US2022331364A1PendingUtilityA1
T cell-antigen coupler with various construct optimizations
Assignee: TRIUMVIRA IMMUNOLOGICS USA INCPriority: Jul 17, 2018Filed: Jun 30, 2022Published: Oct 20, 2022
Est. expiryJul 17, 2038(~12 yrs left)· nominal 20-yr term from priority
C07K 14/82C07K 14/7051A61P 35/00A61K 35/17A61K 40/4215A61K 40/4211A61K 40/4205A61K 40/32A61K 40/31A61K 40/11A61K 2239/59A61K 2239/48A61K 2239/38A61K 2239/31C07K 14/70514C07K 14/70517C07K 16/2803C07K 16/2809C07K 16/32C07K 2317/622C07K 2319/03
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Claims
Abstract
A trifunctional molecule is provided, comprising (i) a target-specific ligand, (ii) a ligand that binds a protein associated with a TCR complex, and (iii) a T cell receptor signaling domain polypeptide. Variants of the molecule are provided, including variants that exhibit optimized surface expression, transduction efficiency, and effector functionality. Variations include, for example, different ligands that bind CD3 epsilon (e.g., OKT3, L2K, F6A, UCHT1 and humanized UCHT1), different signaling domains, and different linkers between domains.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A nucleic acid sequence encoding a CD19 Trifunctional T cell-antigen coupler (CD19-TAC) comprising:
(a) a first polynucleotide encoding a ligand that selectively binds a CD19 antigen; (b) a second polynucleotide encoding a UCHT1 ligand that binds CD3; and (c) a third polynucleotide encoding a TCR signaling domain polypeptide comprising a cytosolic domain and a transmembrane domain; wherein components encoded by (a), components encoded by (b), and components encoded by (c) are fused directly to each other, or joined by at least one linker.
2 . The nucleic acid sequence of claim 1 , wherein the ligand that selectively binds the CD19 antigen is a single chain variable fragment (scFv).
3 . The nucleic acid sequence of any one of claims 1 - 2 , wherein the ligand that selectively binds the CD19 antigen comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 36.
4 . The nucleic acid sequence of any one of claims 1 - 3 , wherein the UCHT1 ligand is a single chain antibody.
5 . The nucleic acid sequence of any one of claims 1 - 4 , wherein the UCHT1 ligand comprises a Y182T mutation.
6 . The nucleic acid sequence of any one of claims 1 - 4 , wherein the UCHT1 ligand is a humanized variant of UCHT1 (huUCHT1).
7 . The nucleic acid sequence of any one of claims 1 - 4 , wherein the UCHT1 ligand is a humanized variant of UCHT1 comprising a Y177T mutation (huUCHT1 (Y177T)).
8 . The nucleic acid sequence of any one of claims 1 - 7 , wherein the UCHT1 ligand comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14, SEQ ID NO: 72, SEQ ID NO: 44, or SEQ ID NO: 46.
9 . The nucleic acid sequence of any one of claims 1 - 8 , wherein the cytosolic domain is a CD4 cytosolic domain and the transmembrane domain is a CD4 transmembrane domain.
10 . The nucleic acid sequence of any one of claims 1 - 9 , wherein the third polynucleotide encodes a polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 18.
11 . The nucleic acid sequence of any one of claims 1 - 10 , wherein the component encoded by (a) and the component encoded by (c) are fused to the component encoded by (b).
12 . The nucleic acid sequence of any one of claims 1 - 10 , wherein the component encoded by (b) and the component encoded by (c) are fused to the component encoded by (a).
13 . The nucleic acid sequence of any one of claims 1 - 10 , wherein at least one linker joins the component encoded by (a) to the component encoded by (b).
14 . The nucleic acid sequence of any one of claim 1 - 10 , or 13 , wherein the at least one linker is a GIS flexible linker (SEQ ID NO: 73), a large protein domain, a long helix structure, or a short helix structure.
15 . The nucleic acid sequence of any one of claim 1 - 10 , or 13 - 14 , wherein the at least one linker comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 30, or SEQ ID NO: 28.
16 . The nucleic acid sequence of any one of claims 1 - 15 , wherein the CD3 is of a TCR complex on a cell expressing the second polynucleotide.
17 . The nucleic acid sequence of any one of claims 1 - 16 , wherein the binding of the CD3 induces activation of a cell expressing the second polynucleotide.
18 . The nucleic acid sequence of any one of claims 1 - 17 , wherein the CD19-TAC comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 63.
19 . The nucleic acid sequence of any one of claims 1 - 17 , wherein the CD19-TAC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 64.
20 . The nucleic acid sequence of any one of claims 1 - 19 , wherein the nucleic acid sequence does not encode a co-stimulatory domain.
