MICROORGANISMS PROGRAMMED TO PRODUCE IMMUNE MODULATORS AND ANTI-CANCER THERAPEUTICS IN TUMOR CELLS
Genetically programmed microorganisms, such as bacteria or virus, pharmaceutical compositions thereof, and methods of modulating and treating cancers are disclosed.
1 . A modified microorganism capable of producing at least one immune initiator and at least one immune sustainer.
2 . The modified microorganism of claim 1 , wherein the immune initiator is capable of enhancing oncolysis, activating antigen presenting cells (APCs), and/or priming and activating T cells.
3 . The modified microorganism of claim 1 or claim 2 , wherein the immune initiator is a therapeutic molecule encoded by at least one gene; a therapeutic molecule produced by an enzyme encoded by at least one gene; at least one enzyme of a biosynthetic or catabolic pathway encoded by at least one gene; at least one therapeutic molecule produced by at least one enzyme of a biosynthetic or catabolic pathway encoded by at least one gene; or a nucleic acid molecule that mediates RNA interference, microRNA response or inhibition, TLR response, antisense gene regulation, target protein binding, or gene editing.
4 . The modified microorganism of any one of claims 1 - 3 , wherein the immune initiator is a cytokine, a chemokine, a single chain antibody, a ligand, a metabolic converter, a T cell co-stimulatory receptor, a T cell co-stimulatory receptor ligand, an engineered chemotherapy, or a lytic peptide.
5 . The modified microorganism of any one of claims 1 - 4 , wherein the immune initiator is a STING agonist, arginine, 5-FU, TNFα, IFNγ, IFNβ1, agonistic anti-CD40 antibody, CD40L, SIRPα, GMCSF, agonistic anti-OXO40 antibody, OXO40L, agonistic anti-4-1BB antibody, 4-1BBL, agonistic anti-GITR antibody, GITRL, anti-PD1 antibody, anti-PDL1 antibody, or azurin.
6 . The modified microorganism of claim 5 , wherein the immune initiator is a STING agonist.
7 . The modified microorganism of claim 6 , wherein the STING agonist is c-diAMP, c-GAMP, or c-diGMP.
8 . The modified microorganism of any one of claims 5 - 7 , wherein the modified microorganism comprises at least one gene sequence encoding an enzyme which produces the immune initiator.
9 . The modified microorganism of claim 8 , wherein the at least one gene sequence encoding the immune initiator is a dacA gene sequence.
10 . The modified microorganism of claim 8 , wherein the at least one gene sequence encoding the immune initiator is a cGAS gene sequence.
11 . The modified microorganism of claim 10 , wherein the cGAS gene sequence is selected from a human cGAS gene sequence, a Verminephrobacter eiseniae cGAS gene sequence, Kingella denitrificans cGAS gene sequence, and a Neisseria bacilliformis cGAS gene sequence.
12 . The modified microorganism of any one of claims 8 - 11 , wherein the at least one gene sequence encoding the immune initiator is integrated into a chromosome of the modified microorganism or is present on a plasmid.
13 . The modified microorganism of any one of claims 8 - 11 , wherein the at least one gene sequence encoding the immune initiator is operably linked to an inducible promoter.
14 . The modified microorganism of claim 13 , wherein the inducible promoter is induced by low oxygen, anaerobic, or hypoxic conditions.
15 . The modified microorganism of claim 5 , wherein the immune initiator is arginine.
16 . The modified microorganism of claim 15 , wherein the microorganism comprises at least one gene sequence encoding at least one enzyme of the arginine biosynthetic pathway.
17 . The modified microorganism of claim 15 , wherein the at least one gene sequence encoding the at least one enzyme of the arginine biosynthetic pathway comprises feedback resistant argA.
18 . The modified microorganism of claim 16 , wherein the at least one gene sequence encoding the at least one enzyme of the arginine biosynthetic pathway is selected from the group consisting of: argA, argB, argC, argD, argE, argF, argG, argH, argI, argJ, carA, and carB.
19 . The modified microorganism of claim 17 or claim 18 , further comprising a deletion or a mutation in an arginine repressor gene (argR).
20 . The modified microorganism of any one of claims 16 - 19 , wherein the at least one gene sequence for the production of arginine is integrated into a chromosome of the modified microorganism or is present on a plasmid.
21 . The modified microorganism of any one of claims 16 - 20 , wherein the at least one gene sequence for the production of arginine is operably linked to an inducible promoter.
