Chimeric antigen receptors targeting epidermal growth factor receptor variant iii

Abstract

The invention provides Chimeric Antigen Receptors (CARs) that specifically bind to EGFRvIII (Epidermal Growth Factor Receptor Variant III). The invention further relates to engineered immune cells comprising such CARs, CAR-encoding nucleic acids, and methods of making thereof, engineered immune cells, and nucleic acids. The invention further relates to therapeutic methods for using these CARs and engineered immune cells for the treatment of EGFRvIII-mediated pathologies, including cancers such as glioblastoma.

1 - 32 . (canceled) 
     
     
         33 . An isolated engineered immune cell comprising a polynucleotide encoding an Epidermal Growth Factor Receptor Variant III (EGFRvIII) specific chimeric antigen receptor (CAR),
 wherein the polynucleotide is under the transcriptional control of a promoter,   wherein the EGFR-specific CAR comprises an EGFRvIII-binding domain, a transmembrane domain, and an intracellular signaling domain, and   wherein the EGFRvIII-binding domain comprises:   a) a heavy chain variable (VH) region comprising three complementarity determining regions of VH complementary determining region 1 (VH CDR1), VH complementary determining region 2 (VH CDR2), and VH complementary determining region 3 (VH CDR3) of 42G9 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 74-76, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 77-78, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 79; and
 a light chain variable (VL) region comprising three complementarity determining regions of VL complementarity determining region 1 (VL CDR1), VL complementarity determining region 2 (VL CDR2), and VL complementarity determining region 3 (VL CDR3) of 42G9 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 156, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 157, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 158; or 
   b) a VH region comprising three complementarity determining regions of VH CDR1, VH CDR2, and VH CDR3 of 32A10 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 80-82, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 83-84, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 85; and
 a VL region comprising three complementarity determining regions of VL CDR1, VL CDR2, and VL CDR3 of 32A10 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 159, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 160, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 161; or 
   c) a VH region comprising three complementarity determining regions of VH CDR1, VH CDR2, and VH CDR3 of 20B9 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 80-82, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 86-87, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 79; and
 a VL region comprising three complementarity determining regions of VL CDR1, VL CDR2, and VL CDR3 of 20B9 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 162, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 163, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 164; or 
   d) a VH region comprising three complementarity determining regions of VH CDR1, VH CDR2, and VH CDR3 of 14C11 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 88-90, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 91-92, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 85; and
 a VL region comprising three complementarity determining regions of VL CDR1, VL CDR2, and VL CDR3 of 14C11 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 165, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 163, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 161; or 
   e) a VH region comprising three complementarity determining regions of VH CDR1, VH CDR2, and VH CDR3 of 30D8 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 109-111, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 112-113, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 114; and
 a VL region comprising three complementarity determining regions of VL CDR1, VL CDR2, and VL CDR3 of 30D8 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 182, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 183, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 184; or 
   f) a VH region comprising three complementarity determining regions of VH CDR1, VH CDR2, and VH CDR3 of 20E12 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 115-117, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 118-119, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 120; and
 a VL region comprising three complementarity determining regions of VL CDR1, VL CDR2, and VL CDR3 of 20E12 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 185, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 186, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 184; or 
   g) a VH region comprising three complementarity determining regions of VH CDR1, VH CDR2, and VH CDR3 of 26B9 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 121-123, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 124-125, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 126; and
 a VL region comprising three complementarity determining regions of VL CDR1, VL CDR2, and VL CDR3 of 26B9 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 187, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 188, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 189; or 
   h) a VH region comprising three complementarity determining regions of VH CDR1, VH CDR2, and VH CDR3 of C6 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 137-139, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 140-141, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 142; and
 a VL region comprising three complementarity determining regions of VL CDR1, VL CDR2, and VL CDR3 of C6 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 195, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 196, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 197; or 
   i) a VH region comprising three complementarity determining regions of VH CDR1, VH CDR2, and VH CDR3 of 12B2 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 74-76, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 102-103, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 104; and
 a VL region comprising three complementarity determining regions of VL CDR1, VL CDR2, and VL CDR3 of 12B2 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 176, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 172, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 177. 
   
