A2/NY-ESO-1 SPECIFIC T CELL RECEPTORS AND USES THEREOF
The application provides genetically modified T cell receptors (TCRs) specific for an epitope from cancer antigen NY-ESO-1. Also provided are related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, and populations of cells, including but not limited to genetically engineered cells, and pharmaceutical compositions. The application further provides the use of such modified T cell receptors (TCRs) and related compositions for cancer immunotherapy (e.g., adoptive cell therapy).
1 . A polynucleotide encoding a modified T cell receptor (TCR), or functional fragment thereof, wherein the modified TCR comprises a single amino acid substitution within a complementary determining region (CDR) 2 of a beta chain of the modified TCR relative to a CDR2 of a beta chain of an unsubstituted wild-type (WT) TCR.
2 . The polynucleotide of claim 1 , wherein outside of the CDR2 region of the beta chain of the modified TCR, or functional fragment thereof, the beta chain sequence of the modified TCR comprises an amino acid sequence at least 80% identical to the beta chain of the unsubstituted WT TCR, or functional fragment thereof.
3 . The polynucleotide of claim 1 or claim 2 , wherein the beta chain of the modified TCR, or functional fragment thereof, comprises the amino acid sequence of the beta chain of the unsubstituted WT TCR, or functional fragment thereof, with the single amino acid substitution in the CDR2 region.
4 . The polynucleotide of any one of claims 1-3 , wherein the beta chain of the unsubstituted WT TCR comprises the amino acid sequence of SEQ ID NO: 1.
5 . The polynucleotide of any one of claims 1-4 , wherein the single amino acid substitution occurs at residues 50, 51, 53, or 55 relative to the WT TCR.
6 . The polynucleotide of any one of claims 1-5 , wherein the single amino acid substitution occurs at residues 53 or 55 relative to the WT TCR.
7 . The polynucleotide of any one of claims 1-6 , wherein the single amino acid substitution is I53E, I53F, I53W, or D55E.
8 . The polynucleotide of any one of claims 1-7 , wherein the modified TCR binds to a cancer antigen at a higher binding affinity than the WT TCR.
9 . The polynucleotide of claim 8 , wherein the cancer antigen is NY-ESO-1 157-165 epitope (SEQ ID NO: 8).
10 . The polynucleotide of claim 8 or claim 9 , wherein the binding affinity of the modified TCR to the cancer antigen is about 5 to about 75 times higher as compared to the binding affinity of the WT TCR to the cancer antigen.
11 . The polynucleotide of claim 10 , wherein the binding affinity of the modified TCR to the cancer antigen is about 10 to about 75 times higher as compared to the binding affinity of the WT TCR to the cancer antigen.
12 . The polynucleotide of claim 11 , wherein the binding affinity of the modified TCR to the cancer antigen is about 25 to about 75 times higher as compared to the binding affinity of the WT TCR to the cancer antigen.
13 . The polynucleotide of claim 12 , wherein the binding affinity of the modified TCR to the cancer antigen is about 40 to about 75 times higher as compared to the binding affinity of the WT TCR to the cancer antigen.
14 . The polynucleotide of claim 13 , wherein the binding affinity of the modified TCR to the cancer antigen is about 40 to about 60 times higher as compared to the binding affinity of the WT TCR to the cancer antigen.
15 . The polynucleotide of claim 14 , wherein the binding affinity of the modified TCR to the cancer antigen is about 40 to about 50 times higher as compared to the binding affinity of the WT TCR to the cancer antigen.
16 . The polynucleotide of claim 8 or claim 9 , wherein the binding affinity of the modified TCR to the cancer antigen is approximately about 50 times higher as compared to the binding affinity of the WT TCR to the cancer antigen.
17 . The polynucleotide of claim 8 or claim 9 , wherein the dissociation constant (K D ) of the modified TCR to the cancer antigen is between about 0.30 and about 4.5 μM.
18 . The polynucleotide of claim 17 , wherein the dissociation constant (K D ) of the modified TCR to the cancer antigen is between about 0.30 and about 2 μM.
19 . The polynucleotide of claim 17 , wherein the dissociation constant (K D ) of the modified TCR to the cancer antigen is between about 2 μM and about 3 μM.
