Chimeric antigen receptors against AXL or ROR2 and methods of use thereof
The present disclosure provides chimeric antigen receptors that bind to Axl and Ror2, and conditionally active chimeric antigen receptors (CARs) that recognize Axl and Ror2. Furthermore, provided herein are nucleic acids encoding these CARs and methods of making and using the CARs, including methods of treating cancer, especially cancers that express Axl and/or Ror2, such as renal cell carcinoma. The present disclosure provides cells genetically modified to produce the CARs.
1. A nucleic acid encoding a chimeric antigen receptor for binding Axl, comprising:
a) an antigen-specific targeting region (ASTR), wherein the ASTR binding to Axl comprises a heavy chain variable region comprising three complementarity determining regions having H1, H2, and H3 sequences and a light chain variable region comprising three complementarity determining regions having L1, L2, and L3 sequences, wherein:
i) the H1 sequence is X1GX2TMN (SEQ ID NO:87);
ii) the H2 sequence is LIKPSNGGTSYNQKFKG (SEQ ID NO:88);
iii) the H3 sequence is GX3YX4SYX5AMDY (SEQ ID NO:89);
iv) the L1 sequence is KASQDVX6SAVA (SEQ ID NO:90);
v) the L2 sequence is WX7X8TRX9T (SEQ ID NO:91); and
vi) the L3 sequence is QEHFSX10PLX11 (SEQ ID NO:92),
wherein X1 is T or W; X2 is Hor A; X3 is Hor D; X4 is E or H; X5 is E or F; X6 is S or V; X7 is A or Q; X8 is S or D; X9 is Hor D; X10 is T or P; and X11 is T or R;
b) a transmembrane domain; and
c) an intracellular activating domain, wherein the transmembrane domain is located between the ASTR and the intracellular activating domain.
2. A nucleic acid encoding a chimeric antigen receptor for binding Axl, comprising:
a) an antigen-specific targeting region (ASTR), wherein the ASTR binding to Axl comprises a heavy chain variable region comprising three complementarity determining regions having H1, H2, and H3 sequences and a light chain variable region comprising three complementarity determining regions having L1, L2, and L3 sequences, wherein:
i) the H1 sequence is X1GX2TMN (SEQ ID NO:87);
ii) the H2 sequence is LIKPSNGGTSYNQKFKG (SEQ ID NO:88);
iii) the H3 sequence is GX3YX4SYX5AMDY (SEQ ID NO:89);
iv) the L1 sequence is KASQDVX6SAVA (SEQ ID NO:90);
v) the L2 sequence is WX7X8TRX9T (SEQ ID NO:91); and
vi) the L3 sequence is QEHFSX10PLX11 (SEQ ID NO:92),
wherein X1 is Tor W; X2 is Hor A; X3 is H; X4 is E; X5 is E; X6 is S or V; X7 is Q; X8 is D; X9 is H; X10 is T or P; and X11 is T or R;
b) a transmembrane domain; and
c) an intracellular activating domain, wherein the transmembrane domain is located between the ASTR and the intracellular activating domain.
3. The nucleic acid of claim 1 , wherein the ASTR binds to the same epitope of Axl as a single-chain variable antibody fragment comprising an antibody heavy chain variable region of SEQ ID NO:79 and an antibody light chain variable region of SEQ ID NO:80.
4. The nucleic acid of claim 1 , wherein the heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:79.
5. The nucleic acid of claim 1 , wherein the light chain variable region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:80.
6. The nucleic acid of claim 5 , wherein the heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:79.
7. The nucleic acid of claim 1 , wherein the ASTR comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 128-129 or 159-161.
8. The nucleic acid of claim 1 , wherein the heavy chain variable region and the light chain variable region are separated by a linker, wherein the linker is between 6 and 100 amino acids in length.
9. The nucleic acid of claim 8 , wherein the linker comprises the amino acid sequence of any one of SEQ ID NOs: 53-55.
10. The nucleic acid of claim 1 , wherein the chimeric antigen receptor further comprises a stalk domain and a co-stimulatory domain, and wherein the chimeric antigen receptor comprises from amino terminus to carboxy terminus, the ASTR, the stalk domain, the transmembrane domain, the co-stimulatory domain, and the intracellular activating domain.
11. The nucleic acid of claim 10 , wherein the stalk domain comprises a CD8 stalk domain or a CD28 stalk domain, and wherein the co-stimulatory domain comprises a CD137 co-stimulatory domain, an ICA co-stimulatory domain comprising proline-to-alanine mutations corresponding to residues 29, 32, and 33 of SEQ ID NO:2, a CD28 co-stimulatory domain, or comprises both the ICA co-stimulatory domain and the CD137 co-stimulatory domain.
12. The nucleic acid of claim 11 , wherein the stalk domain comprises the CD28 stalk domain, wherein the transmembrane domain comprises a CD28 transmembrane domain, and wherein the co-stimulatory domain comprises the ICA co-stimulatory domain.
13. The nucleic acid of claim 1 , wherein the transmembrane domain comprises a CD8 transmembrane domain or a CD28 transmembrane domain, and wherein the intracellular activating domain comprises a CD3Z activating domain.
14. The nucleic acid of claim 1 , wherein the ASTR is an antibody selected from an scFv, an Fab fragment, an Fab′ fragment, an (Fab′) 2 fragment, an Fv fragment, and a divalent single-chain antibody or a diabody.
15. The nucleic acid of claim 1 , wherein the ASTR is a single-chain variable fragment.
16. An isolated recombinant T cell, comprising a genome comprising the nucleic acid of claim 1 , wherein the nucleic acid is operably linked to a promoter active in T cells.
17. The isolated recombinant T cell of claim 16 , wherein the nucleic acid further encodes an elimination domain, wherein the nucleic acid sequence encoding the elimination domain is separated from the nucleic acid sequence encoding the chimeric antigen receptor by a ribosomal skip sequence.
18. A vector, comprising the nucleic acid of claim 1 , wherein the nucleic acid is operably linked to a promoter active in T cells.
19. The vector of claim 18 , wherein the nucleic acid further encodes an elimination domain, wherein the nucleic acid sequence encoding the elimination domain is separated from the nucleic acid sequence encoding the chimeric antigen receptor by a ribosomal skip sequence.
20. The vector of claim 19 , wherein the elimination domain is recognized by a regulatory authority-approved antibody.
21. The vector of claim 18 , wherein the vector is a replication incompetent retroviral particle.
22. The vector of claim 18 , wherein the vector is a lentiviral vector.