytes. It has been discovered that antigen-specific activation of human effector T-cells may induce expression of Foxp3 in a subgroup of the activated effector cells, which in turn develop a regulatory phenotype. These induced regulatory T-cells were shown to be capable of cell-contact-dependent suppression of freshly isolated effector cells (Walker et al., 2003, supra). In mice, prolonged exposure of effector cells to TGF- ⁇ induces Tregs both in vitro and in vivo (Fantini et al., J Immunol. 2004 and 2006, supra). This small, peripherally generated population of inducible Tregs may be central in regulation and containment of ongoing immune response, while the inability to induce such Tregs may be responsible for a propensity to develop autoimmunity.
  • T effector cells were isolated by FACS sorting and cultured for 7 days in the presence of either (1) anti CD3 Ab, (2) murine TGF- ⁇ , (3) mAb to TNP, (4) anti CD3 Ab+TGF- ⁇ , or (5) anti TNP Ab+TGF- ⁇ .
  • Induction of Foxp3 in cells “developing” from these effector T-cells was assessed after seven days of culture using intracellular Foxp3 staining
  • Antigen-specific activation of human effector T-cells leads to inducible expression of Foxp3 in a subgroup of activated effector cells, which in turn develop regulatory phenotype. These induced regulatory T-cells are capable of cell-contact-dependent suppression of freshly isolated effector cells.
  • both in vitro and in vivo induction of Tregs can be achieved with prolonged exposure of effector cells to TGF- ⁇ (Fantini et al., 2004, 2006, supra).
  • the present inventors adopted this technology to induce murine redirected Tregs from redirected effector T-cells (see FIG. 3 ).
  • mice strains were used in the studies described below and are used in various other embodiments of the invention. These include transgenic mouse lines that specifically expresses anti-TNP or anti-Erb B2 TpCRs (bearing CD28-FcR signaling chains) under the control of a CD2 promoter, as well as a transgenic mouse line expressing human CEA (Saha A et al., Immunology 2006, 118:483-496)
  • mice All transgenic mice were back-crossed to Balb/c.
  • Balb/c wild-type mice serve routinely as controls and recipients of adoptively transferred cells.
  • One cell-transfer colitis model is used in immune deficient Rag ⁇ / ⁇ and SCID mice.
  • mice were sensitized with 15 ⁇ l of the haptenating agent 2,4,6-trinitrobenzenesulfonic acid (TNBS, Sigma-Aldrich) at a concentration of 2.5% v/v in 50% ethanol by skin painting on day 1.
  • TNBS 2,4,6-trinitrobenzenesulfonic acid
  • 150 ⁇ l of 1% TNBS in 50% ethanol was administered intrarectally via a 3.5 F catheter under general anesthesia.
  • OXA-induced colitis was induced by sensitizing mice with oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one; Sigma-Aldrich) at a concentration of 3% v/v in 100% ethanol by skin painting on day 1, followed by intrarectal administration of 150 ⁇ l at a concentration of 1% v/v in 50% ethanol on day 8.
  • oxazolone 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one; Sigma-Aldrich
  • CD45RB high (na ⁇ ve) T-cells are transferred to immune deficient mice from syngeneic background (Powrie F et al., J Exp Med. 1994; 179:589-600. This model of mucosal inflammation allows separating T effector and Treg cell function within an inflammatory site.
  • colitis is assessed following induction using the following parameters: degree of colon ulcerations, intestinal and peritoneal adhesions, wall thickness, and degree of mucosal edema. Each parameter is graded on a scale from 0 (completely normal) to 4 (most severe) by two experienced, blinded observers. For histological evaluation of inflammation, distal colon tissue (last 10 cm) is removed and fixed in 10% formaldehyde. Five paraffin sections from each mouse are stained with hematoxylin-eosin using standard techniques.
  • the degree of inflammation is graded semiquantitatively on microscopic cross sections of the colon from 0 to 4 as follows: Grade 0: Normal with no signs of inflammation; Grade 1: very low level of leukocyte infiltration; Grade 2: Low level of leukocyte infiltration; and Grade 3: High level of infiltration with high vascular density, and bowel wall thickening; Grade 4: Transmural infiltrates with loss of goblet cells, high vascular density, wall thickening, and disruption of normal bowel architecture.