20 . The polynucleotide of claim 17 , wherein the dissociation constant (K D ) of the modified TCR to the cancer antigen is between about 3 μM and about 4 μM.
21 . The polynucleotide of claim 17 , wherein the dissociation constant (K D ) of the modified TCR to the cancer antigen is about 0.41 μM.
22 . The polynucleotide of claim 17 , wherein the dissociation constant (K D ) of the modified TCR to the cancer antigen is about 3.89 μM.
23 . The polynucleotide of any one of claims 1-22 , wherein the modified TCR comprises the amino acid sequence of any one of SEQ ID NOs: 2-5, or functional fragment thereof, or an amino acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 2-5.
24 . The polynucleotide of any one of claims 1-23 , wherein the modified TCR is encoded by the nucleotide sequence of any one of SEQ ID NOs: 11-14, or a nucleotide sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 11-14.
25 . The polynucleotide of any one of claims 1-24 , wherein the polynucleotide is operably linked to at least one regulator element for expression of the modified TCR.
26 . The polynucleotide of claim 25 , wherein the at least one regulatory element is a promoter.
27 . The polynucleotide of any one of claims 1-26 , which is a DNA molecule.
28 . The polynucleotide of any one of claims 1-26 , which is an RNA molecule or a derivative thereof.
29 . A recombinant vector comprising the polynucleotide of any one of claims 1-28 , wherein the polynucleotide is operably linked to at least one regulatory element for expression of the modified T cell receptor (TCR).
30 . The vector of claim 29 , which is a viral vector.
31 . The vector of claim 30 , wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, or a vaccinia virus vector.
32 . The vector of claim 29 , wherein the vector is a non-viral vector.
33 . A modified T cell receptor (TCR), comprising a beta chain of the modified TCR, or functional fragment thereof, encoded by the polynucleotide of any one of claims 1-28 .
34 . A modified T cell receptor (TCR), comprising a) a beta chain of the modified TCR, or functional fragment thereof, encoded by the polynucleotide of any one of claims 1-29 and b) an alpha chain, or a functional fragment thereof.
35 . A modified T cell receptor (TCR), comprising CDRs of the beta chain of the modified TCR encoded by the polynucleotide of any one of claims 1-28 .
36 . A modified T cell receptor (TCR), comprising a) a functional fragment of a beta chain of the modified TCR, wherein the functional fragment comprises the CDRs of a beta chain encoded by the polynucleotide of any one of claims 1-28 and b) a functional fragment of an alpha chain, wherein the functional fragment comprises the CDRs of an alpha chain.
37 . The modified TCR of claim 36 , wherein the functional fragment of a) further comprises a constant region of a TCR beta chain and/or the functional fragment of b) further comprises a constant region of a TCR alpha chain.
38 . The modified TCR of claim 37 , wherein either of the constant regions are of human origin.
39 . The modified TCR of claim 37 , wherein either of the constant regions are of mouse origin.
40 . The modified TCR of any one of claims 34-39 , wherein the alpha chain comprises the alpha chain of a WT TCR, or functional fragment thereof.
41 . The modified TCR of claim 40 , wherein the alpha chain of the WT TCR comprises the amino acid sequence of SEQ ID NO: 7.
42 . The modified TCR of any one of claims 33-41 , wherein the modified TCR comprises the amino acid sequence of any one of SEQ ID NOs: 2-5, or fragment thereof, or an amino acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 2-5.
43 . A modified T cell receptor (TCR) comprising an alpha chain comprising the amino acid sequence of SEQ ID NO: 7 and a beta chain comprising the amino acid sequence of any one of SEQ ID NOs: 2-5.
44 . An isolated host cell comprising the modified T cell receptor (TCR) of any one of claims 33-43 .
45 . An isolated host cell comprising the polynucleotide of any one of claims 1-28 .
46 . The isolated host cell of claim 45 , wherein the polynucleotide is operably linked to at least one regulatory element which is capable of mediating expression of the modified T cell receptor (TCR) in the host cell.
47 . An isolated host cell comprising the vector of any one of claims 29-32 .