22 . The modified microorganism of claim 21 , wherein the inducible promoter is induced by low oxygen, anaerobic, or hypoxic conditions.
23 . The modified microorganism of claim 5 , wherein the immune initiator is 5-FU.
24 . The modified microorganism of claim 23 , wherein the microorganism comprises at least one gene sequence encoding an enzyme capable of converting 5-FC to 5-FU.
25 . The modified microorganism of claim 24 , wherein the at least one gene sequence is codA.
26 . The modified microorganism of claim 24 or claim 25 , wherein the at least one gene sequence is integrated into a chromosome of the modified microorganism or is present on a plasmid.
27 . The modified microorganism of any one of claims 24 - 26 , wherein the at least one gene sequence encoding the immune initiator is operably linked to an inducible promoter.
28 . The modified microorganism of claim 27 , wherein the inducible promoter is induced by low oxygen, anaerobic, or hypoxic conditions.
29 . The modified microorganism of claim 1 , wherein the immune sustainer is capable of enhancing trafficking and infiltration of T cells, enhancing recognition of cancer cells by T cells, enhancing effector T cell response, and/or overcoming immune suppression.
30 . The modified microorganism of claim 1 or claim 29 , wherein the immune sustainer is a therapeutic molecule encoded by at least one gene; a therapeutic molecule produced by an enzyme encoded by at least one gene; at least one enzyme of a biosynthetic or catabolic pathway encoded by at least one gene; at least one therapeutic molecule produced by at least one enzyme of a biosynthetic or catabolic pathway encoded by at least one gene; or a nucleic acid molecule that mediates RNA interference, microRNA response or inhibition, TLR response, antisense gene regulation, target protein binding, or gene editing.
31 . The modified microorganism of any one of claim 1 , 29 , or 30 , wherein the immune sustainer is a cytokine, a chemokine, a single chain antibody, a ligand, a metabolic converter, a T cell co-stimulatory receptor, or a T cell co-stimulatory receptor ligand.
32 . The modified microorganism of any one of claim 1 or 29 - 31 , wherein the immune sustainer is a metabolic converter, arginine, a STING agonist, CXCL9, CXCL10, anti-PD1 antibody, anti-PDL1 antibody, anti-CTLA4 antibody, agonistic anti-GITR antibody or GITRL, agonistic anti-OX40 antibody or OX40L, agonistic anti-4-1BB antibody or 4-1BBL, IL-15, IL-15 sushi, IFNγ, or IL-12.
33 . The modified microorganism of claim 32 , wherein the immune sustainer is a metabolic converter.
34 . The modified microorganism of claim 33 , wherein the metabolic converter is at least one enzyme of a kynurenine consumption pathway or at least one enzyme of an adenosine consumption pathway.
35 . The modified microorganism of claim 33 or claim 34 , wherein the microorganism comprises at least one gene sequence encoding the at least one enzyme of the kynurenine consumption pathway.
36 . The modified microorganism of claim 35 , wherein the at least one gene sequence encoding the at least one enzyme of the kynurenine consumption pathway is a kynureninase gene sequence.
37 . The modified microorganism of claim 36 , wherein the at least one gene sequence is kynU.
38 . The modified microorganism of claim 37 , wherein the at least one gene sequence is operably linked to a constitutive promoter.
39 . The modified microorganism of any one of claims 35 - 38 , wherein the at least one gene sequence encoding the at least one enzyme of the kynurenine consumption pathway is integrated into a chromosome of the microorganism or is present on a plasmid.
40 . The modified microorganism of any one of claims 35 - 39 , wherein the microorganism comprises a deletion or a mutation in trpE.
41 . The modified microorganism of claim 32 or claim 33 , wherein the microorganism comprises at least one gene sequence encoding at least one enzyme of an adenosine consumption pathway.
42 . The modified microorganism of claim 41 , wherein the at least one gene sequence encoding the at least one enzyme of the adenosine consumption pathway is selected from add, xapA, deoD, xdhA, xdhB, and xdhC.
43 . The modified microorganism of claim 42 , wherein the at least one gene sequence encoding the at least one enzyme of the adenosine consumption pathway is operably linked to a promoter induced by low oxygen, anaerobic, or hypoxic conditions.
