Generation and isolation of antigen-specific T cells

Abstract

The present invention relates generally to methods for generating, expanding, and isolating antigen-specific T cells. Compositions of antigen-specific T cells activated and expanded by the methods herein are further provided.

1 . A method for expanding a population of antigen-specific T cells comprising: 
 contacting a population of cells wherein at least a portion thereof comprises antigen-specific T cells, with a surface, wherein said surface has attached thereto a first agent and a second agent, wherein said first agent ligates a CD3/TCR complex on said T cells and said second agent ligates an accessory molecule on said T cells, and wherein said ligation by said first and second agent of said T cells induces proliferation of antigen-specific T cells and wherein said surface is present at a ratio of surface to T cells of 1:2 or less.    
     
     
         2 . The method according to  claim 1  wherein said surface is selected from the group consisting of paramagnetic beads, lipids, and cell surfaces.  
     
     
         3 . The method according to  claim 2  wherein said surface comprises paramagnetic beads.  
     
     
         4 . The method according to  claim 3  wherein said beads comprise beads conjugated to an antibody.  
     
     
         5 . The method of  claim 1  wherein said surface is present in a ratio of surface to T cells of about 1:2.5.  
     
     
         6 . The method of  claim 1  wherein said surface is present in a ratio of surface to T cells of about 1:5.  
     
     
         7 . The method of  claim 1  wherein said surface is present in a ratio of surface to T cells of about 1:10.  
     
     
         8 . The method of  claim 1  wherein said surface is present in a ratio of surface to T cells of about 1:25.  
     
     
         9 . The method of  claim 1  wherein said surface is present in a ratio of surface to T cells of about 1:50.  
     
     
         10 . The method of  claim 1  wherein said surface is present in a ratio of surface to T cells of about 1:100.  
     
     
         11 . A method for generating and/or enriching antigen-specific T cells comprising: 
 (a) exposing a first population of cells wherein at least a portion thereof comprises antigen presenting cells to a surface wherein said surface has antigen attached thereto, such that said surface with antigen attached thereto is ingested by said APC;    (b) exposing a second population of cells wherein at least a portion thereof comprises T cells to the population of cells in part (a);    thereby generating and/or enriching antigen-specific T cells.    
     
     
         12 . The method according to  claim 11  wherein said APC are in direct contact with said antigen-specific T cells.  
     
     
         13 . The method according to  claim 12  wherein said APC in direct contact with said antigen-specific T cells are isolated by exposing said APC to a magnetic field.  
     
     
         14 . The method according to  claim 13  wherein said antigen-specific T cells are expanded according to the following method: 
 (a) exposing said T cells to an anti-CD3 antibody which is immobilized on a surface; and  
 (b) stimulating an accessory molecule on the surface of the T cells with an anti-CD28 antibody, wherein said anti-CD28 antibody is immobilized on the same surface as the anti-CD3 antibody;  
 thereby inducing expansion of said antigen-specific T cells.  
 
     
     
         15 . The method according to  claim 14 , further comprising exposing said T cells to IL-15.  
     
     
         16 . The method according to  claim 14 , further comprising exposing said T cells to a natural ligand for CD137.  
     
     
         17 . The method according to  claim 14 , further comprising exposing said T cells to an anti-CD137 antibody.  
     
     
         18 . The method according to  claim 14 , further comprising exposing said T cells to an anti-NKG2D antibody or a natural ligand for NKG2D.  
     
     
         19 - 20 . (Canceled)  
     
     
         21 . The method according to  claim 11  wherein said antigen is selected from the group consisting of protein, glycoprotein, peptides, antibody/antigen complexes, whole tumor or virus-infected cells, fixed tumor or virus-infected cells, heat-killed tumor or virus-infected cells, tumor lysate, non-soluble cell debris, apoptotic bodies, necrotic cells, whole tumor cells from a tumor or a cell line that have been treated such that they are unable to continue dividing, allogeneic cells that have been treated such that they are unable to continue dividing, irradiated tumor cells, irradiated allogeneic cells, natural or synthetic complex carbohydrates, lipoproteins, lipopolysaccharides, transformed cells or cell line, transfected cells or cell line, transduced cells or cell line, and virally infected cells or cell line.  
     
     
         22 . The method according to  claim 11  wherein said antigen is attached to said surface by an antibody/ligand interaction.  
     
