US2022380429A1PendingUtilityA1
T cells expressing membrane-anchored il-12 for the treatment of cancer
Est. expiryOct 7, 2036(~10.2 yrs left)· nominal 20-yr term from priority
A61K 31/675A61K 31/7076C07K 2319/03A61K 31/704A61P 35/00A61K 45/06A61K 38/208A61K 38/00C12N 2740/15043C12N 2501/998C07K 14/5434A61K 35/76C12N 15/86C12N 7/00C12N 5/0636A61K 35/17A61K 40/4224A61K 40/11A61K 2239/55A61K 2239/38A61K 2239/50
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Claims
Abstract
Provided herein are polypeptides comprising membrane-anchored IL-12. Also provided herein are T cells expressing the membrane-anchored IL-12. Further, methods of treating cancer comprising administering T cells expressing membrane-anchored IL-12 are provided herein. Also provided are combination treatments comprising T cells expressing membrane-anchored IL-12 and T cell chemoattractant-inducing chemokines. In addition, methods are provided for activating T cells to express NKG2D and methods of their use in the treatment of cancer.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A membrane-anchored interleukin 12 (IL-12) heterodimer protein comprising:
a) a first polypeptide comprising an IL-12 alpha subunit p35 or a polypeptide at least 90% similar thereto; b) a second polypeptide comprising an IL-12 beta subunit p40 or a polypeptide at least 90% similar thereto; and c) a transmembrane domain fused to a terminus of the first polypeptide and/or the second polypeptide.
2 . The protein of claim 1 , wherein the first polypeptide is fused to the transmembrane domain.
3 . The protein of claim 2 , wherein the transmembrane domain is C-terminal to the first polypeptide.
4 . The protein of claim 1 , wherein the first polypeptide is N-terminal to the second polypeptide.
5 . The protein of claim 1 , wherein the first polypeptide is C-terminal to the second polypeptide.
6 . The protein of claim 1 , wherein the protein comprises from N-terminal to C-terminal the first polypeptide, the transmembrane domain, and the second polypeptide.
7 . The protein of claim 1 , wherein the protein comprises from N-terminal to C-terminal the second polypeptide, the transmembrane domain, and the first polypeptide.
8 . The protein of claim 1 or claim 2 , wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:3.
9 . The protein of claim 1 , wherein element a) is an IL-12 alpha subunit p35 or a polypeptide at least 90% identical thereto and element b) is an IL-12 beta subunit p40 or a polypeptide at least 90% identical thereto.
10 . The protein of claim 1 , further comprising a linker.
11 . The protein of claim 10 , wherein the linker comprises the amino acid sequence GGGGSGGGGSS (SEQ ID NO:5), SGGGGSGGGGSS (SEQ ID NO:6) or GGGGSGGGGS (SEQ ID NO:7).
12 . The protein of claim 10 , wherein the linker is between the IL-12 alpha subunit p35 and the transmembrane domain.
13 . The protein of claim 1 , wherein the first polypeptide comprises an amino acid sequence at least 90% similar to SEQ ID NO:1.
14 . The protein of claim 1 , wherein the first polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO:1.
15 . The protein of claim 1 , wherein the first polypeptide is at least 91%, 92%, 93%, 94%, 95% or 96% similar to SEQ ID NO:1.
16 . The protein of claim 1 , wherein the first polypeptide is at least 91%, 92%, 93%, 94%, 95% or 96% identical to SEQ ID NO:1.
17 . The protein of claim 1 , wherein the second polypeptide comprises an amino acid sequence at least 90% similar to SEQ ID NO:4.
18 . The protein of claim 1 , wherein the second polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO:4.
19 . The protein of claim 1 , wherein the second polypeptide is at least 91%, 92%, 93%, 94%, 95% or 96% similar to SEQ ID NO:4.
20 . The protein of claim 1 , wherein the second polypeptide is at least 91%, 92%, 93%, 94%, 95% or 96% identical to SEQ ID NO:4.
21 . A polynucleotide encoding the protein of any one of claims 1 - 20 .
22 . An expression vector comprising the polynucleotide of claim 21 .
23 . The expression vector of claim 22 , wherein the expression vector is a viral vector.
24 . The expression vector of claim 23 , wherein the viral vector is further defined as a lentiviral vector, retroviral vector, adenoviral vector, or adeno-associated viral vector.
25 . The expression vector of claim 23 , wherein the viral vector is further defined as a lentiviral vector.
26 . A population of T cells engineered to express membrane-anchored IL-12 according to any one of claims 1 - 20 .
27 . The population of claim 26 , wherein the T cells express an expression vector of any one of claims 22 - 25 .
28 . The population of claim 26 , wherein the T cells are tumor infiltrating lymphocytes (TILs), CD8 + T cells and/or CD4 + T cells.