21 . The nucleic acid sequence of any one of claims 1 - 20 , wherein the nucleic acid sequence does not encode an activation domain.
22 . A vector construct comprising:
(a) a nucleic acid sequence of any one of claims 1 - 21 ; and (b) a promoter functional in a mammalian cell.
23 . A T cell comprising the nucleic acid sequence of any one of claims 1 - 21 .
24 . A pharmaceutical composition comprising the T cell of claim 23 , and a pharmaceutically acceptable excipient.
25 . A method of treating a cancer expressing CD19 in an individual in need thereof comprising administering to the individual the pharmaceutical composition of claim 24 .
26 . The method of claim 25 , wherein the cancer is a B cell malignancy.
27 . The method of claim 25 , wherein the cancer is B cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or Non-Hodgkins Lymphoma.
28 . The method of any one of claims 25 - 27 , wherein the pharmaceutical composition is administered transarterially, subcutaneously, intradermally, intratumorally, intranodally, intrameduliary, intramuscularly, intravenously or intraperitoneally.
29 . A nucleic acid sequence encoding a Trifunctional T cell-antigen coupler (Tri-TAC) comprising:
(a) a first polynucleotide encoding a target-specific ligand; (b) a second polynucleotide encoding a ligand that binds a protein associated with a TCR complex; and (c) a third polynucleotide encoding a T cell receptor signaling domain polypeptide; wherein the ligand that binds the protein associated with the TCR complex is selected from OKT3, F6A or L2K.
30 . The nucleic acid sequence of claim 29 , wherein component encoded by (a), component encoded by (b), and component encoded by (c) are fused directly to each other, or joined by at least one linker.
31 . The nucleic acid sequence of any one of claims 29 - 30 , wherein the component encoded by (a) and the component encoded by (b) are directly fused and joined to the component encoded by (c) by a linker.
32 . The nucleic acid sequence of any one of claims 29 - 30 , wherein the component encoded by (b) and the component encoded by (c) are directly fused and joined to the component encoded by (a) by a linker.
33 . The nucleic acid sequence of any one of claims 30 - 32 , wherein the at least one linker is a G 4 S flexible linker (SEQ ID NO: 73), a large protein domain, a long helix structure, or a short helix structure.
34 . The nucleic acid sequence of any one of claims 30 - 33 , wherein the at least one linker has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 30, or SEQ ID NO: 28.
35 . The nucleic acid sequence of any one of claims 29 - 34 , wherein the ligand that binds the protein associated with the TCR complex is OKT3.
36 . The nucleic acid sequence of any one of claims 29 - 35 , wherein the ligand that binds a protein associated with the TCR complex comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 22.
37 . The nucleic acid sequence of any one of claims 29 - 34 , wherein the ligand that binds the protein associated with the TCR complex is F6A.
38 . The nucleic acid sequence of any one of claim 29 - 34 , or 37 , wherein the ligand that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 24.
39 . The nucleic acid sequence of any one of claims 29 - 34 , wherein the ligand that binds the protein associated with the TCR complex is L2K.
40 . The nucleic acid sequence of any one of claim 29 - 34 , or 39 , wherein the ligand that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 26.
41 . The nucleic acid sequence of any one of claims 29 - 40 , wherein the protein associated with the TCR complex is CD3.
42 . The nucleic acid sequence of any one of claims 29 - 41 , wherein the target-specific ligand selectively binds a tumor antigen.
43 . The nucleic acid sequence of any one of claims 29 - 42 , wherein the target-specific ligand is a designed ankyrin repeat (DARPin) polypeptide, or a single chain variable fragment (scFv).
44 . The nucleic acid sequence of any one of claims 29 - 43 , wherein the target-specific ligand selectively binds a CD19 antigen, a HER2 antigen, or a BCMA antigen.
45 . The nucleic acid sequence of any one of claims 29 - 44 , wherein the target-specific ligand selectively binds a HER-2 antigen comprises an antigen binding domain of an antibody selected from Trastuzumab, Pertuzumab, Lapatinib, Neratinib, Ado-trastuzmab Emtansine, Gancotamab, Margetuximab, Timigutuzumab, and Ertumaxomab.
46 . The nucleic acid sequence of any one of claims 29 - 44 , wherein the target-specific ligand selectively binds a BCMA antigen comprises an antigen binding domain of an antibody selected from Belantamab mafodotin, and GSK2857916.
47 . The nucleic acid sequence of any one of claims 29 - 44 , wherein the target-specific ligand comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 36, SEQ ID NO: 8 or SEQ ID NO: 34.
48 . The nucleic acid sequence of any one of claims 29 - 47 , wherein the T cell receptor signaling domain polypeptide comprises a cytosolic domain and a transmembrane domain.
