Masked chimeric antigen receptor specific to tyrosine-protein kinase like 7 (PTK7) and immune cells expressing such
Masked chimeric antigen receptor (CAR) constructs comprising an extracellular antigen binding domain specific tyrosine-protein kinase-like 7 (PTK7), which is linked to a mask peptide that blocks binding of masked CAR from binding to PTK7. Also provided herein are genetically engineered T cells expressing a masked CAR specific to PTK7 and therapeutic uses thereof.
1. A masked chimeric antigen receptor (CAR) specific to tyrosine-protein kinase-like 7 (PTK7), the masked CAR comprising:
(i) an extracellular antigen binding domain, which comprises a single chain variable fragment (scFv) that binds PTK7 and a mask peptide linked to the N-terminus of the scFv via a protease cleavage site; and
(ii) one or more intracellular signaling domains,
wherein the mask peptide comprises the amino acid sequence selected from the group consisting of:
(a)
(SEQ ID NO: 1)
EVAPGKRWFYNHVKQVPHLV,
(b)
(SEQ ID NO: 2)
HEEVHMRPNKLSLTWAYTGPQLR,
and
(c) X 1 CX 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 , in which X 1 is V, W, or absent; X 2 is T, H, or Y; X 3 is M, F, Y, I, or H; X 4 is P, G, or V; X 5 is P, N, S, Y, K, L, V, or A; X 6 is S, T, W, A, H, R, or Q; X 7 is P, T, V, H, I, M, A, F, or W; X 8 is R, M, A, H, V, Y, or absent; X 9 is S, Q, Y, T, P, A, M, or I; X 10 is K, R, I, C, S, Q, H, or absent; X 11 is V, T, R, L, F, W, or A; X 12 is I, F, L, W, or H; and X 13 is C, I, or M.
2. The masked CAR of claim 1 , wherein the mask peptide comprises the amino acid sequence of (c), which is:
(c1)
(SEQ ID NO: 3)
CTMPPSPRSKVIC,
(c2)
(SEQ ID NO: 4)
CTFPNTTMQRTFC,
(c3)
(SEQ ID NO: 5)
CTYPSWVAYIRFC,
(c4)
(SEQ ID NO: 6)
VCTYPPAHRTRFC,
(c5)
(SEQ ID NO: 7)
CTMPYHIHSIGLC,
(c6)
(SEQ ID NO: 8)
WCTIPSSMSIRLC,
(c7)
(SEQ ID NO: 9)
CHIGKRPVPCLWI,
(c8)
(SEQ ID NO: 10)
CYIGLRMVPCFHM,
(c9)
(SEQ ID NO: 11)
CTMPSHAVASFLC,
(c10)
(SEQ ID NO: 12)
CTMPVHTYSQWLC,
(c11)
(SEQ ID NO: 13)
CTYPPRFHMHWLC,
or
(c12)
(SEQ ID NO: 14)
CTHVAQWAIKAFC.
3. The masked CAR of claim 1 , wherein the mask peptide is 13-25 amino acids in length.
4. The masked CAR of claim 1 , wherein the mask peptide is:
(a)
(SEQ ID NO: 1)
EVAPGKRWFYNHVKQVPHLV,
(b)
(SEQ ID NO: 2)
HEEVHMRPNKLSLTWAYTGPQLR,
(c1)
(SEQ ID NO: 3)
CTMPPSPRSKVIC,
(c2)
(SEQ ID NO: 4)
CTFPNTTMQRTFC,
(c3)
(SEQ ID NO: 5)
CTYPSWVAYIRFC,
(c4)
(SEQ ID NO: 6)
VCTYPPAHRTRFC,
(c5)
(SEQ ID NO: 7)
CTMPYHIHSIGLC,
(c6)
(SEQ ID NO: 8)
WCTIPSSMSIRLC,
(c7)
(SEQ ID NO: 9)
CHIGKRPVPCLWI,
(c8)
(SEQ ID NO: 10)
CYIGLRMVPCFHM,
(c9)
(SEQ ID NO: 11)
CTMPSHAVASFLC,
(c10)
(SEQ ID NO: 12)
CTMPVHTYSQWLC,
(c11)
(SEQ ID NO: 13)
CTYPPRFHMHWLC,
or
(c12)
(SEQ ID NO: 14)
CTHVAQWAIKAFC.
