CONSTRUCTION OF CHIMERIC ANTIGEN RECEPTOR TARGETING CD20 ANTIGEN AND ACTIVITY IDENTIFICATION OF ENGINEERED T CELLS THEREOF
Provided are a chimeric antigen receptor targeting CD20 antigen and a preparation method thereof. The extracellular antigen binding domain of the chimeric antigen receptor includes an antibody heavy chain variable region shown in SEQ ID NO: 7 or 9 or 33 and an antibody light chain variable region shown in SEQ ID NO: 11 or 13 or 35, and is capable of killing tumor cells.
1 . An immune cell comprising a chimeric antigen receptor (CAR), the CAR comprising an anti-CD20 antigen binding region which comprises: (i) a heavy chain variable region (V H ) having an amino acid sequence set forth in SEQ ID NO: 7, and (ii) a light chain variable region (V L ) having an amino acid sequence set forth in SEQ ID NO: 11;
wherein V H is located at the N-terminus of V L ,
wherein the anti-CD20 antigen-binding region is a single-chain variable fragment (scFv) that specifically binds CD20,
wherein the CAR further comprises:
(iii) a signal peptide having an amino acid sequence encoded by a nucleic acid sequence having ≥90% identity to the nucleic acid sequence set forth in SEQ ID NO: 28,
(iv) a hinge region having an amino acid sequence encoded by a nucleic acid sequence having ≥90% identity to the nucleic acid sequence set forth in SEQ ID NO: 20,
(v) a transmembrane domain having an amino acid sequence encoded by a nucleic acid sequence having ≥90% identity to the nucleic acid sequence set forth in SEQ ID NO: 22,
(vi) a co-stimulatory region having an amino acid sequence encoded by a nucleic acid sequence having ≥90% identity to the nucleic acid sequence set forth in SEQ ID NO: 24, and
(vii) a cytoplasmic signaling domain having an amino acid sequence encoded by a nucleic acid sequence having ≥90% identity to the nucleic acid sequence set forth in SEQ ID NO: 26.
2 . The immune cell of claim 1 , wherein the CAR comprises from N-terminus to C-terminus: the signal peptide, V H , V L , the hinge region, the transmembrane domain, the co-stimulatory region, and the cytoplasmic signaling domain.
3 . The immune cell of claim 1 , wherein the CAR comprises an amino acid sequence encoded by a nucleic acid sequence having ≥95% identity to the nucleic acid sequence set forth in SEQ ID NO: 6.
4 . The immune cell of claim 1 , wherein the CAR comprises an amino acid sequence encoded by a nucleic acid sequence having ≥98% identity to the nucleic acid sequence set forth in SEQ ID NO: 6.
5 . The immune cell of claim 1 , wherein the immune cell is a T cell.
6 . An immune cell comprising a chimeric antigen receptor (CAR), the CAR comprising an anti-CD20 antigen binding region which comprises: (i) a heavy chain variable region (V H ) having an amino acid sequence set forth in SEQ ID NO: 7, and (ii) a light chain variable region (V L ) having an amino acid sequence set forth in SEQ ID NO: 11;
wherein V H is located at the N-terminus of V L ,
wherein the anti-CD20 antigen-binding region is a single-chain variable fragment (scFv) that specifically binds CD20, and
wherein the CAR comprises an amino acid sequence encoded by a nucleic acid sequence having ≥95% identity to the nucleic acid sequence set forth in SEQ ID NO: 6.
7 . The immune cell of claim 6 , wherein the CAR comprises an amino acid sequence encoded by a nucleic acid sequence having ≥98% identity to the nucleic acid sequence set forth in SEQ ID NO: 6.
8 . The immune cell of claim 6 , wherein the immune cell is a T cell.
9 . A pharmaceutical composition comprising an immune cell which comprises a chimeric antigen receptor (CAR), the CAR comprising an anti-CD20 antigen binding region which comprises: (i) a heavy chain variable region (V H ) having an amino acid sequence set forth in SEQ ID NO: 7, and (ii) a light chain variable region (V L ) having an amino acid sequence set forth in SEQ ID NO: 11;
wherein V H is located at the N-terminus of V L ,
wherein the anti-CD20 antigen-binding region is a single-chain variable fragment (scFv) that specifically binds CD20,
wherein the CAR further comprises:
(iii) a signal peptide having an amino acid sequence encoded by a nucleic acid sequence having ≥90% identity to the nucleic acid sequence set forth in SEQ ID NO: 28,
(iv) a hinge region having an amino acid sequence encoded by a nucleic acid sequence having ≥90% identity to the nucleic acid sequence set forth in SEQ ID NO: 20,
(v) a transmembrane domain having an amino acid sequence encoded by a nucleic acid sequence having ≥90% identity to the nucleic acid sequence set forth in SEQ ID NO: 22,
(vi) a co-stimulatory region having an amino acid sequence encoded by a nucleic acid sequence having ≥90% identity to the nucleic acid sequence set forth in SEQ ID NO: 24, and
(vii) a cytoplasmic signaling domain having an amino acid sequence encoded by a nucleic acid sequence having ≥90% identity to the nucleic acid sequence set forth in SEQ ID NO: 26.
10 . The pharmaceutical composition of claim 9 , wherein the CAR comprises from N-terminus to C-terminus: the signal peptide, V H , V L , the hinge region, the transmembrane domain, the co-stimulatory region, and the cytoplasmic signaling domain.
11 . The pharmaceutical composition of claim 9 , wherein the CAR comprises an amino acid sequence encoded by a nucleic acid sequence having ≥95% identity to the nucleic acid sequence set forth in SEQ ID NO: 6.
12 . The pharmaceutical composition of claim 9 , wherein the CAR comprises an amino acid sequence encoded by a nucleic acid sequence having ≥98% identity to the nucleic acid sequence set forth in SEQ ID NO: 6.
13 . The pharmaceutical composition of claim 9 , wherein the immune cell is a T cell.