     
         23 . The method according to  claim 22  wherein said antibody/ligand interaction comprises an interaction between an antibody/ligand pair selected from the group consisting of anti-MART-1 antibody/MART-1 antigen, anti-WT-1 antibody/WT-1, anti-PR1 antibody /PR1, anti-PR3 antibody /PR3, anti-tyrosinase antibody/tyrosinase antigen, anti-MAGE-1 antibody/MAGE-1 antigen, anti-MUC-1 antibody/MUC-1 antigen, anti-α-fetoprotein antibody/α-fetoprotein antigen, anti-Her2Neu antibody/Her2Neu, anti-HIV gp120 antibody/HIV gp120, anti-influenza HA antibody/influenza HA, anti-CMV pp65/CMV pp65, anti-hepatitis C antibody/hepatitis C proteins, anti-EBV EBNA 3B antibody/EBV EBNA 3B antigen, and anti-human Ig heavy and lignt chains/Ig from a myeloma cancer patient, and anti-human Ig heavy and lignt chains/Ig from a CLL cancer patient.  
     
     
         24 . The method according to  claim 11  wherein said antigen is chemically attached to said surface.  
     
     
         25 . The method according to  claim 11  wherein the attachment of said antigen to said surface comprises a biotin-avidin interaction.  
     
     
         26 . The method according to  claim 11  wherein said population of cells wherein at least a portion thereof comprises APC is derived from a source selected from the group consisting of leukapheresis product, peripheral blood, lymph node, tonsil, thymus, tissue biopsy, tumor, spleen, bone marrow, cord blood, CD34 +  cells, monocytes, and adherent cells.  
     
     
         27 - 29 . (Canceled)  
     
     
         30 . A population of antigen-specific T cells generated according to the method of any one of claims  1 .  
     
     
         31 . A composition comprising the antigen-specific T cells according to  claim 30  and a pharmaceutically acceptable excipient.  
     
     
         32 . A method for stimulating an immune response in a mammal comprising, administering to the mammal the composition of  claim 31 .  
     
     
         33 . A method for reducing the presence of cancer cells in a mammal comprising, exposing the cells to the composition of  claim 31 .  
     
     
         34 . The method of  claim 33  wherein the cancer cells are from a cancer selected from the group consisting of melanoma, non-Hodgkin's lymphoma, Hodgkin's disease, leukemia, plasmocytoma, sarcoma, glioma, thymoma, breast cancer, prostate cancer, colo-rectal cancer, kidney cancer, renal cell carcinoma, pancreatic cancer, esophageal cancer, brain cancer, lung cancer, ovarian cancer, cervical cancer, multiple myeloma, hepatoma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL).  
     
     
         35 . A method for inhibiting the development of a cancer in a mammal, comprising administering to the mammal the composition of  claim 31 .  
     
     
         36 . The method of  claim 35  wherein the cancer cells are from a cancer selected from the group consisting of melanoma, non-Hodgkin's lymphoma, Hodgkin's disease, leukemia, plasmocytoma, sarcoma, glioma, thymoma, breast cancer, prostate cancer, colo-rectal cancer, kidney cancer, renal cell carcinoma, pancreatic cancer, esophageal cancer, brain cancer, lung cancer, ovarian cancer, cervical cancer, multiple myeloma, hepatoma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL).  
     
     
         37 . A method for ameliorating an immune response dysfunction in a mammal comprising administering to the mammal the composition of  claim 31 .  
     
     
         38 . A method for reducing the presence of an infectious organism in a mammal comprising, administering to the mammal the composition of  claim 31 .  
     
     
         39 . The method of  claim 38  wherein said organism is selected from the group consisting of a virus, a single-stranded RNA virus, a single-stranded DNA virus, a double-stranded DNA virus, Human Immunodeficiency Virus (HIV), Hepatitis A, B, or C virus, Herpes Simplex Virus (HSV), Human Papilloma Virus (HPV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), a parasite, a bacterium,  M tuberculosis, Pneumocystis carinii, Candida, Aspergillus.    
     
     
         40 . A method for inhibiting the development of an infectious disease in a mammal, comprising administering to the mammal the composition of  claim 31 .  
     
     
         41 . The method of  claim 40  wherein said organism is selected from the group consisting of a virus, a single-stranded RNA virus, a single-stranded DNA virus, a double-stranded DNA virus, Human Immunodeficiency Virus (HIV), Hepatitis A, B, or C virus, Herpes Simplex Virus (HSV), Human Papilloma Virus (HPV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), a parasite, a bacterium,  M tuberculosis, Pneumocystis carinii, Candida, Aspergillus.