Search

US-12617836-B2 - Tumor environment specific expression of effector genes

US12617836B2US 12617836 B2US12617836 B2US 12617836B2US-12617836-B2

Abstract

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.

Inventors

  • Roi Gazit
  • Angel Porgador

Assignees

  • The National Institute for Biotechnology in the Negev Ltd.

Dates

Publication Date
20260505
Application Date
20220321

Claims (18)

  1. 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. 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. 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. 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. 5 . The TME responsive expression vector of claim 1 , wherein the synthetic promoter comprises at least two TGF-β derived response elements.
  6. 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. 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. 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. 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. 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. 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. 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. 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. 14 . The TME responsive expression vector of claim 13 , comprising the nucleotide sequence set forth in SEQ ID NO: 93.
  15. 15 . An immune effector cell comprising the TME responsive expression vector of claim 1 .
  16. 16 . The immune effector cell of claim 15 , wherein the tumor is a solid tumor.
  17. 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. 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.

Description

FIELD OF INVENTION The present disclosure generally relates to the field of focusing immune cells activity within tumor-microenvironment. For example, the expression of a chimeric antigen receptor (CAR) specifically to tumor tissue that will focus anti-cancer immune cells and spare normal healthy tissues. BACKGROUND Harnessing the immune system to eradicate cancer has proved highly efficient in recent years. The main success is the engineered immune cells, such as Chimeric-Antigen-Receptor T-cells (CAR-T), which are already approved by the FDA for the treatment of few types of cancers that have no other cure. However, although CAR-T can reach tumors and metastasis throughout a patient's body, the specificity of the engineered receptor does not fully distinguish between tumor cells and normal cells, since tumors often are not having an absolutely unique antigen that is not expressed by any other normal cells in the body. As a result, several CAR treatments caused toxic immune response, similar to Graft-vs-Host-Disease (GVHD), and even death that resulted from the CAR treatment during clinical trials. Importantly, unlike antibodies or other treatments, CAR-T are having an extreme potency to identify antigens, even at minute levels, and efficiently attack the cells that express them—both cancerous and normal healthy cells. Therefore, focusing the CAR expression into the tumor-microenvironment may improve specificity and reduce ON-antigen OFF-tumor activities. Attempts to achieve non-constitutive expression of CAR within engineered immune cells have been made. An example includes applying an “ON-OFF switch” within the CAR expression vector, by utilizing a promoter activated only in the presence of an exogenously provided molecule (such as tetracycline/doxycycline). This may allow for turning off CAR expression in case of pronounced adverse symptoms but will turn off also the positive activity of the CAR T-cells against the tumor cells and thus terminates a potent CAR treatment. There remains an unmet need for controlled CAR expression that reduces the risks of its life-threatening “ON-target OFF-tumor”, while allowing effective elimination of tumors. SUMMARY The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other advantages or improvements. The platform includes the tumor environment (TME) responsive expression vectors, including nucleic acid sequences encoding for a synthetic promoter (also referred to herein as “CARTIV” promoters) comprising multiple TME-dependent promoter response elements that is conjugated to effector-genes such as, but not limited to, nucleic acid sequence encoding for Chimeric Antigen Receptor. According to some embodiments, the ligand binding domain of the CAR is capable of specifically binding to Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2-PK), CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), Galactin, Ral-B, Integrin Alpha-V-Beta3 (CD51/CD61), an abnormal p53 protein, or an abnormal RAS protein such as K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene). Each possibility is a separate embodiment. According to some embodiments, the ligand-binding domain of the CAR is capable of specifically binding to Her2. Advantageously, the TME responsive vector is designed such that binding of two or more factors that are present in the TME to the promoter response element induces the expression of the effector-gene. In the absence of such TME factors, the promoter expression is basal. This advantageously ensures minimal- and possibly even down to no expression of effector-mechanisms, such as CAR, in tissue environments different from that of the tumor. Accordingly, these promoters endogenously upregulate the expression of effector mechanisms, such as CARs, in immu