Chimeric antigen receptors comprising a human transferrin epitope sequence
The present invention is directed to a chimeric antigen receptor fusion protein comprising: (i) a single-chain variable fragment (scFv) comprising V H and V L , wherein scFv has an activity against a tumor antigen, (ii) a transmembrane domain, (iii) at least one co-stimulatory domains, and (iv) an activating domain; wherein the CAR further comprises a human transferrin fragment, which is an epitope for an antibody against human transferrin, at N-terminus or C-terminus to scFv, or between V H and V L . Preferred tumor antigens are CD19, CD22 and BCMA. The CD19-TF-CAR-T cells, CD22-TF-CAR-T cells, and BAMA-TF CAR-T cells secrete less cytokines, but they have the same efficacy against cancer target cells when comparing with same CAR without TF.
1. A chimeric antigen receptor (CAR) comprising from N-terminal to C-terminal:
an extracellular domain comprising
a single-chain variable fragment (scFv) comprising VH and VL, wherein scFv binds to a tumor antigen,
a human transferrin fragment having the amino acid sequence of KNPDPWAKNLNEKDY (SEQ ID NO: 1, TF), 2-5 TFs, or 1/2 TF having the amino sequence of KNLNEKDY (SEQ ID NO: 2), wherein the transferrin fragment is C-terminus to the scFv;
a transmembrane domain; and
an intracellular domain comprising:
at least one co-stimulatory domains, and
an activating domain.
2. The CAR according to claim 1 , wherein the tumor antigen is selected from the group consisting of: CD19, CD22, BCMA, VEGFR-2, CD20, CD30, CD25, CD28, CD30, CD33, CD47, CD52, CD56, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, CD133, EGFR, GPC3; PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2/HER-2, ErbB3/HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis A, Lewis Y, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRa, TCRp, TLR7, TLR9, PTCH1, WT-1, Robol, a, Frizzled, OX40, CD79b, and Notch-1-4.
3. The CAR according to claim 2 , wherein the tumor antigen is CD19.
4. The CAR according to claim 3 , wherein (a) the VH has the amino acid sequence of SEQ ID NO: 6 and VL has the amino acid sequence of SEQ ID NO: 4, or (b) the VH has the amino acid sequence of SEQ ID NO: 16 and the VL has the amino acid sequence of SEQ ID NO: 14.
5. The CAR according to claim 2 , wherein the tumor antigen is CD22.
6. The CAR according to claim 5 , wherein the VH has the amino acid sequence of SEQ ID NO: 17 and the VL has the amino acid sequence of SEQ ID NO: 18.
7. The CAR according to claim 2 , wherein the tumor antigen is BCMA.
8. The CAR according to claim 7 , wherein (a) the VH has the amino acid sequence of SEQ ID NO: 22 and the VL has the amino acid sequence of SEQ ID NO: 23, or (b) the VH has the amino acid sequence of SEQ ID NO: 26 and the VL has the amino acid sequence of SEQ ID NO: 27, or (c) the VH has the amino acid sequence of SEQ ID NO: 29 and the VL has the amino acid sequence of SEQ ID NO: 30, or (d) the VH has the amino acid sequence of SEQ ID NO: 31 and VL has the amino acid sequence of SEQ ID NO: 32.
9. The CAR according to claim 1 , wherein the co-stimulatory domain is selected from the group consisting of CD28, 4-1BB, ICOS-1, CD27, OX-40, GITR and DAP10.
10. The CAR according to claim 1 , wherein the co-stimulatory domain is CD28.
11. The CAR according to claim 1 , wherein the human transferrin fragment has the amino acid sequence of SEQ ID NO: 1.
12. The CAR according to claim 11 , which has the amino acid sequence of SEQ ID NO: 11 or at least 95% sequence identity thereof.
13. The CAR according to claim 1 , wherein the human transferrin fragment has the amino acid sequence of 2 TF.
14. The CAR according to claim 13 , which has the amino acid sequence of SEQ ID NO: 13 or at least 95% sequence identity thereof.
15. The CAR according to claim 1 , wherein the human transferrin fragment has the amino acid sequence of SEQ ID NO: 2.
16. The CAR according to claim 15 , which has the amino acid sequence of SEQ ID NO: 12 or at least 95% sequence identity thereof.
17. The CAR according to claim 1 , wherein the scFv comprises from N-terminal to C-terminal the VL, a linker, and the VH, and the linker has the amino acid sequence of SEQ ID NO: 5.