48 . The isolated host cell of any one of claims 44-47 , wherein the host cell is a mammalian cell.
49 . The isolated host cell of any one of claims 44-48 , wherein the host cell is a lymphoid cell.
50 . The isolated host cell of claim 49 , wherein the lymphoid cell is a T cell.
51 . The isolated host cell of claim 49 , wherein the lymphoid cell is a natural killer (NK) cell.
52 . The isolated host cell of any one of claims 48-51 , wherein the host cells are obtained from peripheral blood mononuclear cells (PBMC), tumor draining lymph nodes or tumor infiltrates.
53 . The isolated host cell of any one of claims 44-52 , wherein the host cell has been activated and/or expanded ex vivo.
54 . The isolated host cell of any one of claims 44-53 , wherein the host cell is an allogeneic cell.
55 . The isolated host cell of any one of claims 44-53 , wherein the host cell is an autologous cell.
56 . The isolated autologous host cell of claim 55 , wherein the host cell is isolated from a subject having a disease.
57 . The isolated autologous host cell of claim 56 , wherein the disease is cancer.
58 . The isolated autologous host cell of claim 57 , wherein the cancer presents cancer antigen NY-ESO-1 157-165 epitope (SEQ ID NO: 8) on the surface of its cells.
59 . The isolated autologous host cell of claim 57 or claim 58 , wherein the cancer is myeloma, melanoma, sarcoma, bladder cancer, esophageal cancer, hepatocellular cancer, head and neck cancer, breast cancer, prostate cancer, urinary bladder cancer, skin cancer, lung cancer, ovarian cancer, or brain cancer.
60 . The isolate host cell of any one of claims 44-59 , further engineered to express one or more exogenous molecule.
61 . The isolated host cell of claim 60 , wherein the one or more exogenous molecule is an immune signaling molecule.
62 . The isolated host cell of claim 61 , wherein the immune signaling molecule is a cytokine.
63 . The isolated host cell of claim 61 , wherein the immune signaling molecule is a chemokine.
64 . The isolated host cell of claim 61 , wherein the immune signaling molecule is a growth factor.
65 . The isolated host cell of claim 64 , wherein the growth factor is granulocyte-macrophage colony-stimulating factor (GM-CSF).
66 . An isolated host cell comprising a T cell receptor (TCR), or functional fragment thereof, that binds to a cancer antigen, wherein the host cell is further engineered to express granulocyte-macrophage colony-stimulating factor (GM-CSF).
67 . The isolated host cell of claim 66 , wherein the cancer antigen is NY-ESO-1 157-165 epitope (SEQ ID NO: 8).
68 . The isolated host cell of claim 66 or 67 , wherein the T cell receptor (TCR) is a WT TCR.
69 . The isolated host cell of any one of claims 66-68 , wherein the beta chain of the WT TCR comprises an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 1.
70 . The isolated host cell of any one of claims 66-69 , wherein the alpha chain of the WT TCR comprises the amino acid sequence of SEQ ID NO: 7.
71 . The isolated host cell of any one of claims 65-70 , wherein the amino acid sequence for GM-CSF comprises SEQ ID NO: 21, 34 or 15, or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 21, 34 or 15.
72 . The isolated host cell of any one of claims 65-71 , wherein the nucleotide sequence encoding GM-CSF comprises SEQ ID NO: 22, 35 or 16, or nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 22, 35 or 16.
73 . The isolated host cell of claim 60 or 61 , wherein the one or more exogenous molecule is a soluble receptor.
74 . The isolated host cell of claim 60 or 61 , wherein the one or more exogenous molecule is a ligand.
75 . The isolated host cell of claim 60 or 61 , wherein the one or more exogenous molecule is an antigen binding protein.
76 . The isolated host cell of claim 75 , wherein the antigen binding protein is an antibody, or an antibody fragment.
77 . The isolated host cell of claim 60 , wherein the one or more exogenous molecule is a phosphodiesterase.
78 . The isolated host cell of claim 77 , wherein the phosphodiesterase is PDE4B2.
79 . An isolated host cell comprising a T cell receptor (TCR), or functional fragment thereof, that binds to a cancer antigen, wherein the host cell is further engineered to express PDE4B2.
