Engineering antiviral T cell immunity through stem cells and chimeric antigen receptors
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 that encodes a truncated chimeric antigen receptor (CAR), which comprises a truncated CD4, which has a D1 extracellular domain and lacks at least one extracellular domain selected from D2, D3, and D4, fused to a signaling domain of a CD3 complex ζ-chain and said CAR is 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 of claim 1 , wherein the truncated CD4 CAR has an extracellular domain that binds gp120 expressed on the surface of cells infected with HIV.
7. The recombinant progenitor cell according to claim 1 , wherein the virus is a lentivirus.
8. The recombinant progenitor cell of claim 7 , wherein the lentivirus is a human immunodeficiency virus.
9. The recombinant progenitor cell according to claim 1 , wherein the functional effector cell is a T-cell.
10. The recombinant progenitor cell of claim 9 , wherein the T-cell expresses the truncated CAR on its cell surface.
11. 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.
12. The recombinant progenitor cell of claim 11 , 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.
13. 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.
14. The method of claim 13 , wherein the recombinant progenitor cell is administered to or engrafted in a subject.
15. An engineered functional effector cell made by the method according to claim 13 .
16. The engineered functional effector cell of claim 15 , which expresses a truncated CAR on its cell surface.
17. 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.
18. The recombinant progenitor cell according to claim 1 , wherein the recombinant progenitor cell lacks HLA-restricted T cell receptors.
19. A nucleic acid molecule comprising a sequence encoding a truncated chimeric antigen receptor (CAR), which comprises a truncated CD4 which has a D1 extracellular domain and lacks at least one extracellular domain selected from D2, D3, and D4, fused to the signaling domain of the CD3 complex ζ-chain.
20. The nucleic acid molecule of claim 19 , wherein the nucleic acid molecule encodes a truncated Double CAR, a truncated Triple CD4ζ CAR, CD4D1D2D3CAR, CD4D1D2CAR, or CD4D1CAR.
21. The nucleic acid molecule of claim 19 , wherein the nucleic acid 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.
22. The recombinant progenitor cell of claim 1 , wherein the stem cell is a human stem cell.
23. The nucleic acid molecule of claim 19 , wherein the nucleic acid molecule further comprises a nucleotide sequence encoding C46 fusion inhibitory antiviral peptide.
24. The nucleic acid molecule of claim 19 , wherein the nucleic acid molecule further comprises an antiviral shRNA.
25. An engineered functional effector cell that expresses on its cell surface a truncated chimeric antigen receptor (CAR), which comprises a truncated CD4, which has a D1 extracellular domain and lacks at least one extracellular domain selected from D2, D3, and D4, fused to the signaling domain of the CD3 complex ζ-chain.
26. An engineered functional effector cell that expresses on its cell surface a chimeric antigen receptor (CAR) comprising 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.