Combination gene and radiation therapy for cancer treatment

Claims

1. A recombinant expression system for use in combined gene and radiation therapy of a disease in a recipient mammal, wherein a. the recombinant expression system comprises a first nucleic acid construct encoding a STING target that responds to a STING agonist that recognizes said STING target, but not human STING and its naturally occurring variants, wherein said sequence encoding the STING target is operably linked to elements that ensure the expression and / or translation of said STING target; and / or a second nucleic acid construct containing a therapeutic gene sequence that is operably linked to a STING-responsive promoter or a radiation-inducible promoter; and b. the recombinant expression system is administered to the recipient mammal prior to or simultaneously with, or following, low-dose irradiation of said recipient mammal of the tissue or organ to be transfected or transduced using said recombinant expression system (target tissue or organ); and / or c. after introducing said recombinant expression system in step b), the target tissue or organ of the recipient mammal is exposed to low-dose irradiation and / or a STING agonist that recognizes said target STING, but not human STING and its naturally occurring variants, is administered to the recipient mammal.

2. A recombinant expression system for use according to claim 1, wherein at least one other additional therapy is used, selected from the group consisting of a. a therapy comprising at least one checkpoint inhibitor, preferably selected from the group consisting of an inhibitor of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, BTLA, B7H3, B7H4, TIM3, KIR or A2aR, more preferably selected from the group consisting of an inhibitor of PD-1, PD-L1, PD-L2 or CTLA-4; and / or b. a therapy comprising at least one adoptive cell therapy, preferably selected from adoptive NK cell therapy, adoptive dendritic cell therapy or adoptive T cell therapy, wherein the adoptive T cell therapy is preferably CAR-T cell therapy or TIL therapy; and / or c. therapy including at least one cancer vaccine; and / or d. therapy including at least one exogenous cytokine, and wherein at least one other additional therapy is administered before, simultaneously and / or after the administration according to step b.

3. A recombinant expression system for use according to claim 1 or 2, wherein the disease is selected from the group consisting of cancer, an autoimmune disease, an inflammatory disease, and an infectious disease, preferably a solid cancer selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, thymus cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, Hodgkin's lymphoma, primary liver cancer, metastatic liver cancer, gallbladder cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, Merkel cell carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasma cell tumors, neuroendocrine tumors, cholangiocarcinoma or carcinoid tumors.

4. A recombinant expression system for use according to any one of claims 1 to 3, wherein the therapeutic gene sequence of the second nucleic acid construct encodes a protein or gene selected from the group consisting of cytokines, such as chemokines, interferons, interleukins, lymphokines and tumor necrosis factors; hormones; growth factors; cell surface receptors; enzymes; transcription factors; tumor suppressor proteins; nanobodies; monoclonal antibodies; single-chain variable fragments (scFv); peptides that block the activity of cytokines, nuclear factors or hormones; or is a gene such as shRNA against cytokines, nuclear factors or hormones.

5. A recombinant expression system for use according to claim 4, wherein the protein encoded by the therapeutic gene sequence of the second nucleic acid construct is selected from the group consisting of interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-12 (IL-12), single-chain interleukin-12 (scIL-12), interleukin-15 (IL-15), interleukin-15-sushi (IL-15-sushi), interleukin-21 (IL-21), interleukin-23 (IL-23), granulocyte-macrophage colony-stimulating factor (GM-CSF), type I interferons (IFN-α and IFN-β), IFN-γ, TNF-α, CXCL10, FLT3 ligand; immunostimulatory monoclonal antibody or single-chain variable fragment (scFV) or nanobody neutralizing PD1, PDL1, CTLA4, CD137, TIM3, LAG3; blocking peptides or shRNA against immunosuppressive factors such as TGF-β or IL-10 or FoxP3.

6. A recombinant expression system for use according to any one of the above claims, wherein the second nucleic acid construct comprises at least two therapeutic gene sequences, each encoding an immunomodulatory protein, wherein the first immunomodulatory protein is a single-chain IL-12 comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or 10, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 9 or 10, wherein the polypeptide has the biological activity of interleukin-12, and the second immunomodulatory protein is an IFN-β comprising or consisting of the amino acid sequence of SEQ ID NO: 79, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 79, wherein the polypeptide has the biological activity of IFN-β; or IL-15-sushi, comprising or consisting of the amino acid sequence of SEQ ID NO: 80, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 80, wherein the polypeptide has the biological activity of IL-15-sushi; or CXCL10 comprising or consisting of the amino acid sequence of SEQ ID NO: 81, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 81, wherein the polypeptide has the biological activity of CXCL10.