  • RNA isolation For continuous monitoring of colitis pathology, a newly-developed, high resolution mouse video endoscopic system has been used Becker C et al., Gut. 2005; 54:950-4.
  • the experimental endoscopy system (from Karl Storz, Tuttlingen, Germany) consists of a miniature endoscope (1.9 mm outer diameter), a xenon light source, a triple chip camera, and an air pump. Parameters for grading of colitis include bowel wall thickening, granularity, fecal consistency, fibrin deposition and vascular pattern. Whole colon methylene blue chromoendoscopy staining is used, when appropriate, to visualize crypt pattern. A 3fr. Flexible biopsy forceps is used for biopsy-taking. Biopsies are either placed in formalin for paraffin embedding, sectioning and subsequent immunohistochemistry, frozen in liquid nitrogen for cryosections, or obtained and used for RNA isolation. A typical yield of a biopsy specimen is approximately 2 ⁇ g RNA
  • Redirected Tregs were labeled with the near-infrared (NIR) lipophilic carbocyanine dye 1,1′-dioctadecyl-3,3,3′, 3′-tetramethylindotricarbocyanine iodide (DiR, Invitrogen, USA).
  • NIR near-infrared
  • This dye has absorption and fluorescence maxima at 750 and 782 nm, respectively, enables the safe direct labeling of membranes of human lymphoid cells with very low light absorption and autofluorescence levels in living tissues (Miller M J et al., Proc Natl Acad Sci USA, 2003; 100:2604-9; Kalchenko V et al., submitted for publication, 2007). Additional in vivo visualization of Tregs labeled with carboxy fluorescein diacetate succinimide ester (CFSE) at colonic mucosa was performed by intrarectal insertion of a 300 and 650 ⁇ m diameter confocal microendoscope (Cell Vizio, MKT, Paris, France). This unique modality, previously untested in colitis models, allows repeated in vivo assessment of homing of CFSE-labeled redirected Tregs to the most inner layers of colon tissue following induction of inflammation.
  • CFSE carboxy fluorescein diacetate succinimide ester
  • Colon mRNA expression of selected cytokines is determined to allow assessment of redirected Treg effects on local intestinal immune response. in particular, levels of pro-inflammatory (TNF ⁇ and IFN ⁇ ) and anti-inflammatory cytokines (TGF ⁇ and IL10), as well as levels of the TH 1 transcription factor Tbet and the TH 2 transcription factor GATA-3. Colon cytokine levels are assessed by measuring mRNA expression and protein levels.
  • mice are divided into the following groups: naive mice, colitis-induced mice, and colitis-induced mice adoptively transferred with Tregs (naturally occurring, induced, or redirected, see detailed adoptive transfer experiments herein).
  • the following sets of oligonucleotides and amplification conditions are used:
  • IL-10 and IFN- ⁇ protein expression levels in colon tissue are quantified by a cytofluorimetry-based ELISA system.
  • whole proteins are isolated from colon specimens in the absence of detergent. Proteins (100 ⁇ g) are immediately used for cytokine determination according to manufacturer's instructions.
  • Foxp3 immunofluorescence is performed to estimate in situ the targeting of Treg to diseased colon, using TSA Cy3 and a fluorescence microscope (Olympus).
  • TSA Cy3 and a fluorescence microscope Olympus
  • cryosections are fixed in cold acetone for 10 minutes, followed by sequential incubation with methanol, avidin/biotin (Vector Laboratories, CA), and protein blocking reagent to eliminate nonspecific background staining. Slides are then incubated overnight with primary antibodies specific for Foxp3 (e.g., from Alexis Biochemicals, Lausanne, Switzerland).
  • Tg mice transgenic mice expressing a TNP-specific tripartite chimeric receptor (TpCR) that serve as a source of redirected Treg cells specific for the trinitrophenyl (TNP) hapten.
  • TpCR tripartite chimeric receptor
  • This hapten has served as a “classical” antigen for years in studying both antibodies and T cell-mediated immunity.
