Anti-hla-A2 antibodies and methods of using the same
Provided are humanized anti-HLA-A2 antibodies. In certain aspects, the humanized anti-HLA-A2 antibodies are capable of constituting an antigen binding domain of a chimeric antigen receptor (CAR), where the CAR is capable of being expressed in a human cell such that the CAR specifically binds to HLA-A2. Also provided are CARs that include the humanized anti-HLA-A2 antibodies. Modified cells including the antibodies and CARs, as well as methods of using such modified cells are also provided.
1. A chimeric antigen receptor (CAR) comprising:
a) an extracellular domain comprising a humanized anti-HLA-A2 antibody or an antigen-binding fragment thereof that exhibits reduced binding to one or more HLA-A subtypes selected from the group consisting of HLA-A*25, HLA-A*29, and HLA-A*30 as compared to antibody BB7.2 (ATCC Deposit No. HB-82), wherein said humanized anti-HLA-A2 antibody or antigen-binding fragment comprises
i) a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 61 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 68;
ii) a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 68;
iii) a VH comprising the amino acid sequence of SEQ ID NO: 65 and a VL comprising the amino acid sequence of SEQ ID NO: 68;
iv) a VH comprising the amino acid sequence of SEQ ID NO: 66 and a VL comprising the amino acid sequence of SEQ ID NO: 68;
v) a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 69;
vi) a VH comprising the amino acid sequence of SEQ ID NO: 65 and a VL comprising the amino acid sequence of SEQ ID NO: 69; or
vii) a VH comprising the amino acid sequence of SEQ ID NO: 63 and a VL comprising the amino acid sequence of SEQ ID NO: 70;
b) a transmembrane domain; and
c) a cytoplasmic domain comprising an intracellular signaling domain;
wherein said CAR is capable of being expressed in an immune cell such that said CAR specifically binds to HLA-A2.
2. The CAR of claim 1 , wherein the humanized anti-HLA-A2 antibody or antigen-binding fragment is an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 81, 83, 84, 87, 88 and 91.
3. The CAR of claim 1 , wherein the humanized anti-HLA-A2 antibody or antigen-binding fragment is an scFv comprising the amino acid sequence of SEQ ID NO: 73.
4. The CAR of claim 1 , further comprising a hinge region that comprises a stalk region of CD8α.
5. The CAR of claim 1 , wherein the transmembrane domain is a CD8 transmembrane domain and the intracellular signaling domain comprises a CD28 costimulatory domain and a CD3 zeta primary signaling domain.
6. The CAR of claim 1 , comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 120, 128, 130, 131, 134, 135, 138 and 213.
7. A chimeric antigen receptor (CAR) comprising the amino acid sequence of SEQ ID NO: 213.
8. A nucleic acid molecule encoding the CAR of claim 1 .
9. An immune cell comprising the CAR of claim 1 .
10. The immune cell of claim 9 , wherein the immune cell is a regulatory T cell.
11. A pharmaceutical composition comprising the immune cell of claim 9 and a pharmaceutically acceptable excipient.
12. A method for:
a) preventing or treating organ or tissue transplant rejection in a subject;
b) preventing or treating graft versus host disease (GVHD) in a subject;
c) promoting immune tolerance in a subject in need thereof;
d) inducing tolerance to a transplanted organ or tissue in a subject; or
e) any combination of a)-d);
wherein said method comprises administering an immune cell of claim 9 to the subject.
13. The method of claim 12 , wherein the subject is undergoing or has undergone a hematopoietic stem cell transplant.
14. The CAR of claim 1 , wherein said transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of: CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, the alpha chain of the T cell receptor, the beta chain of the T cell receptor, the gamma chain of the T cell receptor, the delta chain of the T cell receptor, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and any combination thereof.
15. The CAR of claim 1 , wherein said intracellular signaling domain comprises a functional signaling domain of a protein selected from the group consisting of: CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, FcR gamma, FcR alpha, FcR epsilon, CD5, CD22, CD79a, CD79b, and CD66d, and any combination thereof.
16. The CAR of claim 1 , wherein said intracellular signaling domain comprises a functional signaling domain of CD3 zeta.
17. The CAR of claim 1 , wherein said intracellular signaling domain comprises a functional signaling domain and a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, lymphocyte function-associated antigen-1 (LFA-1) (CD11a/CD18), TNFR1 (CD120a/TNFRSF1A), TNFR2 (CD120b/TNFRSF1B), CTLA-4 (CD152), CD95, ICOS (CD278), 4-1BB (CD137), CD2, CD30, CD40, PD-1, CD7, LIGHT, NKG2C, B7-H3, ICAM-1, a ligand that specifically binds to CD83, IL2Ra (CD25), IL6Ra (CD126), IL-7Ra (CD127), IL-13RA1, IL-13RA2, IL-33R(IL1RL1), IL-10RA, IL-10RB, IL-4R, IL-5R (CSF2RB), ARHR, BAFF receptor, IL-21R, TGFbR1, TGFbR2, TGFbR3, common gamma chain, and any combination thereof.
18. The CAR of claim 1 , wherein said intracellular signaling domain comprises a costimulatory domain, wherein said costimulatory domain comprises a functional signaling domain of a protein selected from CD28 and 4-1BB.