Chimeric antigen receptor (CAR) comprising a CD19-binding domain
There is provided a chimeric antigen receptor (CAR) comprising a CD19-binding domain which comprises a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences: CDR1—GY-AFSSS (SEQ ID No. 1); CDR2—YPGDED (SEQ ID No. 2) CDR3—SLLYGDYLDY (SEQ ID No. 3); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1—SASSSVSYMH (SEQ ID No. 4); CDR2—DTSKLAS (SEQ ID No. 5) CDR3—QQWNINPLT (SEQ ID No. 6). There is also provided a cell comprising such a CAR, and the use of such a cell in the treatment of cancer, in particular a B cell malignancy.
1. A chimeric antigen receptor (CAR) comprising a CD19-binding domain which comprises:
a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:
CDR1
(SEQ ID NO. 1)
GYAFSSS;
CDR2
(SEQ ID NO. 2)
YPGDED;
and
CDR3
(SEQ ID NO. 3)
SLLYGDYLDY;
and
b) a light chain variable region (VL) having CDRs with the following sequences:
CDR1
(SEQ ID NO. 4)
SASSSVSYMH;
CDR2
(SEQ ID NO. 5)
DTSKLAS;
and
CDR3
(SEQ ID NO. 6)
QQWNINPLT.
2. The CAR according to claim 1 , wherein the CD19 binding domain comprises a VH domain having the sequence shown as SEQ ID NO: 7 and/or or a VL domain having the sequence shown as SEQ ID NO: 8 or a variant thereof having at least 95% sequence identity.
3. The CAR according to claim 1 , wherein the CD19 binding domain comprises an scFv in the orientation VH-VL.
4. The CAR according to claim 3 , wherein the CD19 binding domain comprises the sequence shown as SEQ ID NO: 9 or a variant thereof having at least 90% sequence identity.
5. The CAR according to claim 1 , wherein CD19-binding domain is connect to a transmembrane domain by a spacer.
6. The CAR according to claim 5 , wherein the spacer comprises one of the following: a human an IgG1 Fc domain; an IgG1 hinge; or a CD8 stalk.
7. A CAR according to claim 1 that comprises an intracellular T-cell signalling domain.
8. The CAR according to claim 7 , wherein the intracellular T-cell signalling domain comprises one or more of the following endodomains: CD28 endodomain; 41 BB endodomain, OX40 endodomain and the CD3-Zeta endodomain.
9. The CAR according to claim 8 , wherein the intracellular T-cell signalling domain comprises:
(i) the 41 BB endodomain and the CD3-Zeta endodomain;
(ii) the OX40 endodomain and the CD3-Zeta endodomain; or
(iii) all of the following endodomains: CD28 endodomain; OX40 and CD3-Zeta endodomain.
10. The CAR according to claim 1 , which comprises the sequence shown as any of SEQ ID NOs: 10 to 15, or a variant thereof which has at least 80% sequence identity and retains the capacity to i) bind CD19 and ii) induce T cell signalling.
11. The CAR according to claim 1 which, when expressed by a T-cell and used to target a CD19-expressing cell, causes lower IFNγ release by the CD19-expressing target cell than that caused by a T-cell expressing a CAR comprising a CD19-binding domain which comprises
a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:
CDR1
(SEQ ID NO. 16)
GVSLPDY;
CDR2
(SEQ ID NO. 17)
WGSET;
and
CDR3
(SEQ ID NO. 18)
HYYYGGSYAMDY;
and
b) a light chain variable region (VL) having CDRs with the following sequences:
CDR1
(SEQ ID NO. 19)
RASQDISKYLN;
CDR2
(SEQ ID NO. 20)
HTSRLHS;
and
CDR3
(SEQ ID NO. 21)
QQGNTLPYT.
12. The CAR according to claim 1 , wherein the CDRs are grafted on to a human or humanised framework.
13. A nucleic acid comprising a nucleic acid sequence which encodes a CAR, wherein the CAR comprises a CD19-binding domain which comprises:
a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:
(SEQ ID NO: 1)
CDR1 - GYAFSSS;
(SEQ ID NO: 2)
CDR2 - YPGDED
(SEQ ID NO: 3)
CDR3 - SLLYGDYLDY;
and
b) a light chain variable region (VL) having CDRs with the following sequences:
(SEQ ID NO: 4)
CDR1 - SASSSVSYMH;
(SEQ ID NO: 5)
CDR2 - DTSKLAS;
and
(SEQ ID NO: 6)
CDR3 - QQWNINPLT.
14. The nucleic acid according to claim 13 , wherein the CD19 binding domain comprises a VH domain having the sequence shown as SEQ ID NO: 7 and/or or a VL domain having the sequence shown as SEQ ID NO: 8 or a variant thereof having at least 95% sequence identity.
15. The nucleic acid according to claim 13 , wherein the CD19 binding domain comprises the sequence shown as SEQ ID NO: 9 or a variant thereof having at least 90% sequence identity.
16. The nucleic acid according to claim 13 , wherein the CAR comprises the sequence shown as any of SEQ ID NOs: 10 to 15, or a variant thereof which has at least 80% sequence identity and retains the capacity to i) bind CD19 and ii) induce T cell signalling.
17. A vector which comprises a nucleic acid according to claim 13 .
18. A cell which comprises a CAR according to claim 1 .
19. The cell according to claim 18 that is a T cell or a natural killer (NK) cell.
20. A cell composition which comprises a plurality of cells according to claim 18 .
21. A method for making a cell which comprises a CAR, the method comprising a step of transducing or transfecting a cell with a vector according to claim 17 .
22. A method for making a composition which comprises a plurality of cells which comprise a CAR, the method comprising a step of transducing or transfecting a sample of cells from a subject ex vivo with a vector according to claim 17 .
23. A pharmaceutical composition which comprises a cell according to claim 18 , or a plurality of said cells, together with a pharmaceutically acceptable carrier, diluent, or excipient.
24. A method of treating a B cell malignancy, the method comprising administering to a subject with the B cell malignancy:
a cell according to claim 18 , or
a pharmaceutical composition which comprises said cell or a plurality of said cells, together with a pharmaceutically acceptable carrier, diluent, or excipient.