  • a chemically reactive form of this hapten, TNBS is a contact sensitizing agent that induces and evokes delayed-type hypersensitivity (DTH) responses as well as inducing colitis in animals, as described herein.
  • DTH delayed-type hypersensitivity
  • TNP-specific Tregs was achieved by the creation of Tg mice that express TNP-specific TpCR that comprises an scFv from the TNP-specific mAb Sp6 mAb linked to a truncated CD28 molecule which was inserted between the scFv and the cytoplasmic part of the FcR ⁇ chain (abbreviated as y herein (see FIG. 1 ).
  • This construct includes the hinge region, transmembrane region, and cytoplasmic region of CD28 but lacks the B7 (ligand) binding site.
  • TpCR/CD28 truncated form of CD28
  • FIG. 1 For the truncated form of CD28 (TpCR/CD28, FIG. 1 ) that does not include the CD28 intracellular signaling domain, the inventors cloned the vector at the same site.
  • a Tg mouse expressing TpCR specific for another, irrelevant antigen (Erb-B2) was used as a control.
  • TpCR For expression of TpCR in T-cells of Tg mice, a construct comprising an anti-TNP (Sp6-derived scFv-CD28- ⁇ was cloned into a human CD2 promoter/enhancer minigene-based vector.
  • Tg mice were generated at the Weizmann Institute's Department for Veterinary Resources by pronuclear microinjection of (BALB/c ⁇ C57BL/6)F 1 fertilized eggs derived from hyperovulated donor females. Founder mice were screened by PCR of DNA from tail samples. Several founder strains were obtained that express high level of the TpCR on their cell surfaces. These were backcrossed for more than nine generations to either BALB/c or C57BL/6 mice to obtain MHC-homogeneous mice.
  • Tregs were isolated using double magnetic bead separation (Miltenyi Biotech) or by fluorescent cell sorting in which fluorescently labeled CD4+CD25+ cells were sorted using the FACSARIA cell sorting system.
  • Treg expression of TNP-specific TpCR was assessed by containing cells for Foxp3 (considered the “gold standard” marker of Tregs) and PE-labeled mAb specific for TNP antibody (generated in the inventors' laboratory). Controls included groups stained with the appropriate isotype controls. As is shown in FIG. 2 , Tregs from TNP-Tg mice, but not from wild-type mice, expressed high levels of TNP-specific TpCR.
  • TNP-Tg Mice Posses Increased Numbers of Foxp3+Tree Population
  • a CD4+CD25+ cell population (represented as the ratio of CD4+CD25+ cells among CD4+T-cells) was elevated modestly in TNP-Tg mice in comparison to control mice (wildtype, ErbB2-Tg and TNP-CD28 null-Tg mice).
  • higher numbers of Foxp3+ cells were observed in TNP-Tg animals compared to the control animals in comparison to all other mouse types ( FIG. 4 ).
  • effector CD4+CD25 ⁇ cells were isolated by cell sorting to a level of 99% purity. Isolated cells were stained for Foxp3 ( FIG. 5 ). As expected, no positive Foxp3 staining was noted in T effector cells from wildtype, ErbB2-Tg and TNP-CD28null-Tg mice. In contrast, TNP-Tg T effector cells featured a significant population of Foxp3+ cells. This observation was further validated in whole spleen cell populations that were co-staining for Foxp3 and CD25 ( FIG. 6 ).
  • Tregs expressing TNP-specific TpCR in the treatment of autoimmunity is verification of their regulatory activity, namely an ability to suppress T effector cell proliferation in a dose-dependent manner. Also examined was whether such Treg activation occurs as a result of TpCR signaling, and whether it was indeed independent of CD28-B7 interaction.
  • CD4+CD25+ Treg cells and CD4+CD25 ⁇ T effector cells from different Tg mouse founders were purified from bulk splenocytes. 10 5 cells were incubated in vitro for 24 h, 48 h or 72 hrs ( FIG. 9 ) and activated non-specifically with anti CD3 and anti-CD28 Abs, or specifically with Fowl gamma globulin-modified TNP (FyG-TNP).