49 . The nucleic acid sequence of claim 48 , wherein the cytosolic domain is a CD4 cytosolic domain and the transmembrane domain is a CD4 transmembrane domain, or wherein the cytosolic domain is a CD8 cytosolic domain and the transmembrane domain is a CD8 transmembrane domain.
50 . The nucleic acid sequence of any one of claims 29 - 49 , further comprising a leader sequence.
51 . The nucleic acid sequence of claim 50 , wherein the leader sequence comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 6, SEQ ID NO: 48, or SEQ ID NO: 50.
52 . The nucleic acid sequence of any one of claims 29 - 51 , wherein the CD3 is of a TCR complex on a cell expressing the second polynucleotide.
53 . The nucleic acid sequence of any one of claims 29 - 52 , wherein the binding of the CD3 induces activation of a cell expressing the second polynucleotide.
54 . The nucleic acid sequence of any one of claims 29 - 53 , wherein the Tri-TAC comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 75, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61.
55 . The nucleic acid sequence of any one of claims 29 - 53 , wherein the Tri-TAC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, or SEQ ID NO: 62.
56 . The nucleic acid sequence of any one of claims 29 - 55 , wherein the nucleic acid sequence does not encode a co-stimulatory domain.
57 . The nucleic acid sequence of any one of claims 29 - 56 , wherein the nucleic acid sequence does not encode an activation domain.
58 . A nucleic acid sequence encoding a Trifunctional T cell-antigen coupler (Tri-TAC) comprising:
(a) a first polynucleotide encoding a target-specific ligand; (b) a second polynucleotide encoding a ligand that binds a protein associated with a TCR complex; and (c) a third polynucleotide encoding a T cell receptor signaling domain polypeptide; wherein the nucleic acid sequence further comprises a leader sequence, and wherein component encoded by (a), component encoded by (b), and component encoded by (c) are fused directly to each other, or joined by at least one linker.
59 . The nucleic acid sequence of claim 58 , wherein the target-specific ligand selectively binds a tumor antigen.
60 . The nucleic acid sequence of any one of claims 58 - 59 , wherein the target-specific ligand is a designed ankyrin repeat (DARPin) polypeptide, or a single chain variable fragment (scFv).
61 . The nucleic acid sequence of any one of claims 58 - 60 wherein the target-specific ligand selectively binds a CD19 antigen, a HER2 antigen, or a BCMA antigen.
62 . The nucleic acid sequence of any one of claims 58 - 61 , wherein the target-specific ligand selectively binds a HER-2 antigen and comprises an antigen binding domain of an antibody selected from Trastuzumab, Pertuzumab, Lapatinib, Neratinib, Ado-trastuzmab Emtansine, Gancotamab, Margetuximab, Timigutuzumab, and Ertumaxomab.
63 . The nucleic acid sequence of any one of claims 58 - 61 , wherein the target-specific ligand selectively binds a BCMA antigen and comprises an antigen binding domain of an antibody selected from Belantamab mafodotin, and GSK2857916.
64 . The nucleic acid sequence of any one of claims 58 - 61 , wherein the target-specific ligand comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 36, SEQ ID NO: 8, SEQ ID NO: 34, SEQ ID NO: 52, or SEQ ID NO: 54.
65 . The nucleic acid sequence of any one of claims 58 - 64 , wherein the ligand that binds the protein associated with the TCR complex is selected from UCHT1, UCHT1 (Y182T), huUCHT1, huUCHT1 (Y177T), OKT3, F6A, or L2K.
66 . The nucleic acid sequence of any one of claims 58 - 65 , wherein the ligand that binds a protein associated with the TCR complex has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14, SEQ ID NO: 72, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26.
67 . The nucleic acid sequence of any one of claims 58 - 66 , wherein the protein associated with the TCR complex is CD3.
68 . The nucleic acid sequence of any one of claims 58 - 67 , wherein the T cell receptor signaling domain polypeptide comprises a cytosolic domain and a transmembrane domain.
69 . The nucleic acid sequence of claim 68 , wherein the cytosolic domain is a CD4 cytosolic domain and the transmembrane domain is a CD4 transmembrane domain, or wherein the cytosolic domain is a CD8 cytosolic domain and the transmembrane domain is a CD8 transmembrane domain.
70 . The nucleic acid sequence of any one of claims 58 - 69 , wherein the leader sequence comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 6, SEQ ID NO: 48, or SEQ ID NO: 50.
71 . The nucleic acid sequence of any one of claims 58 - 70 , wherein the component encoded by (a) and the component encoded by (b) are directly fused and joined to the component encoded by (c) by a linker.
72 . The nucleic acid sequence of any one of claims 58 - 70 , wherein the component encoded by (b) and the component encoded by (c) are directly fused and joined to the component encoded by (a) by a linker.
73 . The nucleic acid sequence of any one of claims 58 - 72 , wherein the at least one linker is a G 4 S flexible linker (SEQ ID NO: 73), a large protein domain, a long helix structure, or a short helix structure.