7. A recombinant expression system for use according to any one of the above claims, wherein the second nucleic acid construct is a bi- or polycistronic expression cassette comprising two or more therapeutic gene sequences, each encoding a different immunomodulatory protein or proteins or RNA with anti-cancer properties, wherein a. The therapeutic gene sequences are separated by a sequence encoding an IRES; and / or b. the therapeutic gene is separated by a sequence encoding the 2A peptide, preferably a sequence encoding the P2A peptide.

8. A recombinant expression system for use according to any one of the above items, wherein the STING-responsive promoter of the second nucleic acid construct is selected from the group consisting of an IFN-β-responsive promoter, a CXCL9-responsive promoter, a CXCL10-responsive promoter, a STAT motif promoter, an IRSF3 motif promoter, an ISRE motif promoter, an NFKB motif promoter, a STAT6 motif promoter, preferably wherein the STING-responsive promoter comprises at least one interferon-stimulated response element (ISRE).

9. A recombinant expression system for use according to any of the above claims, wherein the STING-responsive promoter of the second nucleic acid construct is selected from the list consisting of: a. a nucleotide sequence comprising one to ten copies, preferably two to seven copies, more preferably three to five copies, and particularly preferably 4 copies of a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 11 or identical to SEQ ID NO: 11; b. the nucleotide sequence according to a., wherein the copies are separated by intervals of no more than forty nucleotides in length, preferably of four to thirty-five nucleotides in length, more preferably of ten to thirty nucleotides in length, and particularly preferably of fifteen to twenty-five nucleotides in length; c. the nucleotide sequence according to SEQ ID NO: 12; d. the nucleotide sequence according to SEQ ID NO:

13.

10. A recombinant expression system for use according to any of the above items, wherein the second nucleic acid construct of the expression system, comprising a therapeutic gene sequence, further comprises at least one sequence motif that inhibits transgenic expression of the therapeutic gene sequence in tissues or cells not intended for protein expression, wherein the tissue is preferably the liver, wherein the sequence motif is preferably a microRNA target sequence that is present in large quantities in the liver.

11. A recombinant expression system for use according to claim 10, wherein the liver-extensive microRNA is selected from the group consisting of miR-122, miR-192, miR-199a, miR-101, miR-99a, let7a, let7b, let7c, let7f, preferably wherein the liver-extensive microRNA is miR-122.

12. A recombinant expression system for use according to any of the above claims, wherein the low-dose irradiation is based on photon beams, wherein the photon beam is preferably selected from the group consisting of X-rays or gamma rays, or particle beams, wherein the particle beam is preferably selected from the group consisting of a beta particle, an alpha particle, a neutron, a muon, a pion, a proton or other heavier positive ions.

13. A recombinant expression system for use according to any of the above items, wherein the low-dose irradiation has a total radiation dose of 100 Gy or less, preferably 40 Gy or less, and more preferably 8 Gy or less.

14. A recombinant expression system for use according to any of the above claims, wherein the low-dose radiation is administered using stereotactic radiosurgery (SRS), intraoperative radiation therapy (IORT), stereotactic body radiation therapy (SBRT), single-dose radiation therapy (SDRT), intensity-modulated radiation therapy (IMRT), image-guided radiation therapy (IGRT), stereotactic ablative radiation therapy (SABR) or a form of hypofractionated radiation therapy, preferably performed with linear accelerators (LINAC), gamma knives or proton beam therapy.

15. A recombinant expression system for use according to any of the above items, wherein the time interval between low-dose irradiation b. and subsequent administration of the expression system is from 1 minute to 20 days, preferably from 10 minutes to 2 days, and more preferably from 30 minutes to 24 hours.

16. A recombinant expression system for use according to any of the above claims, wherein the subsequent low-dose irradiation with, carried out after administration of the recombinant expression system, is repeated at intervals of from 1 day to 30 days, preferably from 1 day to 5 days, and more preferably from 1 day to 3 days.