  • T cell proliferation was measured using either the uptake of a dye (tetrazolium salt XTT) or radiolabeled Thymidine.
  • IL2 secretion was measured using XTT staining of the IL-2-dependent CTLL-2 cell line.
  • TNP-Tg Tregs were cocultured in 96-well microplates (0.2 ml) with irradiated antigen presenting cells (APCs) and T effector cells (CD4+CD25 ⁇ ) at 1:1 ratios.
  • APCs irradiated antigen presenting cells
  • CD4+CD25 ⁇ T effector cells
  • Cells in these culture were activated either by (1) immobilized antigen “mimic” (anti-CD3+ anti-CD28) or (2) soluble Concanavalin A (ConA).
  • T cell proliferation was measured as Thymidine uptake and IL2 secretion was measured as growth of cells of the IL-2-dependent CTLL-2 cell line (XTT staining).
  • FIG. 10 shows a ConA experiment.
  • Non-specific (polyclonal) stimulation of Tregs induced these cells to exhibit potent inhibition of T effector cell proliferation and IL2 secretion, irrespective of the origin of the Tregs or the presence of the chimeric receptor.
  • genetic manipulation of Tregs of the type described here preserves their suppressive properties.
  • TNP-loaded APCs provided the Ag presentation ( FIG. 11 ). Comparisons of TNP-specific Treg stimulation was performed, comparing wildtype vs. TNP-Tg Tregs ( FIG. 11 , left panel) or ErbB2-Tg and TNP-Tg Tregs ( FIG. 11 , right panel). In the absence of TNP stimulation, T effector cell proliferation did not occur (left-most bars in both graphs). In contrast, incubation with TNP-modified APC's resulted in:
  • FIG. 12 Co-culture of varying ratios of TNP-specific Tregs and TNP-specific effector T-cells ( FIG. 12 ) demonstrated successful antigen-specific inhibition by Tregs at a ratio of 1 Treg to 8 T effector cells.
  • TNP-modified P815 cells a cell line that does not express B7
  • TNP-loaded genetically modified P815 cells stably expressing the B7 gene
  • Stimulation of TNP-Tg effector T-cells with TNP-P815 cells induced proliferation, which was markedly suppressed by TNP-Tg Tregs.
  • Expression of B7 on these APC's did not promote any further Treg-mediated suppression. It was concluded that maximal Treg suppression occurred independently of B7. Some suppression was also noted with wildtype Tregs.
  • TNBS is a potent inducer of T-cell responses such as DTH/contact sensitization. This reactive hapten also induces autoimmune colitis when applied to the colon of pre-sensitized mice.
  • TpCR-bearing Tregs could suppress autoimmunity
  • the acute TNBS-mediated colitis model was employed. Intra-rectal administration of TNBS leads to its binding to colon proteins, rendering these modified proteins immunogenic so that they elicited a T cell mediated immune response.
  • the suppressive effect of endogenous or exogenously transferred Tregs on autoimmune inflammatory disease was tested in this model.
  • a different hapten, oxazolone (OXA) with similar sensitizing properties and which induces experimental colitis was used as a specificity control in vivo.
  • TNP hapten-mediated colitis was induced in Tg and WT mice by first sensitizing the animals with 150 ⁇ l of the 2,4,6-trinitrobenzenesulfonic acid (TNBS, Sigma-Aldrich) at a concentration of 2.5% in 50% ethanol painted on the skin on day 1. On day 8, the antigen was administered rectally (150 ⁇ l of 1% TNBS in 50% ethanol; high dose colitis). WT mice developed severe colitis within 2-5 days of rectal TNBS administration ( FIG. 14 , left panel). In contrast, 90% of the TNP-Tg mice had normal looking colons ( FIG. 14 , right). Colitis severity scores were as follows:
  • Tregs are thymus derived, express high levels of Foxp3 and suppress activation of effector lymphocytes. Antigen-specific activation of human effector T-cells may induce expression of Foxp3 in a subgroup of the activated effector cells, which in turn develop a regulatory phenotype. These induced regulatory T-cells were shown to be capable of cell-contact-dependent suppression of freshly isolated effector cells (Walker et al., 2003, supra). In mice, it has been demonstrated that prolonged exposure of effector cells to TGF- ⁇ induces Tregs both in vitro and in vivo (Fantini et al., 2004, 2006, supra). This small, peripherally generated population of inducible Tregs may be central in regulation and containment of ongoing immune response, while the inability to induce such Tregs may be responsible for a propensity to develop autoimmunity.
