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

Abstract

A redirected Treg cell is endowed with specificity toward a selected target antigen or ligand. The cell contains a chimeric receptor polypeptide that is expressed in a single, continuous chain, with an extracellular recognition region displayed on the surface of the cell, a transmembrane region and an intracellular signaling region. The extracellular recognition region is specific for the selected target antigen or ligand. The intracellular signaling region includes a combination of T-cell signaling polypeptide moieties, which combination, upon binding of the extracellular recognition region to the selected target antigen or ligand, triggers activation of the redirected Treg cells to cause suppression of T-cell mediated immunity. Such redirected Treg cells may be used to suppress undesired activity of T effector cells thereby mediating an immune or inflammatory response. They are particularly useful in treating T effector cell-mediated diseases, such as inflammatory bowel disease, transplant rejection and GVH disease.

1 . A redirected regulatory T lymphocyte (Treg cell) endowed with specificity toward a selected target antigen or ligand, which cell comprises a chimeric nucleic acid that encodes a chimeric receptor polypeptide that comprises, expressed in a single, continuous chain, an extracellular recognition region, a transmembrane region and an intracellular signaling region, and is expressed in the redirected Treg cell so as to display the extracellular region on the cell surface, wherein
 (a) said extracellular recognition region of said chimeric receptor is specific for the selected target antigen or ligand; and   (b) said intracellular signaling region comprises a combination of T-cell signaling polypeptide moieties, which combination of moieties, upon binding of the extracellular recognition region to the selected target antigen or ligand, triggers activation of the redirected Treg cells to cause suppression of T-cell mediated immunity.   
     
     
         2 . A redirected Treg cell in accordance with  claim 1 , wherein said extracellular recognition region comprises an antibody-derived scFv domain that is specific for a selected antigen. 
     
     
         3 . A redirected Treg cell in accordance with  claim 1 , wherein said extracellular recognition region comprises a member of a ligand-receptor pair, which is specific for the other member of that pair. 
     
     
         4 . A redirected Treg cell in accordance with  claim 1 , wherein said extracellular recognition region does not comprise an MHC protein extracellular domain. 
     
     
         5 . A redirected Treg cell in accordance with  claim 1 , wherein said extracellular recognition region is linked to said transmembrane region through a flexible spacer. 
     
     
         6 . A redirected Treg cell in accordance with  claim 5 , wherein said flexible spacer is a hinge from a molecule of the immunoglobulin superfamily. 
     
     
         7 . A redirected Treg cell in accordance with  claim 1 , wherein said intracellular region comprises a signaling moiety from (i) a polypeptide chain of an antigen-specific receptor of a T-cell and/or (ii) a polypeptide chain of a receptor of a T-cell that has a region comprising an immunoreceptor tyrosine-based activation motif (ITAM). 
     
     
         8 . A redirected Treg cell in accordance with  claim 7 , wherein said intracellular region comprises a signaling moiety from (ii). 
     
     
         9 . A redirected Treg cell in accordance with  claim 7 , wherein said intracellular region comprises a signaling moiety from a polypeptide chain selected from the group consisting of a chain of the TCR/CD3 complex, and the γ chain of an Ig Fc receptor (FcRγ). 
     
     
         10 . A redirected Treg cell in accordance with  claim 9 , wherein said polypeptide chain of an antigen-specific receptor of a T-cell is the CD3-ζ chain or the FcRγ chain. 
     
     
         11 . A redirected Treg cell in accordance with  claim 1 , wherein said intracellular region includes a signaling moiety of a T cell costimulatory-receptor protein. 
     
     
         12 . A redirected Treg cell in accordance with  claim 11 , wherein said costimulatory-receptor protein is at least one protein selected from the group consisting of CD28, OX40, CD40L, 4-1BB and PD-1. 
     
     
         13 . A redirected Treg cell in accordance with  claim 11 , wherein said costimulatory-receptor protein is CD28. 
     
     
         14 . A redirected Treg cell in accordance with  claim 11 , wherein said costimulatory-receptor protein is 4-1BB. 
     
     
         15 . A redirected Treg cell in accordance with  claim 1 , wherein said intracellular region comprises at least two different ones of said costimulatory-receptor protein signaling moieties. 
     
     
         16 . A redirected Treg cell in accordance with  claim 15 , wherein said intracellular region comprises an intracellular signaling moiety from CD28 and from 4-1BB. 
     
     
         17 . A redirected Treg cell in accordance with  claim 1 , wherein said intracellular region comprises a signaling moiety from a T cell cytokine receptor. 
     
     
         18 . A redirected Treg cell in accordance with  claim 17 , wherein said cytokine receptor is the IL-2 receptor. 
     
     
         19 . A redirected Treg cell in accordance with  claim 17 , wherein said cytokine receptor is the TGF-β receptor. 
     