80 . The isolated host cell of claim 79 , wherein the cancer antigen is NY-ESO-1 157-165 epitope (SEQ ID NO: 8).
81 . The isolated host cell of claim 79 or 80 , wherein the T cell receptor (TCR) is a WT TCR.
82 . The isolated host cell of any one of claims 79-81 , wherein the beta chain of the WT TCR comprises an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 1.
83 . The isolated host cell of any one of claims 79-82 , wherein the alpha chain of the WT TCR comprises the amino acid sequence of SEQ ID NO: 7.
84 . The isolated host cell of any one of claims 78-83 , wherein the amino acid sequence of PDE4B2 comprises SEQ ID NO: 27 or 29, or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 27 or 29.
85 . The isolated host cell of any one of claims 78-84 , wherein the nucleotide sequence encoding PDE4B2 comprises SEQ ID NO: 28 or 30, or a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 28 or 30.
86 . The isolated host cell of claim 60 , wherein the one or more exogenous molecule is a cell surface receptor.
87 . The isolated host cell of claim 86 , wherein the cell surface receptor is a chimeric antigen receptor.
88 . The isolated host cell of claim 86 , wherein the cell surface receptor is a T cell receptor that does not bind the cancer antigen NY-ESO-1 157-165 epitope (SEQ ID NO: 8).
89 . A bifunctional molecule comprising the modified T cell receptor (TCR) of claims 33-43 , or a functional fragment thereof, and an immune effector polypeptide that specifically binds to a cell surface protein on a T cell.
90 . The bifunctional molecule of claim 89 , wherein the immune effector polypeptide comprises an antibody, or an antibody fragment.
91 . The bifunctional molecule of claim 89 or 90 , wherein the immune effector polypeptide comprises a single-chain variable fragment (scFv).
92 . The bifunctional molecule of any one of claims 89-91 , wherein the immune effector polypeptide specifically binds to CD3.
93 . The bifunctional molecule of claim 92 , wherein the immune effector polypeptide comprises the antibody or antibody fragment derived from OKT3, UCHT-1, BMA031, or 12F6.
94 . A pharmaceutical composition comprising the host cell of any one of claims 44-88 , or the bifunctional molecule of any one of claims 89-93 , and a pharmaceutically acceptable carrier and/or excipient.
95 . The pharmaceutical composition of claim 94 , wherein the composition is used in adoptive cell transfer therapy.
96 . A method of producing the host cells of any one of claims 44-88 , comprising genetically engineering the host cells with the polynucleotides of any one of claims 1-28 or the vector of any one of claims 29-32 .
97 . A method of genetically engineering host cells to express the modified T cell receptor (TCR), or functional fragment thereof, of any one of claims 33-43 .
98 . The method of claim 97 , comprising genetically engineering the host cells with the polynucleotides of any one of claims 1-28 or the vector of any one of claims 29-32 .
99 . The method of any one of claims 96-98 , wherein the genetic engineering step is conducted via viral gene delivery.
100 . The method of claim 99 , wherein the genetic engineering step is conducted via non-viral gene delivery.
101 . The method of any one of claims 96-100 , wherein the method is conducted ex vivo.
102 . The method of any one of claims 96-101 , wherein the method further comprises activation and/or expansion of the host cells ex vivo.
103 . The method of any one of claims 96-102 , wherein the modified TCR comprises the amino acid sequence of any one of SEQ ID NOs: 2-5, or fragment thereof, or an amino acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 2-5.
104 . The method of any one of claims 96-103 , further comprising genetically engineering the host cells to further express one or more exogenous molecule.
105 . The method of claim 104 , wherein the one or more exogenous molecule is an immune signaling molecule.
106 . The method of claim 104 or 105 , wherein the exogenous molecule is a cytokine, chemokine, growth factor, soluble receptor, ligand, phosphodiesterase, antigen binding protein, or a cell surface receptor.
107 . The method of claim 106 , wherein the growth factor is granulocyte-macrophage colony-stimulating factor (GM-CSF).