44 . The modified microorganism of any one of claims 41 - 43 , wherein the at least one gene sequence encoding the at least one enzyme of the adenosine consumption pathway is integrated into a chromosome of the microorganism or is present on a plasmid.
45 . The modified microorganism of any one of claims 41 - 44 , wherein the modified microorganism comprises at least one gene sequence encoding an enzyme for importing adenosine into the microorganism.
46 . The modified microorganism of claim 45 , wherein the at least one gene sequence encoding the enzyme for importing adenosine into the microorganism is nupC or nupG.
47 . The modified microorganism of claim 32 or claim 33 , immune sustainer is arginine.
48 . The modified microorganism of claim 47 , wherein the microorganism comprises at least one gene sequence encoding at least one enzyme of the arginine biosynthetic pathway.
49 . The modified microorganism of claim 48 , wherein the at least one enzyme of the arginine biosynthetic pathway comprises feedback resistant argA.
50 . The modified microorganism of claim 48 or claim 49 , wherein the at least one gene sequence encoding the at least one enzyme of the arginine biosynthetic pathway is selected from the group consisting of: argA, argB, argC, argD, argE, argF, argG, argH, argI, argJ, carA, and carB.
51 . The modified microorganism of any one of claims 48 - 50 , wherein the at least one gene sequence encoding the at least one enzyme of the arginine biosynthetic pathway is operably linked to a promoter induced by low oxygen, anaerobic, or hypoxic conditions.
52 . The modified microorganism of any one of claims 48 - 51 , wherein the at least one gene sequence encoding the at least one enzyme of the arginine biosynthetic pathway is integrated into a chromosome of the modified microorganism or is present on a plasmid.
53 . The modified microorganism of any one of claims 47 - 52 , further comprising a deletion or a mutation in an arginine repressor gene (argR).
54 . The modified microorganism of claim 32 or claim 33 , wherein the immune sustainer is a STING agonist.
55 . The modified microorganism of claim 54 , wherein the STING agonist is c-diAMP, c-GAMP, or c-diGMP.
56 . The modified microorganism of any one of claims 54 - 55 , wherein the modified microorganism comprises at least one gene sequence encoding an enzyme which produces the STING agonist.
57 . The modified microorganism of claim 56 , wherein the at least one gene sequence encoding the immune sustainer is a dacA gene sequence.
58 . The modified microorganism of claim 56 , wherein the at least one gene sequence encoding the immune sustainer is a cGAS gene sequence.
59 . The modified microorganism of claim 58 , wherein the cGAS gene sequence is selected from a human cGAS gene sequence, a Verminephrobacter eiseniae cGAS gene sequence, Kingella denitrificans cGAS gene sequence, and a Neisseria bacilliformis cGAS gene sequence.
60 . The modified microorganism of any one of the previous claims, wherein the modified microorganism is a bacterium or a yeast.
61 . The modified microorganism of any one of the previous claims, wherein the modified microorganism is an E. coli bacterium.
62 . The modified microorganism of any one of the previous claims, wherein the modified microorganism is an E. coli Nissle bacterium.
63 . The modified microorganism of any one of the previous claims, wherein the modified microorganism comprises at least one mutation or deletion in a gene which results in one or more auxotrophies.
64 . The modified microorganism of claim 63 , wherein the at least one deletion or mutation is in a dapA gene and/or a thyA gene.
65 . The modified microorganism of any one of the previous claims, comprising a phage deletion.
66 . A composition comprising at least one modified microorganism capable of producing an immune initiator, and an immune sustainer.
67 . The composition of claim 66 , wherein the at least one modified microorganism is capable of producing the immune intiator and the immune sustainer.
68 . The composition of claim 66 , wherein the at least one modified microorganism is capable of producing the immune initiator, and at least a second modified microorganism is capable of producing the immune sustainer.
69 . The composition of claim 66 , wherein the immune sustainer is not produced by a modified microorganism in the composition.
70 . A composition comprising at least one modified microorganism capable of producing an immune sustainer, and an immune initiator.
71 . The composition of claim 70 , wherein the at least one modified microorganism is capable of producing the immune initiator and the immune sustainer.
72 . The composition of claim 70 , wherein the at least one modified microorganism is capable of producing the immune sustainer, and at least a second modified microorganism is capable of producing the immune intiator.
73 . The composition of claim 70 , wherein the immune initiator is not produced by a modified microorganism in the composition.