     
         20 . A redirected Treg cell in accordance with  claim 1 , wherein said intracellular signaling region includes a signal-transducing enzyme that (a) is an enzyme in the signal transduction pathway of an antigen-specific receptor of a T-cell or (b) is an enzyme with corresponding specificity and activity as the enzyme of (a), derived from a non-T-cell lymphocyte. 
     
     
         21 . A redirected Treg cell in accordance with  claim 20 , wherein said signal-transducing enzyme is a member of the Syk-kinase family. 
     
     
         22 . A redirected Treg cell in accordance with  claim 1 , wherein said chimeric nucleic acid further includes a nucleotide sequence that will cause the redirected Treg to express Foxp3. 
     
     
         23 . A redirected Treg cell in accordance with  claim 1 , wherein said selected target antigen or ligand is one that is present or expressed at a site or target tissue of an autoimmune or inflammatory response mediated by effector T-cells. 
     
     
         24 . A redirected Treg cell in accordance with  claim 23 , wherein said autoimmune or inflammatory response comprises an autoimmune response or disease, an allograft or xenograft rejection, or graft-vs. host (GVH) disease. 
     
     
         25 . A redirected Treg cell in accordance with  claim 1 , wherein said target antigen or ligand is an autoantigen or an antigen that is cross-reactive with an autoantigen. 
     
     
         26 . A redirected Treg cell in accordance with  claim 25 , wherein the autoantigen is a pathogenic antigen in the pathophysiology of said autoimmune disease. 
     
     
         27 . The redirected Treg cell of  claim 1 , wherein said autoimmune or inflammatory response and said target antigen or ligand are selected from the group consisting of:
 (a) inflammatory bowel disease (IBD), wherein said antigen or ligand is one that is expressed in diseased colon or ileum;   (b) rheumatoid arthritis, wherein said antigen or ligand is an epitope of collagen or an antigen present in joints;   (c) Type I diabetes mellitus or autoimmune insulitis, wherein said antigen or ligand is a pancreatic β cell antigen;   (d) multiple sclerosis, wherein said antigen or ligand is a myelin basic protein antigen, MOG-1, MOG-2 or another neuronal antigen;   (e) autoimmune thyroiditis, wherein said antigen or ligand is a thyroid antigen;   (f) autoimmune gastritis, wherein said antigen or ligand is a gastric antigen;   (g) autoimmune uveitis or uveoretinitis, wherein said antigen or ligand is S-antigen or another uveal or retinal antigen;   (h) autoimmune orchitis, wherein said antigen or ligand is a testicular antigen;   (i)) autoimmune oophoritis, wherein said antigen or ligand is an ovarian antigen;   (j) psoriasis; wherein said antigen or ligand is a keratinocyte antigen or another dermal or epidermal antigen;   (k) vitiligo, where said antigen or ligand is a melanocyte antigen;   (l) autoimmune prostatitis, wherein said antigen or ligand is a prostate antigen;   (m) any undesired immune response, wherein said antigen or ligand is an activation antigen expressed on T effector cells present at the site of the undesired immune response;   (n) tissue rejection, wherein said antigen or ligand is the MHC molecule having the haplotype of the transplanted tissue, or a portion of that MHC molecule; and   (o) an inflammatory condition, wherein said antigen or ligand is one that is expressed on nonlymphoid cells of the hemopoietic lineage that participate in inflammation.   
     
     
         28 . A redirected Treg cell in accordance with  claim 27 , wherein said autoimmune disease is IBD and said antigen or ligand is carcinoembryonic antigen (CEA), or an antigen of intestinal bacterial flora. 
     
     
         29 . A redirected Treg cell in accordance with  claim 27 , wherein the activation antigen of (m) expressed on T effector cells is CD69 or CD107a. 
     
     
         30 . A redirected Treg cell in accordance with  claim 27 , wherein said target antigen or ligand of (o) is one that is expressed on a dendritic cell, macrophage/monocyte, granulocyte or eosinophil present at said inflammation site. 
     
     
         31 . 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) a Treg cell according to  claim 1 , and   (b) a pharmaceutically and immunologically acceptable carrier, excipient or diluent.   
     
     
         32 . A method of producing the redirected Treg cell of  claim 1 , comprising:
 (a) obtaining from a subject and, optionally enriching or isolating and propagating, a population of lymphocytes or T-cells;   (b) inducing a Treg cell phenotype in said cells by suitably stimulating or activating the cells by exposure to TGF-β or another cytokine that induces Foxp3 expression thereby inducing the Treg phenotype;   (c) before or after step (b), transducing the cells ex vivo with an expression vector encoding said chimeric receptor to be expressed on said Treg cell; and   (d) optionally, growing or expanding the cells obtained as above in vitro.   
     
