Engineered immune cells targeting BCMA and their uses thereof
The present invention provides an engineered immune cell targeting BCMA and use thereof. In particular, the present invention provides a CAR specifically targeting BCMA, the CAR comprising an antigen-binding domain which is an S-derived scFv, an antibody heavy chain variable region as shown in SEQ ID NO: 9 and an antibody light chain variable region as shown in SEQ ID NO: 10. The present invention also provides a CAR-T cell comprising the CAR, a double CAR- and CAR-T cell comprising the S-derived scFv, and related use thereof. Compared to CAR-T cells constructed using other scFvs, the constructed CAR-T cell of the present invention has a better killing effect and tumor elimination capability.
1 . A bispecific chimeric antigen receptor (CAR) comprising a first antigen binding domain directed against B-cell maturation antigen (BCMA) and a second antigen binding domain directed against CD19, wherein:
(i) the first antigen binding domain comprises:
a heavy chain variable region for BCMA (V H,BCMA ) having a complementarity determining region 1 (HCDR1) comprising the amino acids corresponding to amino acids 31 to 35 of SEQ ID NO: 9, a HCDR2 comprising the amino acids corresponding to amino acids 50 to 66 of SEQ ID NO: 9, and a HCDR3 comprising the amino acids corresponding to amino acids 99 to 110 of SEQ ID NO: 9; and
a light chain variable region for BCMA (V L,BCMA ) having a complementarity determining region 1 (LCDR1) comprising the amino acids corresponding to amino acids 24 to 34 of SEQ ID NO: 10, a LCDR2 comprising the amino acids corresponding to amino acids 50 to 56 of SEQ ID NO: 10, and a LCDR3 comprising the amino acids corresponding to amino acids 89 to 97 of SEQ ID NO: 10; and
(ii) the second antigen binding domain comprises a heavy chain variable region for CD19 (VH, CD19);
and a light chain variable region for CD19 (VL, CD19) wherein a polypeptide of the bispecific CAR comprises a structure as shown in formula (III) or (III′):
V L,CD19 −SCFV−V H,CD19 (III); or
V H,CD19 −SCFV−V L,CD19 (III′);
wherein “-” is each independently a linker peptide or a peptide bond; and wherein scFv corresponds to the first antigen binding domain against BCMA, comprising V L,BCMA and V H,BCMA .
2 . The bispecific CAR according to claim 1 , wherein the V H,CD19 comprises a HCDR1 comprising the amino acids corresponding to amino acids 31 to 35 of SEQ ID NO: 11, a HCDR2 comprising the amino acids corresponding to amino acids 50 to 65 of SEQ ID NO: 11, and a HCDR3 comprising the amino acids corresponding to amino acids 98 to 109 of SEQ ID NO: 11.
3 . The bispecific CAR according to 1 , wherein the V L,CD19 comprises a LCDR1 comprising the amino acids corresponding to amino acids 24 to 34 of SEQ ID NO: 12, a LCDR2 comprising the amino acids corresponding to amino acids 50 to 56 of SEQ ID NO: 12, and a LCDR3 comprising the amino acids corresponding to amino acids 89 to 97 of SEQ ID NO: 12.
4 . The bispecific CAR according to claim 1 , wherein
the V H,CD19 comprises a HCDR1 comprising the amino acids corresponding to amino acids 31 to 35 of SEQ ID NO: 11, a HCDR2 comprising the amino acids corresponding to amino acids 50 to 65 of SEQ ID NO: 11, and a HCDR3 comprising the amino acids corresponding to amino acids 98 to 109 of SEQ ID NO: 11; and
the V L,CD19 comprises a LCDR1 comprising the amino acids corresponding to amino acids 24 to 34 of SEQ ID NO: 12, a LCDR2 comprising the amino acids corresponding to amino acids 50 to 56 of SEQ ID NO: 12, and a LCDR3 comprising the amino acids corresponding to amino acids 89 to 97 of SEQ ID NO: 12.
5 . The bispecific CAR according to claim 4 , having the following formulae IIc-d:
L-V L,CD19 -V H,BCMA -V L,BCMA -V H,CD19 -H-TM-C-CD3ζ (IIc); or
L-V L,CD19 -V H,BCMA -I-V L,BCMA -V H,CD19 -H-TM-C-CD3ζ (IId);
wherein in the formulae (IIc-d):
“-” is each independently a linker peptide or a peptide bond;
L is absent or a signal peptide sequence;
I is a flexible linker;
H is absent or a hinge region;
TM is a transmembrane domain;
C is a costimulatory signal molecule; and
CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.