17. A recombinant expression system for use according to any one of the above, wherein after administration of the recombinant expression system comprising the first nucleic acid construct, a human-insensitive STING agonist is administered to the recipient mammal, preferably selected from the group consisting of 5,6-dimethylxanthenone-4-acetic acid (DMXAA), 7-bromo-DMXAA, 7-iodo-DMXAA, 7-hydroxy-DMXAA, 7-formyl-DMXAA, 7-hydroxymethyl-DMXAA, 7-(2-hydroxyethyl)-DMXAA, flavone-8-acetic acid (FAA), 2,7-bis(2-dimethylaminoethoxy)fluoren-9-one (Tilorone) and 10-carboxymethyl-9-acridanone (CMA), and more preferably, wherein the STING agonist is DMXAA.

18. A recombinant expression system for use in combined gene and radiation therapy of a disease in a recipient mammal, wherein a. the recombinant expression system consists of a nucleic acid construct encoding at least two therapeutic gene sequences that are operably linked to a STING-responsive promoter or a radiation-inducible promoter; and b. the recombinant expression system is administered to the recipient mammal prior to or simultaneously with, or following, low-dose irradiation of said recipient mammal of the tissue or organ to be transfected or transduced using said recombinant expression system (target tissue or organ); and / or c. after introducing said recombinant expression system in step b), the target tissue or organ of the recipient mammal is exposed to low-dose irradiation.

19. A recombinant expression system for use according to claim 18, wherein at least one other additional therapy is used, selected from the group consisting of a. a therapy comprising at least one checkpoint inhibitor, preferably selected from the group consisting of an inhibitor of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, BTLA, B7H3, B7H4, TIM3, KIR or A2aR, more preferably selected from the group consisting of an inhibitor of PD-1, PD-L1, PD-L2 or CTLA-4; and / or b. a therapy comprising at least one adoptive cell therapy, preferably selected from NK cell adoptive therapy, dendritic cell adoptive therapy or adoptive T cell therapy, wherein the adoptive T cell therapy is preferably CAR-T cell therapy or TIL therapy; and / or c. therapy including at least one cancer vaccine; and / or d. therapy including at least one exogenous cytokine, and wherein at least one other additional therapy is administered before, simultaneously and / or after the administration according to step b.

20. A recombinant expression system for use according to claim 18 or 19, wherein the disease is selected from the group consisting of cancer, an autoimmune disease, an inflammatory disease, and an infectious disease, preferably a solid cancer selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, thymus cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, Hodgkin's lymphoma, primary liver cancer, metastatic liver cancer, gallbladder cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, Merkel cell carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasma cell tumors, neuroendocrine tumors, cholangiocarcinoma or carcinoid tumors.

21. A recombinant expression system for use according to any one of claims 18-20, wherein the therapeutic gene sequences of the nucleic acid construct encode a protein selected from the group consisting of cytokines, such as chemokines, interferons, interleukins, lymphokines and tumor necrosis factors; hormones; growth factors; cell surface receptors; enzymes; transcription factors; tumor suppressor proteins; nanobodies; monoclonal antibodies; single-chain variable fragments (scFv); peptides that block the activity of cytokines, nuclear factors or hormones.

22. A recombinant expression system for use according to claim 21, wherein the proteins encoded by the therapeutic gene sequences of the nucleic acid construct are selected from the group consisting of interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-12 (IL-12), single-chain interleukin-12 (scIL-12), interleukin-15 (IL-15), interleukin-15-sushi (IL-15-sushi), interleukin-21 (IL-21), interleukin-23 (IL-23), granulocyte-macrophage colony-stimulating factor (GM-CSF), type I interferons (IFN-α and IFN-β), IFN-γ, TNF-α, CXCL10, FLT3 ligand; immunostimulatory monoclonal antibody or single-chain variable fragment (scFV) or nanobody neutralizing PD1, PDL1, CTLA4, CD137, TIM3, LAG3; blocking peptides or shRNA against immunosuppressive factors such as TGF-β or IL-10 or FoxP3.

23. A recombinant expression system for use according to any one of claims 18-22, wherein the nucleic acid construct comprises at least two therapeutic gene sequences, each encoding an immunomodulatory protein, wherein the first immunomodulatory protein is a single-chain IL-12 comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or 10, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 9 or 10, wherein the polypeptide has the biological activity of interleukin-12, and the second immunomodulatory protein is an IFN-β comprising or consisting of the amino acid sequence of SEQ ID NO: 79, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 79, wherein the polypeptide has the biological activity of IFN-β; or IL-15-sushi, comprising or consisting of the amino acid sequence of SEQ ID NO: 80, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 80, wherein the polypeptide has the biological activity of IL-15-sushi, or CXCL10 comprising or consisting of the amino acid sequence of SEQ ID NO: 81, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 81, wherein the polypeptide has the biological activity of CXCL10.