     
         33 . A method of producing the redirected Treg cell of  claim 1 , comprising:
 (a) obtaining from a subject and, optionally enriching or isolating and propagating, a population of lymphocytes or T-cells;   (b) transducing the cells ex vivo with a vector encoding the chimeric receptor;   (c) before, after, or concomitantly with step (b), transducing the cells ex vivo with a recombinant nucleic acid expression construct encoding Foxp3; and   (d) optionally, growing or expanding the cells obtained as above in vitro.   
     
     
         34 . A method of suppressing undesired activity of T effector cells in mediating an immune or inflammatory response, comprising delivering to a site of T effector cells to be suppressed an amount of Treg cells according to  claim 1  effective to suppress said T effector cell activity. 
     
     
         35 . The method of  claim 34  wherein said Treg cells are administered systemically. 
     
     
         36 . The method of  claim 34  wherein the redirected Treg cells are administered regionally or locally to a site of inflammation. 
     
     
         37 . The method of  claim 34  wherein said undesired activity of T effector cells is an autoimmune inflammatory response or disorder, rejection of a transplant or GVH disease. 
     
     
         38 . A method for treating or ameliorating symptoms of a disease or condition in a mammalian subject that is mediated by undesired activity of T effector cells, said method comprising administering to said subject in need thereof an effective amount of Treg cells in accordance with  claim 1 , wherein said target antigen or ligand is one that is present at the site of said undesired T effector cell activity. 
     
     
         39 . The method of  claim 38  wherein the redirected Treg cells are produced by obtaining a population of T-cells from the mammalian subject to be treated, transfecting said cells with said chimeric nucleic acid and causing the cells to express Foxp3 by
 (i) stimulating the transfected cells to induce Foxp3 expression, or   (ii) introducing an exogenous Foxp3-encoding construct into the transfected cells.   
     
     
         40 . The method of  claim 38  wherein the redirected Treg cells are produced by obtaining a mixed population of T-cells from the mammalian subject to be treated, enriching or purifying Treg cells from the mixed population of T-cells on the basis of the Treg cells' expression of CD4 and CD25 and/or Foxp3, and transfecting said enriched or purified Treg cells with said chimeric nucleic acid. 
     
     
         41 . The method of  claim 40  wherein the redirected Treg cells are produced by:
 (a) obtaining
 (i) peripheral blood mononuclear cells, 
 (ii) peripheral blood lymphocytes, 
 (iii) T-cells enriched or purified from (i) or (ii), or 
 (iv) a subset of T-cells enriched or purified from (iii); 
   (b) exposing the cells obtained in (a), ex vivo, to an amount of TGF-β or other Treg-inducing cytokine or agent that is effective to induce expression of Foxp3 and convert T-cells to a Treg phenotype,   (c) optionally, culturing and expanding said exposed cells of (a); and   (d) before after or between said steps (a) and (b), transfecting said cells with said chimeric nucleic acid.   
     
     
         42 . The method of  claim 38  wherein said disease or condition comprises an autoimmune response or disease, an allograft or xenograft rejection, or graft-vs. host (GVH) disease. 
     
     
         43 . The method of  claim 38 , wherein said disease or condition is IBD and said target antigen or ligand is CEA, or an antigen of intestinal bacterial flora. 
     
     
         44 . A method of  claim 38 , wherein said target antigen or ligand is LPS. 
     
     
         45 . A method of  claim 44 , wherein said extracellular recognition region comprises an LPS-binding domain of MD-2 or an LPS-binding domain of CD-14 or both an LPS-binding domain of MD-2 and an LPS binding domain of CD-14. 
     
     
         46 . The method of  claim 38 , further including the step of, either before, concomitantly with, or after administration of the redirected Treg cells, introducing to a subject to the site or target tissue of said immune or inflammatory response, an exogenous antigen or ligand and said target antigen or ligand is said exogenous antigen or ligand. 
     
     
         47 . A chimeric DNA molecule comprising:
 (a) a first DNA segment comprising a sequence encoding an extracellular recognition region specific for a selected target antigen or ligand, which region does not comprise an MHC protein extracellular domain, said selected target antigen or ligand being one that is present or expressed at a site or tissue of a pathogenic or undesired immune response mediated by effector T-cells;   (b) a second DNA segment comprising a sequence encoding a transmembrane region; and   (c) a third DNA segment comprising a sequence encoding an intracellular signaling region comprising a combination of T-cell signaling polypeptide moieties, which combination of moieties trigger activation of the Treg cells to cause suppression of T-cell mediated immunity when the chimeric DNA is transfected into a Treg cell and said extracellular recognition region binds to the selected target antigen or ligand,   wherein, upon transfection of the chimeric DNA into a Treg cell, the Treg cell expresses said extracellular recognition region on its surface, said transmembrane region and said intracellular signaling region in one single, continuous chain.   
     
     
         48 . A chimeric receptor polypeptide encoded by the chimeric DNA molecule of  claim 47 .