74 . The nucleic acid sequence of any one of claims 58 - 73 , wherein the at least one linker has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 30, or SEQ ID NO: 28.
75 . The nucleic acid sequence of any one of claims 58 - 74 , wherein the protein associated with the TCR complex is CD3 and the CD3 is of a TCR complex on a cell expressing the second polynucleotide.
76 . The nucleic acid sequence of any one of claim 75 , wherein the binding of the CD3 induces activation of a cell expressing the second polynucleotide.
77 . The nucleic acid sequence of any one of claims 58 - 76 , wherein the Tri-TAC comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 75, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61.
78 . The nucleic acid sequence of any one of claims 58 - 76 , wherein the Tri-TAC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, or SEQ ID NO: 62.
79 . The nucleic acid sequence of any one of claims 58 - 78 , wherein the nucleic acid sequence does not encode a co-stimulatory domain.
80 . The nucleic acid sequence of any one of claims 58 - 79 , wherein the nucleic acid sequence does not encode an activation domain.
81 . A polypeptide encoded by the nucleic acid sequence of any one of claim 1 - 21 , or 29 - 80 .
82 . A vector construct comprising:
(a) a nucleic acid sequence of any one of claims 29 - 80 ; and (b) a promoter functional in a mammalian cell.
83 . A T cell comprising the nucleic acid sequence of any one of claims 29 - 80 .
84 . A pharmaceutical composition comprising the T cell of claim 83 , and a pharmaceutically acceptable excipient.
85 . A method of treating a cancer in an individual in need thereof, comprising administering to the individual a pharmaceutical composition of claim 84 .
86 . The method of claim 85 , wherein the individual is a mammal.
87 . The method of any one of claims 85 - 86 , wherein the cancer is a solid cancer or a liquid cancer.
88 . The method of any one of claims 85 - 87 , wherein the cancer is a lung cancer, a breast cancer, multiple myeloma, glioblastoma, gastric cancer, ovarian cancer, stomach cancer, colorectal cancer, urothelial cancer, endometrial cancer, or a colon cancer.
89 . The method of any one of claim 85 - 86 , wherein the cancer comprises a CD19 expressing cancer cell.
90 . The method of any one of claim 85 - 86 , or 89 , wherein the cancer is a B cell malignancy.
91 . The method of any one of claim 85 - 86 , or 89 , wherein the cancer is B cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or Non-Hodgkins Lymphoma.
92 . The method of any one of claim 85 - 86 , wherein the cancer comprises a HER-2 expressing cancer cell.
93 . The method of any one of claim 85 - 86 , or 92 , wherein the cancer is breast cancer, bladder cancer, pancreatic cancer, ovarian cancer, or stomach cancer.
94 . The method of any one of claim 85 - 86 , wherein the cancer comprises a BCMA expressing cancer cell.
95 . The method of any one of claim 85 - 86 , or 94 , wherein the cancer is leukemia, lymphoma, or multiple myeloma.
96 . The method of any one of claims 85 - 95 , wherein the pharmaceutical composition is administered to the individual transarterially, subcutaneously, intradermally, intratumorally, intranodally, intrameduliary, intramuscularly, intravenously or intraperitoneally.
97 . The method of any one of claims 85 - 96 , wherein the pharmaceutical composition is in a unit dose form.
98 . The method of any one of claims 85 - 97 , wherein the pharmaceutical composition comprises about 0.5-2×10 9 T cells.
99 . The method of any one of claims 85 - 98 , wherein the pharmaceutical composition is administered daily, weekly, bi-weekly, monthly, bi-month or yearly.
100 . A nucleic acid sequence encoding a HER2 Trifunctional T cell-antigen coupler (HER2-TAC) comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 65, SEQ ID NO: 67, or SEQ ID NO: 75.
101 . The nucleic acid sequence of claim 100 , wherein the HER2-TAC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 76.
102 . The nucleic acid sequence of any one of claims 100 - 101 , wherein the nucleic acid sequence does not encode a co-stimulatory domain.
103 . The nucleic acid sequence of any one of claims 100 - 102 , wherein the nucleic acid sequence does not encode an activation domain.
104 . A nucleic acid sequence encoding a BCMA Trifunctional T cell-antigen coupler (BCMA-TAC) comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61.
105 . The nucleic acid sequence of claim 104 , wherein the BCMA-TAC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, or SEQ ID NO: 62.
106 . The nucleic acid sequence of any one of claims 104 - 105 , wherein the nucleic acid sequence does not encode a co-stimulatory domain.
107 . The nucleic acid sequence of any one of claims 104 - 106 , wherein the nucleic acid sequence does not encode an activation domain.Join the waitlist — get patent alerts
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