Chimeric antigen receptors targeting tumor antigens

Abstract

Nucleic acid constructs encoding a chimeric antigen receptor (CAR) and a truncated human epidermal growth factor receptor (huEGFRt) are described. The encoded CARs include a tumor antigen-specific monoclonal antibody, such as a glypican-3 (GPC3)-specific, a GPC2-specific or a mesothelin-specific monoclonal antibody, fused to a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory domain and a CD3ζ signaling domain. Isolated host cells, such as isolated T cells that co-express the disclosed CARs and huEGFRt are also described. T cells transduced with the disclosed CAR constructs can be used for cancer immunotherapy.

1 . An isolated host cell co-expressing a chimeric antigen receptor (CAR) and a truncated human epidermal growth factor receptor (huEGFRt), wherein:
 the CAR comprises an antigen-specific antibody or antigen-binding fragment thereof, an extracellular hinge region, a transmembrane domain, an intracellular co-stimulatory domain and an intracellular signaling domain; and   the huEGFRt comprises a Domain III, a Domain IV and a transmembrane domain from human EGFR, but lacks an epidermal growth factor (EGF)-binding domain and a cytoplasmic domain.   
     
     
         2 . The isolated host cell of  claim 1 , wherein the extracellular hinge region comprises a CD8a hinge region or a CD28 hinge region. 
     
     
         3 . The isolated host cell of  claim 1 , wherein the transmembrane domain comprises a CD8a transmembrane domain or a CD28 transmembrane domain. 
     
     
         4 . The isolated host cell of  claim 1 , wherein the intracellular co-stimulatory domain comprises a 4-1BB, CD28, ICOS, OX40, CD27 or DAP10 co-stimulatory domain. 
     
     
         5 . The isolated host cell of  claim 1 , wherein the intracellular signaling domain comprises a CD3ζ or an FcεRIγ signaling domain. 
     
     
         6 . The isolated host cell of  claim 1 , wherein the extracellular hinge region comprises a CD8a hinge region, the transmembrane domain comprises a CD8α transmembrane domain, the intracellular co-stimulatory domain comprises a 4-1BB co-stimulatory domain and the intracellular signaling domain comprises a CD3ζ signaling domain. 
     
     
         7 . The isolated host cell of  claim 6 , wherein:
 the CD8α hinge region comprises the amino acid sequence of SEQ ID NO: 4;   the CD8α transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6;   the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 8; and/or   the CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO: 10.   
     
     
         8 . The isolated host cell of  claim 1 , wherein the huEGFRt comprises the amino acid sequence of SEQ ID NO: 14. 
     
     
         9 . The isolated host cell of  claim 1 , wherein the antigen-binding fragment is a single-chain variable fragment (scFv) or a single-domain antibody. 
     
     
         10 . The isolated host cell of  claim 9 , wherein the antibody or antigen-binding fragment specifically binds a tumor antigen. 
     
     
         11 . The isolated host cell of  claim 10 , wherein the tumor antigen is glypican-3 (GPC3), glypican-2 (GPC2) or mesothelin. 
     
     
         12 . The isolated host cell of  claim 11 , wherein the tumor antigen is GPC3, and the amino acid sequence of the antigen-binding fragment comprises the variable heavy (VH) domain complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 26 and the variable light (VL) domain CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 28. 
     
     
         13 . The isolated host cell of  claim 12 , wherein the amino acid sequence of the antibody-binding fragment comprises residues 25-269 of SEQ ID NO: 16. 
     
     
         14 . The isolated host cell of  claim 11 , wherein tumor antigen is GPC3, and the amino acid sequence of the antigen-binding fragment comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 30. 
     
     
         15 . The isolated host cell of  claim 14 , wherein the amino acid sequence of the antibody-binding fragment comprises residues 25-140 of SEQ ID NO: 18. 
     
     
         16 . The isolated host cell of  claim 11 , wherein the tumor antigen is GPC2, and amino acid sequence of the antigen-binding fragment comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 32. 
     
     
         17 . The isolated host cell of  claim 16 , wherein the amino acid sequence of the antibody-binding fragment comprises residues 25-144 of SEQ ID NO: 20. 
     
     
         18 . The isolated host cell of  claim 1 , wherein the cell is a T lymphocyte. 
     
     
         19 . The isolated host cell of  claim 18 , wherein the T lymphocyte is an autologous T lymphocyte or an allogeneic T lymphocyte. 
     
     
         20 . A composition comprising the isolated host cell of  claim 1 , and a pharmaceutically acceptable carrier.