5. The masked CAR of claim 1 , wherein the mask peptide is removable by protease cleavage at the protease cleavage site.
6. The masked CAR of claim 1 , wherein the protease cleavage site is a cleavage site of a matrix metalloproteinase (MMP).
7. The masked CAR of claim 6 , wherein the protease cleavage site is a MMP14 cleavage site, which comprises the motif of PLGLA (SEQ ID NO: 15).
8. The masked CAR of claim 1 , wherein the mask peptide is linked to the protease cleavage site via a first peptide linker.
9. The masked CAR of claim 1 , wherein the protease cleavage site is linked to the N-terminus of the heavy chain or the light chain of the anti-PTK7 antibody via a peptide linker.
10. The masked CAR of claim 8 , wherein the first peptide linker is a G/S peptide linkers.
11. The masked CAR of claim 8 , wherein the mask peptide is linked to the scFv that binds PFK7 in a formula of: M-L 1 -P-L 2 -scFv, in which M represents the mask peptide, L 1 and L 2 represents the first and second peptide linkers, and P represents the protease cleavage site.
12. The masked CAR of claim 1 , wherein the scFv that binds PTK7 comprises a heavy chain variable domain (V H ), which comprises a V H CDR1 sequence of SEQ ID NO: 23, a V H CDR2 sequence of SEQ ID NO: 24, and a V H CDR3 sequence of SEQ ID NO: 25; and/or wherein the anti-PTK7 antibody comprises a light chain variable domain (V L ), which comprises a V L CDR1 sequence of SEQ ID NO: 26, a V L CDR2 sequence of SEQ ID NO: 27 and a V L CDR3 sequence of SEQ ID NO: 28.
13. The masked CAR of claim 12 , wherein the scFv that binds PTK7 comprises a V H sequence of SEQ ID NO: 29 and a V L sequence of SEQ ID NO: 30.
14. The masked CAR of claim 12 , wherein the extracellular antigen binding domain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 120-134.
15. The masked CAR of claim 1 , wherein the one or more intracellular signaling domains comprises a co-stimulatory domain, a CD3CΘ cytoplasmic signaling domain, or a combination thereof.
16. The masked CAR of claim 15 , wherein the co-stimulatory domain is a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain.
17. The masked CAR of claim 15 , which further comprises a transmembrane domain located between the extracellular antigen binding domain and the one or more intracellular signaling domains.
18. The masked CAR of claim 17 , wherein the transmembrane domain is a CD8 transmembrane domain.
19. The masked CAR of claim 1 , which further comprises a signal peptide at the N-terminus of the masked CAR.
20. The masked CAR of claim 1 , which comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 106-119.
21. A nucleic acid, comprising a nucleotide sequence encoding a masked CAR of claim 1 .
22. A genetically engineered T cell, comprising the nucleic acid of claim 21 and expressing the masked CAR encoded by the nucleic acid.
23. The genetically engineered T cell of claim 22 , wherein the T cell further comprises a disrupted TRAC gene, a disrupted B2M gene, or a combination thereof.
24. The genetically engineered T cell of claim 21 , wherein the T cell comprises a disrupted TRAC gene, in which the nucleic acid encoding the masked CAR is inserted, thereby disrupting expression of the TRAC gene.
25. The genetically engineered T cell of claim 23 , wherein the T cell comprises a disrupted TRAC gene, which comprises a deletion of a fragment comprising the nucleic acid sequence of SEQ ID NO: 40.
26. The genetically engineered T cell of claim 25 , wherein the nucleic acid encoding the masked CAR is inserted at the site of the deletion in the disrupted TRAC gene.
27. The genetically engineered T cell of claim 26 , wherein the nucleic acid encoding the masked CAR replaces a fragment comprising SEQ ID NO: 40 in the disrupted TRAC gene.
28. A population of genetically engineered T cells, comprising T cells that express the masked CAR of claim 1 .
29. A method for producing genetically engineered CAR-T cells, comprising:
(a) delivering to T cells a nucleic acid encoding a masked CAR set forth in claim 1 ; and
(b) producing genetically engineered CAR-T cells expressing the masked CAR.
30. A population of genetically engineered T cells, produced by a method of claim 29 .