Generation of ctl lines with specificity against multiple tumor antigens or multiple viruses

Abstract

The present invention encompasses methods and compositions for the generation and use of cytotoxic T lymphocytes that target multiple viruses or that are specific for multiple tumor antigens. In specific embodiments, the generation methods employ use of certain cytokines to promote proliferation and reduce cell death in an activated T cell population and/or that employ a particular bioreactor having a gas permeable membrane.

What is claimed is: 
     
         1 . A polyclonal population of cytotoxic T-lymphocytes (CTLs) that recognize at least one antigen from two or more viruses, said population comprising CD4+ T-lymphocytes and CD8+ T-lymphocytes, wherein the CTLs have been stimulated by dendritic cells (DCs) or PBMCs contacted with more than one library of peptides, which combine to represent at least two different antigens and at least two different viruses, wherein the peptides in each library overlap in sequence to span at least a part of a viral antigen. 
     
     
         2 . A composition comprising cytotoxic T-lymphocytes (CTLs) that recognize at least one antigen from two or more viruses, said composition comprising CD4+ T-lymphocytes and CD8+ T-lymphocytes, wherein the CTLs have been stimulated by dendritic cells (DCs) or PBMCs contacted with more than one library of peptides, which combine to represent at least two different antigens and at least two different viruses, wherein the peptides in each library overlap in sequence to span at least a part of a viral antigen. 
     
     
         3 . A composition comprising cytotoxic T-lymphocytes (CTLs) that recognize at least one antigen from two or more viruses, wherein the CTLs express αβ T cell receptors and wherein the CTLs have been stimulated by dendritic cells (DCs) or PBMCs contacted with more than one library of peptides, which combine to represent at least two different antigens and at least two different viruses, wherein the peptides in each library overlap in sequence to span at least a part of a viral antigen. 
     
     
         4 . A composition comprising MHC-restricted cytotoxic T-lymphocytes (CTLs) that recognize at least one antigen from two or more viruses, wherein the CTLs have been stimulated by dendritic cells (DCs) or PBMCs contacted with more than one library of peptides, which combine to represent at least two different antigens and at least two different viruses, wherein the peptides in each library overlap in sequence to span at least a part of a viral antigen. 
     
     
         5 . The composition of any one of  claims 1 - 4 , wherein the viruses are selected from the group consisting of EBV, CMV, Adenovirus, BK, HHV6, RSV, Influenza, Parainfluenza, Bocavirus, Coronavirus, LCMV, Mumps, Measles, Metapneumovirus, Parvovirus B, Rotavirus, and West Nile Virus. 
     
     
         6 . The composition of any one of  claims 1 - 5 , wherein the more than one library of peptides comprises at least four libraries of peptides, two of which libraries span separate antigens from a first virus and two of which libraries span separate antigens from a second different virus. 
     
     
         7 . The composition of any one of  claims 1 - 6 , wherein at least one of the viruses is
 a. adenovirus;   b. EBV;   c. CMV   d. HHV6;   e. RSV;   f. Influenza; or   g. BKV.   
     
     
         8 . The composition of any one of  claims 1 - 7 , wherein the viruses comprise at least adenovirus, EBV, and CMV. 
     
     
         9 . The composition of  claim 8 , wherein the viruses further comprises at least two, three, or four additional viruses. 
     
     
         10 . The composition of  claim 9 , wherein the additional viruses are selected from HHV6, RSV, influenza, and BKV. 
     
     
         11 . The composition of  claim 9 , wherein the additional viruses consist of HHV6 and BKV. 
     
     
         12 . The composition of any one of  claims 1 - 11 , wherein the CTLs are cultured ex vivo in the presence of both IL-7 and IL-4. 
     
     
         13 . The of any one of  claims 1 - 12 , wherein the multivirus CTLs have expanded sufficiently within 9-18 days of culture that they are ready for administration to a patient. 
     
     
         14 . The composition of any one of  claims 1 - 13 , wherein the CTLs exhibit one or more property selected from
 a. negligible alloreactivity;   b. negligible alloreactivity after a single stimulation   c. less activation induced cell death of antigen-specific T cells harvested from a patient than corresponding antigen-specific T cells harvested from the same patient, but not cultured in the presence of both IL-7 and IL-4;   d. viability of greater than 70%.   
     
     
         15 . The composition of any one of  claims 1 - 14 , wherein the composition is negative for bacteria and fungi for at least 7 days in culture; exhibit less than 5 EU/ml of endotoxin, and are negative for  mycoplasma.    
     
     
         16 . The composition of any one of  claims 1 - 15 , wherein the CTLs were generated by contacting DCs and PBMCs at a ratio of 1:20, wherein the DCs have been contacted with at least two pepmixes; wherein each of the pepmixed spans at least one viral antigen; and wherein at least two of the antigens that the pepmixes span are from different viruses. 
     
     
         17 . The composition of any one of  claims 1 - 16 , wherein the pepmixes were chemically synthesized and are, optionally >90% pure. 
     
     
         18 . A polyclonal population of cytotoxic T-lymphocytes (CTLs) that recognize at least one antigen from each of an adenovirus, EBV, CMV, HHV6 and BKV, said population comprising CD4+ T-lymphocytes and CD8+ T-lymphocytes, wherein the CTLs have been stimulated by dendritic cells (DCs) or PBMCs contacted with more than one library of peptides, which combine to represent at least two different antigens and at least two different viruses, wherein the peptides in each library overlap in sequence to span at least a part of a viral antigen. 
     
     
         19 . A composition comprising cytotoxic T-lymphocytes (CTLs) that recognize at least one antigen from each of an adenovirus, EBV, CMV, HHV6 and BKV, wherein the CTLs express αβ T cell receptors and wherein the CTLs have been stimulated by dendritic cells (DCs) or PBMCs contacted with more than one library of peptides, which combine to represent at least two different antigens and at least two different viruses, wherein the peptides in each library overlap in sequence to span at least a part of a viral antigen. 
     
     
         20 . A composition comprising MHC-restricted cytotoxic T-lymphocytes (CTLs) that recognize at least one antigen from each of an adenovirus, EBV, CMV, HHV6 and BKV, wherein the CTLs have been stimulated by dendritic cells (DCs) or PBMCs contacted with more than one library of peptides, which combine to represent at least two different antigens and at least two different viruses, wherein the peptides in each library overlap in sequence to span at least a part of a viral antigen. 
     
     
         21 . A method of treating or preventing a viral infection comprising administering to a subject in need thereof the composition of any one of  claims 1 - 20 . 
     
     
         22 . The method of  claim 21 , wherein between 5×10 6  and 5×10 7  CTL/m 2  administered to the subject. 
     
     
         23 . The method of  claim 21  or  22 , wherein the subject is immunocompromised. 
     
     
         24 . The method of any one of  claims 21 - 23 , wherein the subject
 a. has received a solid organ transplantation;   b. has received chemotherapy;   c. has an HIV infection;   d. has a genetic immunodeficiency; and/or   e. has received an allogeneic stem cell transplant.   
     
     
         25 . The method of any one of  claims 21 - 24 , wherein the composition is administered to the patient a plurality of times. 
     
     
         26 . The method of any one of  claims 21 - 25 , wherein the administration of the composition effectively treats or prevents a viral infection in the subject, wherein the viral infection is selected from the group consisting of an EBV infection, CMV infection, BV infection, adenoviral infection, an HHV6 infection, and a combination thereof.