     
     
         34 . The engineered immune cell of  claim 33 , wherein the EGFRvIII-binding domain is a single-chain variable fragment (scFv). 
     
     
         35 . The engineered immune cell of  claim 34 , wherein the scFv comprises a VH region and a VL region selected from the group consisting of:
 i) the VH region comprising the amino acid sequence of SEQ ID NO: 9 and the VL region comprising the amino acid sequence of SEQ ID NO: 10 (42G9);   ii) the VH region comprising the amino acid sequence of SEQ ID NO: 11 and the VL region comprising the amino acid sequence of SEQ ID NO: 12 (32A10);   iii) the VH region comprising the amino acid sequence of SEQ ID NO: 13 and the VL region comprising the amino acid sequence of SEQ ID NO: 14 (20B9);   iv) the VH region comprising the amino acid sequence of SEQ ID NO: 15 and the VL region comprising the amino acid sequence of SEQ ID NO: 16 (14C11);   v) the VH region comprising the amino acid sequence of SEQ ID NO: 37 and the VL region comprising the amino acid sequence of SEQ ID NO: 38 (30D8)   vi) the VH region comprising the amino acid sequence of SEQ ID NO: 39 and the VL region comprising the amino acid sequence of SEQ ID NO: 40 (20E12);   vii) the VH region comprising the amino acid sequence of SEQ ID NO: 41 and the VL region comprising the amino acid sequence of SEQ ID NO: 42 (26B9);   viii) the VH region comprising the amino acid sequence of SEQ ID NO: 48 and the VL region comprising the amino acid sequence of SEQ ID NO: 49 (C6); and   ix) the VH region comprising the amino acid sequence of SEQ ID NO: 30 and the VL region comprising the amino acid sequence of SEQ ID NO: 31 (12B2).   
     
     
         36 . The engineered immune cell of  claim 33 , wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-57 and 59-61. 
     
     
         37 . The engineered immune cell of  claim 33 , wherein the engineered immune cell expresses at its cell surface membrane the EGFRvIII-specific CAR. 
     
     
         38 . The engineered immune cell of  claim 33 , wherein the intracellular signaling domain comprises a CD3zeta signaling domain. 
     
     
         39 . The engineered immune cell of  claim 33 , wherein the intracellular signaling domain is a first intracellular signaling domain and the CAR comprises a second intracellular signaling domain. 
     
     
         40 . The engineered immune cell of  claim 39 , wherein the first intracellular signaling domain comprises a CD3zeta signaling domain and the second intracellular signaling domain comprises a 4-1BB signaling domain. 
     
     
         41 . The engineered immune cell of  claim 33 , wherein the EGFRvIII-specific CAR comprises a second extracellular ligand-binding domain, wherein the second extracellular ligand-binding domain is not specific for EGFRvIII. 
     
     
         42 . The engineered immune cell of  claim 33 , wherein the transmembrane domain of the EGFRvIII-specific CAR comprises a transmembrane domain from the α chain of the high-affinity IgE receptor (FcεRI), and
 wherein the engineered immune cell comprises a second polynucleotide encoding a polypeptide comprising a transmembrane domain from the γ or β chain of FcεRI. 
 
     
     
         43 . The engineered immune cell of  claim 33 , wherein the engineered immune cell further comprises a polynucleotide encoding a CAR which is not specific for EGFRvIII. 
     
     
         44 . The engineered immune cell of  claim 33 , wherein the engineered immune cell comprises a polynucleotide encoding a suicide polypeptide. 
     
     
         45 . The engineered immune cell of  claim 33 , wherein the engineered immune cell comprises a disruption of one or more endogenous genes selected from the group consisting of genes encoding TCRα, TCRβ, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), and programmed death-1 (PD-1). 
     
     
         46 . The engineered immune cell of  claim 33 , wherein the engineered immune cell is obtained from a healthy donor. 
     
     
         47 . The engineered immune cell of  claim 33 , wherein the engineered immune cell is obtained from a patient. 
     
     
         48 . The engineered immune cell of  claim 33 , wherein the engineered immune cell is derived from an inflammatory T-lymphocyte, a cytotoxic T-lymphocyte, a regulatory T-lymphocyte, a memory T-lymphocyte, a helper T-lymphocyte, a natural killer T-lymphocyte, or a natural killer cell. 
     