  • T effector cells were isolated by FACS sorting and cultured for 7 days in the presence of either (1) anti CD3 Ab, (2) murine TGF ⁇ , (3_mAb to TNP, (4) anti-CD3 Ab+TGF- ⁇ , or (5) anti-TNP Ab+TGF- ⁇ .
  • Induction of Foxp3 in cells “developing” from these effector T-cells was assessed after seven days of culture using intracellular Foxp3 staining ( FIG. 18 ).
  • induction of Tregs in this manner permits the generation of large populations of TpCR-bearing Tregs that can be used in cell-based therapy of autoimmunity.
  • TNP-Tg Tregs are responsible for the resistance of TNP-Tg mice to TNBS colitis and to evaluate their therapeutic capacity in autoimmunity.
  • Wildtype, TNP-Tg and Erb-b2-Tg Tregs were isolated and administered in varying numbers to wildtype mice a day after induction of TNBS colitis.
  • adoptive transfer of large numbers of Tregs of any origin caused nonspecific attenuation of TNBS colitis. This is believed to result from the presence of a sufficiently large population of pre-activated Tregs that can exert their suppressive activity in the absence of antigen stimulation or specificity.
  • FIG. 21 shows marked bowel shortening, a manifestation of colon inflammation (in WT and ErbB2-Tg, but not in TNP-Tg mice).
  • FIG. 22 shows the severe transmural inflammation, necrosis, mucosal bleeding and loss of normal architecture in colons of WT mice with TNBS colitis that had received control (WT and ErbB2-Tg), but not in TNP-Tg colons.
  • Tregs were isolated and stained with the fluorescent intracellular dye, carboxyfluorescein diacetate succinimidyl ester (CFSE). Following staining, 10 6 Tregs were administered intraperitoneally (ip) to control WT mice or to WT mice in which TNBS colitis had been induced 12 hours earlier. Sixteen hours after this treatment, mice were sacrificed and lamina intestinal lymphocytes isolated from their colons. The protocol used was described above to isolate lamina limba lymphocytes.
  • CFSE carboxyfluorescein diacetate succinimidyl ester
  • Tregs for adoptive therapy of autoimmune inflammation of the Treg in diseased organs, where they are expected to exert their suppressive effects.
  • WT and TNP-Tg Tregs were labeled with CFSE and transferred to WT mice 24 hours following induction of TNBS colitis. While very small numbers of CFSE-labeled WT Tregs were observed in cell extracted from colonic lamina intestinal of na ⁇ ve or TNBS colitis-induced mice, a nine-fold increase in TNP-Tg Tregs was noted ( FIG. 23 ).
  • TNP-Tg Treg localization of in the living animal was employed (Xenogen, Alameda Calif.). Wildtype and TNP-Tg Tregs, 1.5 ⁇ 10 6 , labeled with the near-infrared lipophilic carbocyanine dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR, Invitrogen USA), were administered ip to WT mice with or without TNBS colitis, who were monitored daily with the IVIS whole body CCCD camera ( FIG. 24 ).
  • DiR near-infrared lipophilic carbocyanine dye
  • TNP-Tg Treg-administered to WT mice with TNBS colitis featured a distinct abdominal fluorescent signal for up to a week following cell transfer, substantially stronger than that of wildtype Tregs at all time points.
  • TNP-Tg Tregs reach the inner colonic mucosal layer, the location where most of TNBS-induced mucosal damage takes place, the Cell Vizio confocal microendo-scopy system was employed (Cell Vizio, MKT, Paris, France).
  • An intrarectally-inserted 650 ⁇ m diameter confocal microendoscope enabled visualization of CFSE-labeled cells in up to a 150 ⁇ m bowel wall thickness ( FIG. 25 ).
