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 - 62 . (canceled)
63 . A humanized anti-HLA-A2 antibody or an antigen-binding fragment thereof, that exhibits reduced binding to one or more HLA-A subtype selected from one or more of HLA-A*25, HLA-A*29, HLA-A*30 as compared to a BB7.2 antibody, wherein said antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 61-66, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 67-71.
64 . The humanized anti-HLA-A2 antibody or an antigen-binding fragment of claim 63 , comprising:
a) a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 61 and a light chain variable domain (VL) sequence comprising the amino acid sequence of SEQ ID NO: 68;
b) a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 68;
c) 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;
d) 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;
e) 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;
f) a VH comprising the amino acid sequence of SEQ ID NO: 61 and a VL comprising the amino acid sequence of SEQ ID NO: 69
g) 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;
h) 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;
i) 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;
j) a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 70; or
k) a VH comprising the amino acid sequence of SEQ ID NO: 65 and a VL comprising the amino acid sequence of SEQ ID NO: 70.
65 . The humanized anti-HLA-A2 antibody of claim 63 , wherein said antibody is an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 72-91.
66 . The humanized anti-HLA-A2 antibody of claim 63 , wherein said antibody is capable of constituting an antigen binding domain of a chimeric antigen receptor (CAR), wherein said CAR is capable of being expressed in a T regulatory cell (Treg) such that said CAR specifically binds to HLA-A2.
67 . A chimeric antigen receptor (CAR) comprising:
(i) an extracellular domain comprising the humanized anti-HLA-A2 antibody of claim 63 ;
(ii) a transmembrane domain; and
(iii) 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.
68 . The CAR of claim 67 , comprising:
(i) an extracellular domain comprising a humanized anti-HLA-A2 antibody or antigen-binding fragment, wherein said anti-HLA-A2 antibody or antigen-binding fragment is an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 72-91
(ii) a hinge region comprising a stalk region of CD8a,
(iii) a transmembrane domain of CD28; and
(iv) an intracellular signaling domain comprising a functional signaling domain of CD3 zeta and a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of CD28 and 4-1BB (CD137).
69 . A nucleic acid encoding the CAR of claim 67 .
70 . An expression vector comprising a nucleic acid encoding the CAR of claim 67 , wherein said expression vector is selected from an expression plasmid, a cloning vector, a minicircle, a minivector, a double minute chromosome, a retroviral and lentiviral vector constructs.
71 . A modified immune cell, comprising a nucleic acid encoding the CAR of claim 67 .
72 . The modified immune cell of claim 71 , further comprising the CAR.
73 . The modified immune cell of claim 71 , wherein the nucleic acid encoding the CAR is part of an expression vector selected from an expression plasmid, a cloning vector, a minicircle, a minivector, a double minute chromosome, a retroviral and lentiviral vector constructs.
74 . The modified immune cell of claim 71 , further comprising a suicide gene system.
75 . The modified immune cell of claim 71 , wherein said modified immune cell is a T regulatory cell (Treg).
76 . The modified immune cell of claim 73 , further comprising a suicide gene system.
77 . The modified immune cell of claim 73 , wherein said modified immune cell is a T regulatory cell (Treg).
78 . A pharmaceutical composition comprising a plurality of the modified immune cell of claim 71 and a pharmaceutically acceptable carrier, diluent or excipient.
79 . A pharmaceutical composition comprising a plurality of the modified immune cell of claim 73 and a pharmaceutically acceptable carrier, diluent or excipient.
80 . 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 the subject;
c) promoting immune tolerance in the subject in need thereof;
d) inducing tolerance to a transplanted organ or tissue in the subject; or
e) any combination of a)-d);
the method comprising administering to said subject the pharmaceutical composition of claim 78 .
81 . The method of claim 80 , wherein said pharmaceutical composition is administered to the subject at a dosage of 1×10 4 to 1×10 9 cells/kg body weight or at a dosage of at least 10 4 cells.
82 . The method of claim 80 , wherein said pharmaceutical composition is administered to the subject with an initial administration and with one or more subsequent administration.
83 . The method of claim 80 , wherein said pharmaceutical composition is administered to the subject at the same time as, before, or after transplantation of a transplant into the subject.
84 . The method of claim 80 , wherein said pharmaceutical composition is administered to the subject in combination with another active agent, wherein said another active agent is an immunosuppressive agent.
85 . The method of claim 84 , wherein said pharmaceutical composition is administered to the subject before, at the same time or after the administration of an immunosuppressive agent.
86 . The method of claim 84 , wherein the immunosuppressive agent is selected from the group consisting of calcineurin inhibitors such as cyclosporine, tacrolimus, azathioprine, methotrexate, methoxsalen, rapamycin, mycophenolate mofetil, mycophenolic acid, mycophenolate sodium, 6-mercaptopurine, 6-thioguanine, rituximab, mTOR inhibitors such as sirolimus, everolimus, basiliximab, daclizumab, belatacept, alemtuzumab, muromonab-CD3, anti-thymocyte globulin, glucorticosteroids, or adrenocortical steroids such as prednisone and prednisolone, and any combination thereof.
87 . The method of claim 84 , wherein said pharmaceutical composition is administered in a subject for reducing the amount of an immunosuppressive agent received by the subject.
88 . A kit comprising:
(a) the modified immune cell according to claim 71 , and
(b) a pharmaceutically acceptable buffer.
89 . The kit of claim 88 , further comprising at least one immunosuppressive agent.