6 . The bispecific CAR according to claim 5 , wherein the signal peptide sequence, L, comprises a signal peptide from CD8, CD28, GM-CSF, CD4, or CD137.
7 . The bispecific CAR according to claim 5 , wherein the signal peptide sequence, L, comprises the amino acid sequence of SEQ ID NO: 16.
8 . The bispecific CAR according to claim 5 , wherein the hinge region, H, comprises the hinge region from CD8 CD28, or CD137.
9 . The bispecific CAR according to claim 5 , wherein the hinge region, H, comprises the amino acid sequence of one of SEQ ID NO: 8, 37, or 38.
10 . The bispecific CAR according to claim 5 , wherein the transmembrane domain, TM, comprises a transmembrane domain from CD28, CD3 epsilon, CD45, CD4 CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.
11 . The bispecific CAR according to claim 5 , wherein the transmembrane domain, TM, comprises the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 6.
12 . The bispecific CAR according to claim 5 , wherein the costimulatory signal molecule, C, comprises a costimulatory signal molecule from OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, or TLR2.
13 . The bispecific CAR according to claim 6 , wherein the costimulatory signal molecule, C, comprises the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 5.
14 . The bispecific CAR according to claim 5 , wherein the cytoplasmic signaling sequence, CD3, comprises the amino acid sequence of SEQ ID NO: 3.
15 . The bispecific CAR according to claim 5 , wherein the linker peptide, “-”, or flexible linker, I, if present, each independently comprise the amino acid sequence of SEQ ID NO: 19 or one to six copies of the amino acid sequence of SEQ ID NO: 40.
16 . The bispecific CAR according to claim 6 , having the formula IId, wherein the flexible linker, I, comprises the amino acid sequence of SEQ ID NO: 19.
17 . The bispecific CAR according to claim 5 , having the formula IIc, wherein
L comprises the amino acid sequence of SEQ ID NO: 16;
V L,CD19 comprises the amino acid sequence of SEQ ID NO: 12;
V H,BCMA comprises the amino acid sequence of SEQ ID NO: 9;
V L,BCMA comprises the amino acid sequence of SEQ ID NO: 10;
V H,CD19 comprises the amino acid sequence of SEQ ID NO: 11;
H comprises the amino acid sequence of SEQ ID NO: 38;
TM comprises the amino acid sequence of SEQ ID NO: 7;
C comprises the amino acid sequence of SEQ ID NO: 5; and
CD32 comprises the amino acid sequence of SEQ ID NO: 3.
18 . The bispecific CAR according to claim 5 , having the formula IId, wherein
L comprises the amino acid sequence of SEQ ID NO: 16;
V L,CD19 comprises the amino acid sequence of SEQ ID NO: 12;
V H,BCMA comprises the amino acid sequence of SEQ ID NO: 9;
I comprises the amino acid sequence of SEQ ID NO: 19;
V L,BCMA comprises the amino acid sequence of SEQ ID NO: 10;
V H,CD19 comprises the amino acid sequence of SEQ ID NO: 11;
H comprises the amino acid sequence of SEQ ID NO: 38;
TM comprises the amino acid sequence of SEQ ID NO: 7;
C comprises the amino acid sequence of SEQ ID NO: 5; and
CD3ζ comprises the amino acid sequence of SEQ ID NO: 3.
19 . A pharmaceutical composition, comprising:
the bispecific CAR according to claim 4 ; and
a pharmaceutically acceptable carrier, diluent, or excipient.
20 . A pharmaceutical composition, comprising:
the bispecific CAR according to claim 5 ; and
a pharmaceutically acceptable carrier, diluent, or excipient.
21 . A bispecific chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO: 41.
22 . A pharmaceutical composition, comprising:
the bispecific CAR according to claim 21 ; and
a pharmaceutically acceptable carrier, diluent, or excipient.
23 . An engineered immune cell comprising the bispecific CAR according to claim 4 .
24 . The engineered immune cell according to claim 23 , wherein the engineered immune cell is allogeneic to a subject.
25 . An engineered immune cell comprising the bispecific CAR according to claim 5 .
26 . The engineered immune cell according to claim 25 , wherein the engineered immune cell is allogeneic to a subject.
27 . An engineered immune cell comprising the bispecific CAR according to claim 21 .
28 . The engineered immune cell according to claim 27 , wherein the engineered immune cell is allogeneic to a subject.
29 . A polynucleotide encoding the bispecific CAR according to claim 4 .
30 . A polynucleotide encoding the bispecific CAR according to claim 5 .
31 . A polynucleotide encoding the bispecific CAR according to claim 17 .
32 . A polynucleotide encoding the bispecific CAR according to claim 21 .