Chimeric antigen receptors based on single-domain antibodies and methods of use thereof

Abstract

The present application provides single-domain antibodies, and chimeric antigen receptors comprising one or more antigen binding domains each comprising a single-domain antibody. Further provided are engineered immune effector cells (such as T cells) comprising the chimeric antigen receptors. Pharmaceutical compositions, kits and methods of treating cancer are also provided.

1 .- 43 . (canceled) 
     
     
         44 . A chimeric antigen receptor (CAR) comprising a polypeptide comprising:
 (a) an extracellular antigen binding domain comprising a first single-domain antibody (sdAb) specifically binding to a first antigen and a second single-domain antibody (sdAb) specifically binding to a second antigen, wherein each of the first and second sdAb is a V H H domain;   (b) a transmembrane domain; and   (c) an intracellular signaling domain.   
     
     
         45 . The CAR of  claim 44 , wherein the first antigen is different from the second antigen. 
     
     
         46 . The CAR of  claim 44 , wherein the first sdAb is located at the N-terminus of the second sdAb. 
     
     
         47 . The CAR of  claim 44 , wherein the first sdAb is located at the C-terminus of the second sdAb. 
     
     
         48 . The CAR of  claim 44 , wherein the first antigen and the second antigen are selected from the group consisting of CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. 
     
     
         49 . The CAR of  claim 48 , wherein the first sdAb is an anti-BCMA sdAb. 
     
     
         50 . The CAR of  claim 48 , wherein the first sdAb is an anti-CD38 sdAb. 
     
     
         51 . The CAR of  claim 48 , wherein the first sdAb is an anti-CD19 sdAb. 
     
     
         52 . The CAR of  claim 48 , wherein the first sdAb is an anti-CD20 sdAb. 
     
     
         53 . The CAR of  claim 48 , wherein the first sdAb is an anti-CD22 sdAb. 
     
     
         54 . The CAR of  claim 48 , wherein the first sdAb is an anti-BCMA sdAb, and the second sdAb is an anti-CD38 sdAb. 
     
     
         55 . The CAR of  claim 48 , wherein the first sdAb is an anti-BCMA sdAb, and the second sdAb is an anti-CD19 sdAb. 
     
     
         56 . The CAR of  claim 48 , wherein the first sdAb is an anti-CD19 sdAb, and the second sdAb is an anti-CD20 sdAb. 
     
     
         57 . The CAR of  claim 48 , wherein the first sdAb is an anti-CD19 sdAb, and the second sdAb is an anti-CD22 sdAb. 
     
     
         58 . The CAR of  claim 44 , wherein the first antigen is the same as the second antigen. 
     
     
         59 . The CAR of  claim 58 , wherein the first sdAb and the second sdAb specifically bind to the same epitope. 
     
     
         60 . The CAR of  claim 58 , wherein the first sdAb and the second sdAb specifically bind to the different epitopes. 
     
     
         61 . The CAR of  claim 58 , wherein the first sdAb and the second sdAb bind to BCMA. 
     
     
         62 . The CAR of  claim 44 , wherein the first sdAb and/or the second sdAb are camelid, chimeric, human, or humanized. 
     
     
         63 . The CAR of  claim 44 , wherein the first sdAb and the second sdAb are directly fused to each other via a peptide bond. 
     
     
         64 . The CAR of  claim 44 , wherein the first sdAb and the second sdAb are fused to each other via a peptide linker. 
     
     
         65 . The CAR of  claim 64 , wherein the peptide linker is no more than 50 amino acids long. 
     
     
         66 . The CAR of  claim 44 , wherein the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. 
     
     
         67 . The CAR of  claim 44 , wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. 
     
     
         68 . The CAR of  claim 67 , wherein the primary intracellular signaling domain is derived from CD3ζ. 
     
     
         69 . The CAR of  claim 44 , wherein the intracellular signaling domain comprises a co-stimulatory signaling domain. 
     
     
         70 . The CAR of  claim 69 , wherein the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, Ligands of CD83 and combinations thereof. 
     
     
         71 . The CAR of  claim 44 , further comprising a hinge domain located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain. 
     
     
         72 . The CAR of  claim 44 , further comprising a signal peptide located at the N-terminus of the polypeptide. 
     
     
         73 . An isolated nucleic acid comprising a nucleic acid sequence encoding the CAR of  claim 44 . 
     
     
         74 . A vector comprising the isolated nucleic acid of  claim 73 . 
     
     
         75 . An engineered immune effector cell comprising the CAR of  claim 44 . 
     
     
         76 . The engineered immune effector cell of  claim 75 , wherein the immune effector cell is a T cell. 
     
     
         77 . A pharmaceutical composition comprising the engineered immune effector cell of  claim 75 , and a pharmaceutically acceptable carrier. 
     
     
         78 . A method of treating cancer in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of  claim 77 .