74 . A pharmaceutically acceptable composition comprising the modified microorganism of any one of claims 1 - 65 or the composition of any one of claims 66 - 73 , and a pharmaceutically acceptable carrier.
75 . The pharmaceutically acceptable composition of claim 74 , wherein the composition is formulated for intratumoral administration.
76 . A kit comprising the pharmaceutically acceptable composition of claim 74 or claim 75 , and instructions for use thereof.
77 . A method of treating cancer in a subject, the method comprising administering to the subject the pharmaceutically acceptable composition of claim 74 or claim 75 , thereby treating cancer in the subject.
78 . A method of inducing and sustaining an immune response in a subject, the method comprising administering to the subject the pharmaceutically acceptable composition of claim 74 or claim 75 , thereby inducing and sustaining the immune response in the subject.
79 . A method of inducing an abscopal effect in a subject having a tumor, the method comprising administering to the subject the pharmaceutically acceptable composition of claim 74 or claim 75 , thereby inducing the abscopal effect in the subject.
80 . A method of inducing immunological memory in a subject having a tumor, the method comprising administering to the subject the pharmaceutically acceptable composition of claim 74 or claim 75 , thereby inducing the immunological memory in the subject.
81 . A method of inducing partial regression of a tumor in a subject, the method comprising administering to the subject the pharmaceutically acceptable composition of claim 74 or claim 75 , thereby inducing the partial regression of the tumor in the subject.
82 . The method of claim 81 , wherein the partial regression is a decrease in size of the tumor by at least about 10%, at least about 25%, at least about 50%, or at least about 75%.
83 . A method of inducing complete regression of a tumor in a subject, the method comprising administering to the subject the pharmaceutically acceptable composition of claim 74 or claim 75 , thereby inducing the complete regression of the tumor in the subject.
84 . The method of claim 83 , wherein the tumor is not detectable in the subject after administration of the pharmaceutically acceptable composition.
85 . A method of treating cancer in a subject, the method comprising
administering a first modified microorganism to the subject, wherein the first modified microorganism is capable of producing an immune initiator; and
administering a second modified microorganism to the subject, wherein the second modified microorganism is capable of producing an immune sustainer,
thereby treating cancer in the subject.
86 . A method of inducing and sustaining an immune response in a subject, the method comprising
administering a first modified microorganism to the subject, wherein the first modified microorganism is capable of producing an immune initiator; and
administering a second modified microorganism to the subject, wherein the second modified microorganism is capable of producing an immune sustainer,
thereby inducing and sustaining the immune response in the subject.
87 . The method of claim 85 or claim 86 , wherein the administering steps are performed at the same time; wherein administering of the first modified microorganism to the subject occurs before administering of the second modified microorganism to the subject; or wherein administering of the second modified microorganism to the subject occurs before administering of the first modified microorganism to the subject.
88 . A method of treating cancer in a subject, the method comprising
administering a first modified microorganism to the subject, wherein the first modified microorganism is capable of producing an immune initiator; and
administering an immune sustainer to the subject,
thereby treating cancer in the subject.
89 . A method of inducing and sustaining an immune response in a subject, the method comprising
administering a first modified microorganism to the subject, wherein the first modified microorganism is capable of producing an immune initiator; and
administering an immune sustainer to the subject,
thereby inducing and sustaining the immune response in the subject.
90 . The method of claim 88 or claim 89 , wherein the administering steps are performed at the same time; wherein administering of the first modified microorganism to the subject occurs before administering of the immune sustainer to the subject; or wherein administering of the immune sustainer to the subject occurs before administering of the first modified microorganism to the subject.
91 . A method of treating cancer in a subject, the method comprising
administering an immune initiator to the subject; and
administering a first modified microorganism to the subject, wherein the first modified microorganism is capable of producing an immune sustainer,
thereby treating cancer in the subject.
92 . A method of inducing and sustaining an immune response in a subject, the method comprising
administering an immune initiator to the subject; and
administering a first modified microorganism to the subject, wherein the first modified microorganism is capable of producing an immune sustainer,
thereby inducing and sustaining the immune response in the subject.
93 . The method of claim 91 or claim 92 , wherein the administering steps are performed at the same time; wherein the administering of the first modified microorganism to the subject occurs before the administering of the immune initiator to the subject; or wherein the administering of the immune initiator to the subject occurs before the administering of the first modified microorganism to the subject.
94 . The method of any one of claims 77 - 93 , wherein the administering is intratumoral injection.