     
         49 . A method of engineering an immune cell comprising:
 introducing into an immune cell a polynucleotide encoding an EGFRvIII specific CAR,   wherein the polynucleotide is under the transcriptional control of a promoter,   wherein the EGFR-specific CAR comprises an EGFRvIII-binding domain, a transmembrane domain, and an intracellular signaling domain, and   wherein the EGFRvIII-binding domain comprises:   a) a heavy chain variable (VH) region comprising three complementarity determining regions of VH complementary determining region 1 (VH CDR1), VH complementary determining region 2 (VH CDR2), and VH complementary determining region 3 (VH CDR3) of 42G9 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 74-76, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 77-78, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 79; and
 a light chain variable (VL) region comprising three complementarity determining regions of VL complementarity determining region 1 (VL CDR1), VL complementarity determining region 2 (VL CDR2), and VL complementarity determining region 3 (VL CDR3) of 42G9 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 156, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 157, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 158; or 
   b) a VH region comprising three complementarity determining regions of VH CDR1, VH CDR2, and VH CDR3 of 32A10 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 80-82, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 83-84, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 85; and
 a VL region comprising three complementarity determining regions of VL CDR1, VL CDR2, and VL CDR3 of 32A10 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 159, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 160, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 161; or 
   c) a VH region comprising three complementarity determining regions of VH CDR1, VH CDR2, and VH CDR3 of 20B9 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 80-82, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 86-87, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 79; and
 a VL region comprising three complementarity determining regions of VL CDR1, VL CDR2, and VL CDR3 of 20B9 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 162, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 163, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 164; or 
   d) a VH region comprising three complementarity determining regions of VH CDR1, VH CDR2, and VH CDR3 of 14C11 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 88-90, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 91-92, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 85; and
 a VL region comprising three complementarity determining regions of VL CDR1, VL CDR2, and VL CDR3 of 14C11 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 165, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 163, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 161; or 
   e) a VH region comprising three complementarity determining regions of VH CDR1, VH CDR2, and VH CDR3 of 30D8 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 109-111, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 112-113, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 114; and
 a VL region comprising three complementarity determining regions of VL CDR1, VL CDR2, and VL CDR3 of 30D8 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 182, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 183, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 184; or 
   f) a VH region comprising three complementarity determining regions of VH CDR1, VH CDR2, and VH CDR3 of 20E12 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 115-117, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 118-119, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 120; and
 a VL region comprising three complementarity determining regions of VL CDR1, VL CDR2, and VL CDR3 of 20E12 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 185, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 186, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 184; or 
   g) a VH region comprising three complementarity determining regions of VH CDR1, VH CDR2, and VH CDR3 of 26B9 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 121-123, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 124-125, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 126; and
 a VL region comprising three complementarity determining regions of VL CDR1, VL CDR2, and VL CDR3 of 26B9 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 187, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 188, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 189; or 
   h) a VH region comprising three complementarity determining regions of VH CDR1, VH CDR2, and VH CDR3 of C6 arranged sequentially in the N terminus to C terminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 137-139, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 140-141, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 142; and
 a VL region comprising three complementarity determining regions of VL CDR1, VL CDR2, and VL CDR3 of C6 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 195, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 196, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 197; or 
   i) a VH region comprising three complementarity determining regions of VH CDR1 VH CDR2, and VH CDR3 of 12B2 arranged sequentially in the N terminus to C terminus direction of VH CDR1 VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ ID NOs: 74-76, VH CDR2 comprises the amino acid sequence of one of SEQ ID NOs: 102-103, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 104; and
 a VL region comprising three complementarity determining regions of VL CDR1, VL CDR2, and VL CDR3 of 12B2 arranged sequentially in the N terminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 176, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 172, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 177, thereby generating the engineered immune cell. 
   
     
     
         50 . A pharmaceutical composition comprising the engineered immune cell of  claim 33 . 
     
     
         51 . A method of treating a condition associated with malignant cells expressing EGFRvIII in a subject comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of  claim 50 . 
     
     
         52 . The method of  claim 51 , wherein the condition is a cancer.