Engineering Antiviral T Cell Immunity through Stem Cells and Chimeric Antigen Receptors

Abstract

The HIV-specific cytotoxic T lymphocyte (CTL) response is a critical component in controlling HIV replication and is an important part of the ultimate failure to eradicate the virus. Disclosed herein are methods for genetically enhancing the HIV-specific CTL response to allow long-term viral suppression or viral clearance. Human hematopoietic stem cells (HSCs) were genetically modified such that they differentiate into mature CTLs that will kill HIV infected cells. As disclosed herein, the functional effector cells are not human leukocyte antigen (HLA)-restricted. As disclosed herein, stem cells are transduced with non-HLA restricted chimeric antigen receptors (CARs) that allow the recognition of HIV or HIV-infected cells when expressed by a CTL. These CARs are hybrid molecules that contain an extracellular HIV recognition domain and an intracellular TCR-zeta signaling domain. The CTL response may be enhanced through the targeting of T cell inhibitory receptors. The methods and compositions disclosed herein may be used to engineer antiviral immunity and HIV-specific CTL responses in vivo. Also disclosed herein are methods and compositions for the treatment of chronic viral infections such as HIV.

1 . A recombinant progenitor cell which comprises
 a stem cell transduced with a vector containing a nucleic acid molecule which encodes a chimeric antigen receptor (CAR) specific for a virus or an epitope thereof, wherein the recombinant progenitor cell is capable of differentiating into a functional effector cell.   
     
     
         2 . The recombinant progenitor cell of  claim 1 , wherein the nucleic acid molecule is contained within a CAR construct. 
     
     
         3 . The recombinant progenitor cell of  claim 1 , wherein the stem cell is a hematopoietic stem cell or a hematopoietic progenitor cell. 
     
     
         4 . The recombinant progenitor cell of  claim 3 , wherein the stem cell is a memory T stem cell. 
     
     
         5 . The recombinant progenitor cell according to  claim 1 , wherein the vector is a lentiviral vector. 
     
     
         6 . The recombinant progenitor cell according to  claim 1 , wherein the chimeric antigen receptor comprises a CD4 extracellular and transmembrane domains and a CD3 zeta signaling domain (CD4ζ). 
     
     
         7 . The recombinant progenitor cell of  claim 6 , wherein the CD4 extracellular domain binds gp120 expressed on the surface of cells infected with HIV. 
     
     
         8 . The recombinant progenitor cell according to  claim 1 , wherein the virus is a lentivirus. 
     
     
         9 . The recombinant progenitor cell of  claim 8 , wherein the lentivirus is a human immunodeficiency virus. 
     
     
         10 . The recombinant progenitor cell according to  claim 1 , wherein the functional effector cell is a T-cell. 
     
     
         11 . The recombinant progenitor cell of  claim 10 , wherein the T-cell expresses CD4ζ CAR on its cell surface. 
     
     
         12 . The recombinant progenitor cell according to  claim 1 , wherein the vector further comprises one or more genetic sequences which protect the recombinant progenitor cell from infection by the virus. 
     
     
         13 . The recombinant progenitor cell of  claim 12 , wherein the genetic sequences are selected from the group consisting of: sh1005, sh516, and a nucleic acid molecule encoding C46, and the virus is a human immunodeficiency virus. 
     
     
         14 . A method of producing a functional effector cell which comprises
 differentiating or developing the recombinant progenitor cell of  claim 1  and then maturing it into the functional effector cell.   
     
     
         15 . The method of  claim 14 , wherein the recombinant progenitor cell is administered to or engrafted in a subject. 
     
     
         16 . (canceled) 
     
     
         17 . An engineered functional effector cell made by the method according to  claim 14 . 
     
     
         18 . The engineered functional effector cell of  claim 17 , which expresses CD4ζ CAR on its cell surface. 
     
     
         19 . A method of inhibiting, reducing, or treating a viral infection in a subject which comprises administering the recombinant progenitor cell according to  claim 1  or a functional effector cell matured therefrom to the subject. 
     
     
         20 . The recombinant progenitor cell according to  claim 1 , wherein the recombinant progenitor cell lacks HLA-restricted T cell receptors. 
     
     
         21 . A nucleic acid molecule which comprises a sequence encoding CD4 fused to the signaling domain of the CD3 complex ζ-chain. 
     
     
         22 . (canceled) 
     
     
         23 . The nucleic acid molecule of  claim 21 , wherein the molecule is CD4ζ CAR, Double CAR C46, Triple CD4ζ CAR, CD4D1D2D3CAR, CD4D1D2CAR, or CD4D1CAR. 
     
     
         24 . The nucleic acid molecule of  claim 21 , wherein the molecule contains a nucleotide sequence encoding a single chain antibody having an amino acid sequence selected from the group consisting of SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:14; and SEQ ID NO:15.