24. A recombinant expression system for use according to any one of claims 18 to 23, wherein the nucleic acid construct is a bi- or polycistronic expression cassette comprising two or more therapeutic gene sequences, each encoding a different immunomodulatory protein or proteins or RNA with anti-cancer properties, wherein a. The therapeutic gene sequences are separated by a sequence encoding an IRES; and / or b. the therapeutic gene sequences are separated by a sequence encoding the 2A peptide, preferably a sequence encoding the P2A peptide.

25. A recombinant expression system for use according to any one of claims 18-24, wherein the STING-responsive promoter of the nucleic acid construct is selected from the group consisting of an IFN-β-responsive promoter, a CXCL9-responsive promoter, a CXCL10-responsive promoter, a STAT motif promoter, an IRSF3 motif promoter, an ISRE motif promoter, an NFKB motif promoter, a STAT6 motif promoter, preferably wherein the STING-responsive promoter comprises at least one interferon-stimulated response element (ISRE).

26. A recombinant expression system for use according to any one of claims 18-25, wherein the STING-responsive promoter of the nucleic acid construct is selected from the list consisting of: a. a nucleotide sequence comprising one to ten copies, preferably two to seven copies, more preferably three to five copies, and particularly preferably 4 copies of a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 11 or identical to SEQ ID NO: 11; b. the nucleotide sequence according to a., wherein the copies are separated by intervals of no more than forty nucleotides in length, preferably of four to thirty-five nucleotides in length, more preferably of ten to thirty nucleotides in length, and particularly preferably of fifteen to twenty-five nucleotides in length; c. the nucleotide sequence according to SEQ ID NO: 12; d. the nucleotide sequence according to SEQ ID NO:

13.

27. A recombinant expression system for use according to any one of claims 18-26, wherein the nucleic acid construct of the expression system comprising two therapeutic gene sequences further comprises at least one sequence motif that inhibits transgenic expression of the therapeutic gene sequences in tissues or cells not intended for protein expression, wherein the tissue is preferably the liver, wherein the sequence motif is preferably a microRNA target sequence that is present in large quantities in the liver.

28. A recombinant expression system for use according to claim 27, wherein the liver-extensive microRNA is selected from the group consisting of miR-122, miR-192, miR-199a, miR-101, miR-99a, let7a, let7b, let7c, let7f, preferably wherein the liver-extensive microRNA is miR-122.

29. A recombinant expression system for use according to any one of claims 18-28, wherein the low-dose irradiation is based on photon beams, wherein the photon beam is preferably selected from the group consisting of X-rays or gamma rays, or particle beams, wherein the particle beam is preferably selected from the group consisting of a beta particle, an alpha particle, a neutron, a muon, a pion, a proton or other heavier positive ions.

30. A recombinant expression system for use according to any one of claims 18 to 29, wherein the low-dose irradiation has a total radiation dose of 100 Gy or less, preferably 40 Gy or less, and more preferably 8 Gy or less.

31. The recombinant expression system of any one of claims 18-30, wherein the low-dose radiation is administered using stereotactic radiosurgery (SRS), intraoperative radiation therapy (IORT), stereotactic body radiation therapy (SBRT), single-dose radiation therapy (SDRT), intensity-modulated radiation therapy (IMRT), image-guided radiation therapy (IGRT), stereotactic ablative radiation therapy (SABR) or a form of hypofractionated radiation therapy, preferably performed with linear accelerators (LINAC), gamma knives or proton beam therapy.

32. A recombinant expression system for use according to any one of claims 18 to 31, wherein the time interval between low-dose irradiation b. and subsequent administration of the expression system is from 1 minute to 20 days, preferably from 10 minutes to 2 days, and more preferably from 30 minutes to 24 hours.

33. A recombinant expression system for use according to any one of claims 18 to 32, wherein the subsequent low-dose irradiation of c., carried out after administration of the recombinant expression system, is repeated at intervals of from 1 day to 30 days, preferably from 1 day to 5 days, and more preferably from 1 day to 3 days.