Treatment of cancer using humanized anti-CD19 chimeric antigen receptor
The invention provides compositions and methods for treating diseases associated with expression of CD19. The invention also relates to chimeric antigen receptor (CAR) specific to CD19, vectors encoding the same, and recombinant T cells comprising the CD19 CAR. The invention also includes methods of administering a genetically modified T cell expressing a CAR that comprises a CD19 binding domain.
1. An isolated chimeric antigen receptor (CAR) molecule comprising a single chain antibody or single chain antibody fragment which comprises an anti-CD19 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a primary intracellular signaling domain and a costimulatory domain, wherein:
(i) the anti-CD19 binding domain comprises a light chain complementary determining region 1 (LC CDR1) of SEQ ID NO: 25, a light chain complementary determining region 2 (LC CDR2) of SEQ ID NO: 26, and a light chain complementary determining region 3 (LC CDR3) of SEQ ID NO: 27, and a heavy chain complementary determining region 1 (HC CDR1) of SEQ ID NO: 19, a heavy chain complementary determining region 2 (HC CDR2) of SEQ ID NO: 21, 22, or 23, and a heavy chain complementary determining region 3 (HC CDR3) of SEQ ID NO: 24;
(ii) the primary intracellular signaling domain comprises a native intracellular signaling domain of CD3-zeta, or a variant thereof having at least one modification but not more than 20 modifications of the amino acid sequence of SEQ ID NO: 43; and
(iii) the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, ICAM-1, LFA-1, 4-1BB, and ICOS.
2. The isolated CAR molecule of claim 1 , wherein the anti-CD19 binding domain is a scFv.
3. The isolated CAR molecule of claim 1 , wherein the anti-CD19 binding domain comprises a light chain variable region listed in any of SEQ ID NOs: 1-12 and a heavy chain variable region listed in any of SEQ ID NOs: 1-12.
4. The isolated CAR molecule of claim 1 , wherein the anti-CD19 binding domain comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region listed in any of SEQ ID NOs: 1-12, or an amino acid sequence with at least 95% identity to an amino acid sequence of a light chain variable region listed in any of SEQ ID NOs: 1-12.
5. The isolated CAR molecule of claim 1 , wherein the anti-CD19 binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a heavy chain variable region listed in any of SEQ ID NOs: 1-12, or an amino acid sequence with at least 95% identity to an amino acid sequence of a heavy chain variable region listed in any of SEQ ID NOs: 1-12.
6. The isolated CAR molecule of claim 1 , wherein the anti-CD19 binding domain comprises a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, or an amino acid sequence with at least 95% identity thereto.
7. The isolated CAR molecule of claim 1 , wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
8. The isolated CAR molecule of claim 1 , wherein the transmembrane domain comprises:
(i) the amino acid sequence of SEQ ID NO: 15; or
(ii) an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 15.
9. The isolated CAR molecule of claim 7 , wherein the anti-CD19 binding domain is connected to the transmembrane domain by a hinge region.
10. The isolated CAR molecule of claim 9 , wherein the hinge region comprises the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:102, or an amino acid sequence with at least 95% identity thereto.
11. The isolated CAR molecule of claim 1 , wherein the costimulatory domain comprises:
(i) the amino acid sequence of SEQ ID NO: 16; or
(ii) an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 16.
12. The isolated CAR molecule of claim 1 , wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta.
13. The isolated CAR molecule of claim 1 , wherein the intracellular signaling domain comprises:
(i) the amino acid sequence of SEQ ID NO: 16 and/or the amino acid sequence of SEQ ID NO:17;
(ii) the amino acid sequence of SEQ ID NO:16 and/or the amino acid sequence of SEQ ID NO:43;
(iii) an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO:16 and/or the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:43, or an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:16 and/or the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:43; or
(iv) the amino acid sequence of SEQ ID NO: 16 and the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO:43, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.
14. The isolated CAR molecule of claim 1 , further comprising a leader sequence.
15. The isolated CAR molecule of claim 14 , wherein the leader sequence comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:13.
16. An anti-CD19 binding domain comprising a light chain complementary determining region 1 (LC CDR1) of SEQ ID NO: 25, a light chain complementary determining region 2 (LC CDR2) of SEQ ID NO: 26, and a light chain complementary determining region 3 (LC CDR3) of SEQ ID NO: 27, and a heavy chain complementary determining region 1 (HC CDR1) of SEQ ID NO: 19, a heavy chain complementary determining region 2 (HC CDR2) of SEQ ID NO: 21, 22, or 23, and a heavy chain complementary determining region 3 (HC CDR3) of SEQ ID NO: 24.
17. The anti-CD19 binding domain of claim 16 , wherein the anti-CD19 binding domain comprises a light chain variable region and a heavy chain variable region of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.
18. The anti-CD19 binding domain of claim 16 , wherein the anti-CD19 binding domain comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12 or an amino acid sequence with at least 95% identity to an amino acid sequence of a light chain variable region provided in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a heavy chain variable region provided in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, or an amino acid sequence with at least 95% identity to an amino acid sequence of a heavy chain variable region provided in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.
19. The isolated CAR molecule of claim 1 , wherein the HC CDR2 comprises the amino acid sequence of SEQ ID NO: 21.
20. The isolated CAR molecule of claim 1 , wherein the HC CDR2 comprises the amino acid sequence of SEQ ID NO: 22.
21. The isolated CAR molecule of claim 1 , wherein the HC CDR2 comprises the amino acid sequence of SEQ ID NO: 23.
22. The isolated CAR molecule of claim 1 , comprising the amino acid sequence of any of SEQ ID NOs: 31-42, or an amino acid sequence with at least 95% identity thereto, without the leader sequence of SEQ ID NO: 13.
23. The isolated CAR molecule of claim 1 , comprising the amino acid sequence of SEQ ID NO: 32, without the leader sequence of SEQ ID NO: 13.
24. The isolated CAR molecule of claim 1 , comprising, from N-terminus to C-terminus:
an anti-CD19 binding domain comprising the amino acid sequence of SEQ ID NO: 2,
a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 15,
a costimulatory domain comprising the amino acid sequence of SEQ ID NO: 16, and
a primary intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 17.
25. An isolated chimeric antigen receptor (CAR) molecule comprising, from N-terminus to C-terminus:
an anti-CD19 binding domain comprising the amino acid sequence of SEQ ID NO: 2,
a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 15,
a costimulatory domain comprising the amino acid sequence of SEQ ID NO: 16, and
a primary intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 43.
26. An isolated chimeric antigen receptor (CAR) molecule comprising a single chain antibody or single chain antibody fragment which comprises an anti-CD19 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a primary intracellular signaling domain and a costimulatory domain, wherein:
(i) the anti-CD19 binding domain comprises a light chain complementary determining region 1 (LC CDR1) of SEQ ID NO: 25, a light chain complementary determining region 2 (LC CDR2) of SEQ ID NO: 26, and a light chain complementary determining region 3 (LC CDR3) of SEQ ID NO: 27, and a heavy chain complementary determining region 1 (HC CDR1) of SEQ ID NO: 19, a heavy chain complementary determining region 2 (HC CDR2) of SEQ ID NO: 21, 22, or 23, and a heavy chain complementary determining region 3 (HC CDR3) of SEQ ID NO: 24;
(ii) the primary intracellular signaling domain comprises a native intracellular signaling domain of FcR gamma, or a functional fragment thereof; and
(iii) the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, ICAM-1, LFA-1, 4-1BB, and ICOS.