  • TNP-Tg Tregs Specific to a Bystander Antigen (TNBS) Cures Colitis Mediated by a Pathogenic (Oxazolone) Antigen
  • IBD inflammatory bowel disease
  • WT and TNP-Tg mice were pre-sensitized to oxazolone only. A mixture of oxazolone and low doses of TNBS were introduced intrarectally. As is shown in FIG.
  • TNP-Tg mice administered TNBS+oxazolone featured normal-appearing colonic mucosa with scattered areas of mild colitis ( FIG. 26 b , box II).
  • colons of concomitantly TNBS-treated and oxazolone-treated WT mice featured severe colitis, as opposed to the near-normal colons in TNP-Tg mice ( FIGS. 26 c and 26 d , respectively.)
  • FIG. 27 The results are shown in FIG. 27 .
  • the upper half of the Figure shows the GFP-only controls, whereas the lower half of the Figure shows GFP-Foxp3 constructs.
  • the two-paneled rectangles in the Figure show light microscopic (left half) and fluorescence microscopic (right half) images of the same material (to visualize and localize the GFP).
  • chimeric receptor made of the full length MD2 protein (SEQ ID NO:5) or the CD14 protein (SEQ ID NO:4) was confirmed by the ability of transduced cells, which expressed the extracellular region of the CR on their surface, to bind the ligand of MD2 and CD14, bacterial LPS, which was provided in biotinylated form and revealed by secondary binding of fluorescent avidin.
  • Nucleic acid constructs and vectors that encode extracellular regions that comprise an anti-LPS antibody domain have been made and others can be made.
  • Such vectors express extracellular polypeptide domains that are shown to bind LPS, for example in an assay using biotinylated LPS and detectably labeled (e.g., fluorescently labeled) avidin. See also Example XVI above.
  • Nucleic acid constructs and vectors that encode extracellular regions that comprise a LPS-binding nonantibody polypeptide have been made (e.g., SEQ ID NO:6-11, 13 and 14). Such constructs include bicistronic ones that also comprise Foxp3. Other such constructs can be made using the method described above along with methods well-known in the art. Such constructs (such as SEQ ID NO: 13 and 14) include those encoding full length CD14 (SEQ ID NO:4) or MD2 (SEQ ID NO:5) protein, and constructs encoding LPS-binding motifs therefrom (such as SEQ ID NO:6-9) and combinations (such as SEQ ID NO:10 and 11). The constructs that are made include those with CD28-FcR ⁇ intracellular stimulatory/costimulatory regions and those that utilize others of the type disclosed herein.
  • Treg cells are redirected as described herein using the above constructs, including those that have been made and tested and those that can be made.
  • Treg cells are administered into subjects suffering from IBD, such as ulcerative colitis.
  • Treg cells are administered in numbers in accordance with the above examples, or in numbers that are readily determined to be effective by those skilled in the art using only routine experimentation, and via routes of administration as exemplified above and disclosed throughout this document.
  • These redirected Treg cells that express an LPS binding antibody region or another LPS-binding moiety on their surface (CD14, MD2, fragments thereof, or combinations of these) as part of their CR's are able to reduce the symptoms, intensity, severity and duration of the IBD in the subject to a significant degree compared to untreated control subjects or control subjects administered with control Tregs.

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LT2126054T (lt) 2016-10-10
PT2126054T (pt) 2016-10-12
HUE030386T2 (en) 2017-05-29
PL2126054T3 (pl) 2017-01-31
SI2126054T1 (sl) 2016-12-30
HRP20161191T1 (hr) 2016-11-18
WO2008095141A2 (en) 2008-08-07
EP3097923B1 (en) 2022-07-27
US11326147B2 (en) 2022-05-10
IL200104A0 (en) 2010-04-15
CY1118124T1 (el) 2017-06-28
ES2926805T3 (es) 2022-10-28
EP3097923A1 (en) 2016-11-30
EP2126054B1 (en) 2016-07-06
ES2595307T3 (es) 2016-12-29
WO2008095141A3 (en) 2008-12-11
EP2126054A2 (en) 2009-12-02
US20200056152A1 (en) 2020-02-20

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