Tumor environment specific expression of effector genes
A Tumor Micro-Environment (TME) responsive expression vector including a nucleic acid sequence of a synthetic promoter, comprising two or more promoter-response-elements inducing expression of an immune-effector gene.
1 . A Tumor Micro-Environment (TME) responsive expression vector comprising:
a nucleic acid sequence encoding a synthetic promoter, said promoter comprising two or more different TME dependent promoter response elements (PRE)s; and
a nucleic acid sequence encoding an effector gene,
wherein said TME responsive expression vector is designed such that binding of two or more TME factors present in the TME to the PREs induces expression of the effector-gene, and in the absence of binding of the two or more TME factors to the PREs essentially no effector gene is expressed; and wherein the two or more different PREs comprise at least one TGF-β derived PRE having the nucleotide sequence set forth in SEQ ID NO: 48 and at least one response element other than a TGF-β derived PRE.
2 . The TME responsive expression vector of claim 1 , wherein the at least one TME dependent PRE other than a TGF-β derived PRE is selected from the list consisting of: interferon-gamma-(IFN-γ) PRE, TGF-β PRE, Nuclear Factor kappa-B (NF-κB) PRE, hypoxia PRE, Heat shock protein 70 (HSP-70) PRE, IL-6 PRE, IL-1 PRE, IL-8 PRE, IL-11 PRE, IL-12 PRE, IL-15 PRE, IL-18 PRE, IL-17 PRE, IL-21 PRE, IL-35 PRE, GM-CSF PRE, Hepatic Growth Factor (HGF) PRE, Aryl Hydrogen Receptor (AhR) PRE or any combination thereof, activated within an inflammatory TME.
3 . The TME responsive expression vector of claim 1 , wherein the at least one TME dependent PRE other than a TGF-β derived PRE is selected from an NF-κB PRE, an IL-6 PRE, an IL-6 derived PRE, and an IFN-γ PRE.
4 . The TME responsive expression vector of claim 3 , wherein the IL-6 derived PRE has a nucleotide sequence set forth in SEQ ID NO: 49 or 52.
5 . The TME responsive expression vector of claim 1 , wherein the synthetic promoter comprises at least two TGF-β derived response elements.
6 . The TME responsive expression vector of claim 1 , wherein the synthetic promoter comprises a consensus nucleotide sequence set forth in any one of SEQ ID NO: 53-55.
7 . The TME responsive expression vector of claim 1 , wherein the two or more different TME dependent PREs comprise a TGF-β derived PRE, a Nuclear Factor kappa-B (NF-κB) PRE, and an hypoxia PRE.
8 . The TME responsive expression vector of claim 7 , wherein the hypoxia PRE is downstream of the TGF-β derived PRE and the Nuclear Factor kappa-B (NF-κB) PRE.
9 . The TME responsive expression vector of claim 7 , wherein the synthetic promoter comprises a nucleic acid sequence having at least 80% sequence homology to a nucleic acid selected from the nucleic acid sequences set forth in SEQ ID Nos 1-40 or any combination thereof.
10 . The TME responsive expression vector of claim 9 , wherein the synthetic promoter comprises a nucleic acid sequence having at least 80% sequence homology to a nucleic acid sequence set forth in SEQ ID NO: 21.
11 . The TME responsive expression vector of claim 1 , wherein binding of two or more TME factors to the two or more different TME dependent PREs induces a higher expression level of the effector gene than binding to a single TME dependent PRE.
12 . The TME responsive expression vector of claim 1 , wherein the vector is selected from a DNA vector, a plasmid, a lentivirus vector, an adenoviral vector, or a retrovirus vector.
13 . The TME responsive expression vector of claim 1 , wherein the effector gene is a chimeric antigen receptor (CAR) capable of specifically binding to Her2 (CAR-Her2).
14 . The TME responsive expression vector of claim 13 , comprising the nucleotide sequence set forth in SEQ ID NO: 93.
15 . An immune effector cell comprising the TME responsive expression vector of claim 1 .
16 . The immune effector cell of claim 15 , wherein the tumor is a solid tumor.
17 . The immune effector cell of claim 15 , wherein the effector gene is a chimeric antigen receptor (CAR) capable of specifically binding to Her2 (CAR-Her2).
18 . The TME responsive expression vector of claim 1 , wherein the two or more different PREs comprise a TGF-β derived PRE, a Nuclear Factor kappa-B (NF-κB) PRE, and an IL-6 PRE.