108 . The method of claim 107 , wherein the amino acid sequence for GM-CSF comprises SEQ ID NO: 21, 34 or 15, or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 21, 34 or 15.
109 . The method of claim 107 or 108 , wherein the nucleotide sequence encoding GM-CSF comprises SEQ ID NO: 22, 35 or 16, or nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 22, 35 or 16.
110 . The method of claim 106 , wherein the phosphodiesterase is PDE4B2.
111 . The method of claim 110 , wherein the amino acid sequence of PDE4B2 comprises SEQ ID NO: 27 or 29, or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 27 or 29.
112 . The method of claim 110 or 111 , wherein the nucleotide sequence encoding PDE4B2 comprises SEQ ID NO: 28 or 30, or a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 28 or 30.
113 . The method of claim 106 , wherein the antigen binding protein is an antibody, or an antibody fragment.
114 . The method of claim 106 , wherein the cell surface receptor is a chimeric antigen receptor or a T cell receptor that does not bind the cancer antigen NY-ESO-1 157-165 epitope (SEQ ID NO: 8).
115 . The method of any one of claims 96-114 , wherein the host cell is a mammalian cell.
116 . The method of any one of claims 96-115 , wherein the host cell is a lymphoid cell.
117 . The method of claim 116 , wherein the lymphoid cell is a T cell.
118 . The method of claim 116 , wherein the lymphoid cell is a natural killer (NK) cell.
119 . The method of any one of claims 115-118 , wherein the host cells are obtained from peripheral blood mononuclear cells (PBMC), tumor draining lymph nodes or tumor infiltrates.
120 . The method of any one of claims 96-119 , wherein the host cell has been activated and/or expanded ex vivo.
121 . The method of any one of claims 96-120 , wherein the host cell is an allogeneic cell.
122 . The method of any one of claims 96-120 , wherein the host cell is an autologous cell.
123 . The method of any one of claims 96-122 , wherein the host cell is isolated from a subject having a disease.
124 . The method of claim 123 , wherein the disease is cancer.
125 . The method of claim 124 , wherein the cells of the cancer present cancer antigen NY-ESO-1 157-165 epitope (SEQ ID NO: 8) on their surface.
126 . The method of claim 124 or claim 125 , wherein the cancer is myeloma, melanoma, sarcoma, bladder cancer, esophageal cancer, hepatocellular cancer, head and neck cancer, breast cancer, prostate cancer, urinary bladder cancer, skin cancer, lung cancer, ovarian cancer, or brain cancer.
127 . A method for stimulating or enhancing an immune response in a mammal in need thereof comprising administering to said mammal an effective amount of the lymphoid cells comprising the modified T cell receptors (TCRs) of any one of claims 33-43 , the host cells of any one of claims 44-88 , the bifunctional molecule of any one of claims 89-93 , the composition of claim 94 or claim 95 , or the host cells produced by the method of any one of claims 96-126 .
128 . A method of treatment of a cancer in a subject in need thereof, comprising administering to a subject an effective amount of the lymphoid cells comprising the modified T cell receptors (TCRs) of any one of claims 33-43 , the host cells of any one of claims 44-88 , the bifunctional molecule of any one of claims 89-93 , the composition of claim 94 or claim 95 , or the host cells produced by the method of any one of claims 96-126 .
129 . The method of claim 128 , wherein cells of the cancer present cancer antigen NY-ESO-1 157-165 epitope (SEQ ID NO: 8) on their surface.
130 . The method of claim 128 or claim 129 , wherein the cancer is myeloma, melanoma, sarcoma, bladder cancer, esophageal cancer, hepatocellular cancer, head and neck cancer, breast cancer, prostate cancer, urinary bladder cancer, skin cancer, lung cancer, ovarian cancer, or brain cancer.
131 . The method of any one of claims 128-130 , the method comprising:
a) isolating T cells the subject or mammal;
b) genetically modifying said T cells ex vivo with the polynucleotide of any one of claims 1-28 or the vector of any one of claims 29-32 ;
c) optionally, expanding and/or activating said T cells before, after or during step b); and
d) introducing the genetically modified T cells into the subject or mammal.
132 . The method of any one of claims 128-131 , wherein the subject or mammal is human.