Methods and compositions for expanding t regulatory cells

Abstract

The present invention provides methods and compositions for expanding Treg cells ex vivo or in vivo using one or more conjugates comprising a costimulatory moiety that stimulates at least one of three signals involved in Treg cell development and/or using dendritic cells pulsed with antigens and modified to display TGF-β, or hematopoetic stem cells or bone marrow cells modified to display TGF-β. The methods and compositions are useful, for example, in the treatment and prevention of autoimmune disease, including Type 1 diabetes and in preventing foreign graft rejection, as well as to establish mixed chimerism, induce tolerance to autoantigens, alloantigens or xenoantigens, beta cell regeneration, prevention of foreign graft rejection, and treatment of a genetically inherited hematopoietic disorder.

1 . A combination comprising:
 (A) one or more conjugates selected from the group consisting of:
 (a) a first conjugate comprising (i) a first conjugate member comprising a 4-1BBL polypeptide and (ii) second conjugate member comprising a first member of a binding pair; 
 (b) a second conjugate comprising (i) a first conjugate member comprising a CD80 polypeptide and (ii) a second conjugate member comprising a first member of a binding pair; and 
 (c) a third conjugate comprising (i) a first conjugate member comprising a TGF-β polypeptide and (ii) a second conjugate member comprising a first member of said binding pair; 
   and   (B) one or more conjugates selected from the group consisting of:
 (a′) a fourth conjugate comprising (i) a first conjugate member comprising an anti-CD3 antibody and (ii) a second conjugate member comprising a second member of said binding pair; 
 (b′) a fifth conjugate comprising (i) a first conjugate member comprising a cytokine and (ii) a second conjugate member comprising a second member of said binding pair; 
 (c′) a sixth conjugate comprising (i) a first conjugate member comprising an antigen and (ii) a second conjugate member comprising a second member of said binding pair; and 
 (d′) a seventh conjugate comprising (i) a first conjugate member comprising an anti-CD28 antibody and (ii) a second conjugate member comprising a second member of said binding pair. 
   
     
     
         2 . The combination of  claim 1 , wherein at least one of said first, second or third conjugates comprises a fusion polypeptide comprising said first conjugate member and said second conjugate member. 
     
     
         3 . The combination of  claim 1 , wherein said cytokine is selected from the group consisting of IL-2, IL-4, or IL-7. 
     
     
         4 . The combination of  claim 1 , wherein said antigen is an autoantigen. 
     
     
         5 . The combination of  claim 1 , wherein said antigen is selected from the group consisting of insulin, collagen, myelin basic protein, MHC/antigen complexes, glutamic acid decarboxylase (GAD), an islet cell autoantigen (ICA), and autoantigen NRP-A7. 
     
     
         6 . The combination of  claim 1 , wherein said conjugates are provided in separate compositions. 
     
     
         7 . The combination of  claim 1 , wherein said conjugates are provided in a single composition. 
     
     
         8 . A method of expanding Treg cells comprising contacting a population of Treg cells with
 (A) one or more conjugates selected from the group consisting of:
 (a) a first conjugate comprising (i) a first conjugate member comprising a 4-1BBL polypeptide and (ii) second conjugate member comprising a first member of a binding pair; 
 (b) a second conjugate comprising (i) a first conjugate member comprising a CD80 polypeptide and (ii) a second conjugate member comprising a first member of a binding pair; and 
 (c) a third conjugate comprising (i) a first conjugate member comprising a TGF-β polypeptide and (ii) a second conjugate member comprising a first member of said binding pair; 
   and   (B) one or more conjugates selected from the group consisting of:
 (a′) a fourth conjugate comprising (i) a first conjugate member comprising an anti-CD3 antibody and (ii) a second conjugate member comprising a second member of said binding pair; 
 (b′) a fifth conjugate comprising (i) a first conjugate member comprising a cytokine and (ii) a second conjugate member comprising a second member of said binding pair; 
 (c′) a sixth conjugate comprising (i) a first conjugate member comprising an antigen and (ii) a second conjugate member comprising a second member of said binding pair; and 
 (d′) a seventh conjugate comprising (i) a first conjugate member comprising an anti-CD28 antibody and (ii) a second conjugate member comprising a second member of said binding pair. 
   
     
     
         9 . The method of  claim 8 , wherein said contacting is effected (a) ex vivo and/or (b) in vivo. 
     
     
         10 . The method of  claim 8 , wherein said population of Treg cells comprises Treg cells selected from the group consisting of CD4+ cells, CD25+ cells, and FoxP3+ cells. 
     
     
         11 . The method of  claim 8 , further comprising administering to said patient a composition comprising foreign cells displaying TGF-β, wherein said foreign cells are selected from the group consisting of splenocytes, pancreatic islet tissue, and bone marrow cells. 
     
     
         12 . The method of  claim 8 , wherein said antigen is selected from the group consisting of insulin, collagen, myelin basic protein, MHC/antigen complexes, glutamic acid decarboxylase (GAD), an islet cell autoantigen (ICA), and autoantigen NRP-A7. 
     
     
         13 . A method of obtaining pulsed dendritic cells displaying TGF-β comprising:
 (a) pulsing immature dendritic cells with an antigen, to obtained pulsed dentritic cells; 
 (b) contacting said pulsed dendritic cells with a bifunctional molecule comprising a first member of a binding pair and a molecule that binds to the surface of said cells to form modified pulsed dendritic cells; and 
 (c) contacting said modified pulsed dendritic cells with a conjugate comprising TGF-β and a second member of said binding pair to form pulsed dendritic cells displaying TGF-β. 
 
     
     
         14 . The method  claim 13 , wherein said antigen is selected from the group consisting of collagen; myelin basic protein; and a diabetic autoantigen, wherein said diabetic autoantigen is further selected from the group consisting of a glutamic acid decarboxylase (GAD), an islet cell autoantigen (ICA), and autoantigen NRP-A7. 
     
     
         15 . A population of antigen-pulsed dendritic cells displaying TGF-β. 
     
     
         16 . A method of expanding Treg cells in a patient comprising administering to said patient a composition comprising antigen-pulsed dendritic cells displaying TGF-β. 
     
     
         17 . A method of obtaining hematopoietic stem cells or bone marrow cells displaying TGF-β comprising:
 (a) contacting hematopoietic stem cells or bone marrow cells with a bifunctional molecule comprising a first member of a binding pair and a molecule that binds to the surface of said cells to form modified cells; and 
 (b) contacting said modified cells with a conjugate comprising TGF-β and a second member of said binding pair to form cells displaying TGF-β. 
 
     
     
         18 . A method of expanding Treg cells in a patient comprising administering to said patient a composition comprising hematopoietic stem cells displaying TGF-β or bone marrow cells displaying TGF-β. 
     
     
         19 . A population of cells displaying TGF-β, wherein said cells are selected from bone marrow cells and hematopoietic stem cells. 
     
     
         20 . The population of cells displaying TGF-β of  claim 19 , made by a method comprising
 (a) contacting cells with a bifunctional molecule comprising a first member of a binding pair and a molecule that binds to the surface of said cells to form modified cells, wherein said cells are selected from bone marrow cells and hematopoietic stem cells; and 
 (b) contacting said modified cells with a conjugate comprising TGF-β and a second member of said binding pair to form cells displaying TGF-β.