29 . The population of claim 26 , wherein the T cells are CD8 + T cells.
30 . The population of claim 26 , wherein the T cells are tumor-specific T cells.
31 . The population of claim 26 , wherein the T cells are further engineered to express a T cell receptor (TCR) or chimeric antigen receptor (CAR) having antigenic specificity for a tumor-associated antigen.
32 . The population of claim 31 , wherein the (CAR) comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising a tumor-associated antigen binding region.
33 . The population of claim 32 , wherein the antigen binding region is an F(ab′)2, Fab′, Fab, Fv, or scFv.
34 . The population of claim 32 , wherein the intracellular signaling domain is a T-lymphocyte activation domain.
35 . The population of claim 32 , wherein the intracellular signaling domain comprises CD3ξ, CD28, OX40/CD134, 4-1BB/CD137, FcεRIγ, ICOS/CD278, ILRB/CD122, IL-2RG/CD132, DAP molecules, CD70, cytokine receptor, CD40, Toll-like receptor 9, or a combination thereof.
36 . The population of claim 32 , wherein the transmembrane domain comprises CD28 transmembrane domain, IgG4Fc hinge, Fc regions, CD4 transmembrane domain, the CD3ξ transmembrane domain, cysteine mutated human CD3ξ domain, CD16 transmembrane domain, CD8 transmembrane domain, or erythropoietin receptor transmembrane domain.
37 . A method for producing the population of T cells of any one of claims 26 - 36 comprising obtaining a starting population of T cells and introducing a vector expressing membrane-anchored IL-12, thereby generating a population of T cells expressing membrane-anchored IL-12.
38 . The method of claim 37 , wherein the vector is an expression vector comprises a polynucleotide of claim 21 .
39 . The method of claim 38 , wherein the expression vector is a viral vector.
40 . The method of claim 39 , wherein the viral vector is further defined as a lentiviral vector.
41 . The method of claim 37 , wherein membrane-anchored IL-12 is under the control of two constitutive promoters.
42 . The method of claim 41 , wherein the constitutive promoters are cytomegalovirus (CMV).
43 . The method of claim 37 , wherein introducing comprises performing electroporation.
44 . The method of claim 37 , wherein introducing comprises performing viral transduction.
45 . The method of claim 37 , further comprising activating the T cells with anti-CD3 and CD80-Fc recombinant protein.
46 . The method of claim 45 , wherein activating is about 1-2 days.
47 . The method of claim 45 , wherein activating increases NKG2D expression on the T cells.
48 . A method of treating a cancer in a subject comprising administering an effective amount of T cells engineered to express membrane-anchored IL-12 to the subject.
49 . The method of claim 48 , wherein the T cells are the T cells of any one of claims 26 - 36 .
50 . The method of claim 48 , wherein the membrane-anchored IL-12 is according to any one of claims 1 - 20 .
51 . The method of claim 48 , wherein the subject is a human.
52 . The method of claim 48 , wherein the T cells are autologous T cells.
53 . The method of claim 48 , wherein the T cells are engineered to express membrane-anchored IL-12 by lentiviral transduction.
54 . The method of claim 53 , wherein there is low or essentially no T cell accumulation in the subject's lungs after administering the T cells engineered to express membrane-anchored IL-12.
55 . The method of claim 53 , wherein lentiviral transduction results in a reduced risk of cytokine response syndrome (CRS), reduced systemic toxicity, and/or increased effectiveness of treatment.
56 . The method of claim 48 , further comprising lymphodepletion of the subject prior to administration of the T cells.
57 . The method of claim 56 , wherein lymphodepletion comprises administration of cyclophosphamide and/or fludarabine.
58 . The method of claim 48 , wherein the method further comprises administering at least one additional therapeutic agent.
59 . The method of claim 58 , wherein the at least one additional therapeutic agent is chemotherapy, immunotherapy, surgery, radiotherapy, or biotherapy.
60 . The method of claim 59 , wherein the chemotherapy is selected from the group consisting of cyclophosphamide, methotrexate, fluorouracil, doxorubicin, vincristine, ifosfamide, cisplatin, gemcytabine, busulfan, ara-C, and combinations thereof.
61 . The method of claim 59 , wherein the chemotherapy is doxorubicin or cyclophosphamide.
62 . The method of claim 59 , wherein the chemotherapy is administered prior to the T cells.
63 . The method of claim 59 , wherein the chemotherapy is administered 15 to 25 hours prior to the T cell therapy.
64 . The method of claim 58 , wherein the T cells and/or at least one additional therapeutic agent is administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion.
65 . The method of claim 48 , wherein the cancer is colon cancer or lung cancer.
66 . The method of claim 48 , wherein administration of the T cells expressing membrane-anchored IL-12 does not induce IFNγ or induces a lower level of IFNγ as compared to administration of T cells with wild-type IL-12
67 . The method of claim 66 , wherein the IFNγ is measured in a serum sample.
68 . The method of claim 48 , wherein administering the T cells induces expression of CXCL9, CXCL10 and/or CCL17.
69 . The method of claim 48 , wherein administering the T cells induces expression of NKG2D and/or NKG2D ligands.
70 . The method of claim 48 , wherein administering the T cells induces expression of costimulatory receptor CD28 and/or CD80.
71 . The method of claim 48 , wherein administering the T cells decreases expression of an immune checkpoint inhibitor.
72 . The method of claim 71 , wherein the immune checkpoint inhibitor is PD-1 or PD-L1.
73 . The method of claim 58 , wherein the T cells and/or at least one additional therapy is administered more than once.
74 . The method of claim 58 , wherein the T cells penetrate to or near the center of a tumor within the subject.
75 . An in vitro method for generating NKG2D-positive CD8 + T cells comprising:
(a) obtaining a starting population of T cells; and
(b) culturing the starting population of T cells in the presence of anti-CD3 and CD80 for a period of time sufficient to induce NKG2D expression, thereby generating NKG2D + CD8 + T cells.
76 . The method of claim 75 , wherein the culturing is further defined as (i) pre-treating the starting population of T cells to anti-CD3 and (ii) treating the T cells with CD80.
77 . The method of claim 75 , wherein the starting population of T cells are CD28-positive.
78 . The method of claim 75 , wherein the starting population of T cells are TILs, CD8 + T cells and/or CD4 + T cells.
79 . The method of claim 75 , wherein the starting population of T cells are CD8 + T cells.
80 . The method of claim 76 , wherein pre-treating with anti-CD3 is for 12-48 hours.
81 . The method of claim 76 , wherein culturing in the presence of CD80 is for 1-5 days.
82 . The method of claim 75 or 76 , wherein anti-CD3 is further defined as anti-CD3 microbeads.
83 . The method of claim 75 or 76 , wherein CD80 is further defined as CD80-Fc recombinant protein.
84 . The method of claim 75 , wherein treating with CD80 results in phosphorylation of STAT3.
85 . The method of claim 75 , wherein the T cells are further engineered to express a TCR or CAR having antigenic specificity for a tumor-associated antigen.
86 . The method of claim 85 , wherein the CAR comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising a tumor-associated antigen binding region.
87 . The method of claim 86 , wherein the antigen binding region is an F(ab′)2, Fab′, Fab, Fv, or scFv.
88 . The method of claim 86 , wherein the intracellular signaling domain is a T-lymphocyte activation domain.
89 . The method of claim 86 , wherein the intracellular signaling domain comprises CD3ξ, CD28, OX40/CD134, 4-1BB/CD137, FcεRIγ, ICOS/CD278, ILRB/CD122, IL-2RG/CD132, DAP molecules, CD70, cytokine receptor, CD40, Toll-like receptor 9, or a combination thereof.
90 . The method of claim 86 , wherein the transmembrane domain comprises CD28 transmembrane domain, IgG4Fc hinge, Fc regions, CD4 transmembrane domain, the CD3ξ transmembrane domain, cysteine mutated human CD3ξ domain, CD16 transmembrane domain, CD8 transmembrane domain, or erythropoietin receptor transmembrane domain.
91 . A method of treating a cancer in a subject comprising administering an effective amount of NKG2D + CD8 + T cells of any one of claims 75 - 90 to the subject.
92 . The method of claim 91 , wherein the T cells express membrane-anchored IL-12 according to any one of claims 1 - 20 .
93 . The method of claim 91 , wherein the subject is a human.
94 . The method of claim 91 , wherein the T cells are autologous T cells.
95 . The method of claim 91 , wherein the method further comprises administering at least one additional therapeutic agent.
96 . The method of claim 95 , wherein the at least one additional therapeutic agent is chemotherapy, immunotherapy, surgery, radiotherapy, or biotherapy.
97 . The method of claim 96 , wherein the chemotherapy is selected from the group consisting of cyclophosphamide, methotrexate, fluorouracil, doxorubicin, vincristine, ifosfamide, cisplatin, gemcytabine, busulfan, ara-C, and combinations thereof.
98 . The method of claim 97 , wherein the chemotherapy is doxorubicin or cyclophosphamide.
99 . The method of claim 97 or 98 , wherein the chemotherapy is administered prior to the T cells.
100 . The method of claim 99 , wherein the chemotherapy is administered 15 to 25 hours prior to the T cell therapy.
101 . The method of claim 95 , wherein the T cells and/or at least one additional therapeutic agent is administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion.
102 . The method of claim 91 , wherein the cancer is colon cancer or lung cancer.Join the waitlist — get patent alerts
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