Modified microbial toxin receptor for delivering agents into cells

Patent metadata

Field	Value
Publication number	US-9708374-B2
Application number	US-201514625386-A
Country	US
Kind code	B2
Filing date	Feb 18, 2015
Priority date	Feb 23, 2012
Publication date	Jul 18, 2017
Grant date	Jul 18, 2017

Abstract

We described a novel system of targeted cell therapy with a protein toxin, such as anthrax toxin, that has been modified to re-direct it to a desired cell target instead of its natural cell target. The system can be used for, e.g., targeted killing of undesired cells in a population of cells, such as cancer or overly active immune system cells.

First claim

We claim:

1. An altered binary toxin system for delivery of a therapeutic protein to a target cell comprising:
a fusion protein comprising a receptor-ablated pore-forming AB toxin unit fused to a non-toxin-associated receptor-binding ligand specific for the target cell, wherein the receptor-ablated pore-forming AB toxin unit comprises an anthrax toxin protective antigen (PA) with a domain 4 mutation that ablates receptor binding; and
a complementary toxin unit comprising anthrax toxin lethal factor (LF) or the amino terminal portion of anthrax toxin lethal factor (LF N ).

2. The altered binary toxin system of claim 1 , wherein the PA is PA N682AD683A .

3. The altered binary toxin system of claim 1 , wherein the non-toxin associated receptor-binding ligand specific for the target cell is an epidermal growth factor-1 or epidermal growth factor 2.

4. The altered binary toxin system of claim 1 , wherein the therapeutic protein is the catalytic domain of diphtheria toxin (DTA), ricin, shiga toxin, or pseudomonas exotoxin A.

5. The altered binary toxin system of claim 1 , wherein the non-toxin-associated receptor-binding ligand specific for the target cell binds to a receptor selected from epidermal growth factor receptors HER1, HER2, HER3 or HER4; vascular endothelial growth factor receptors VEGFR-1, VEGFR-2 or VEGFR-3; insulin-like growth factor 1 receptor; fibroblast growth factor receptors; thrombospondin 1 receptor; estrogen receptors; urokinase receptors; progesterone receptors; testosterone receptors; carcinoembryonic antigens; prostate-specific antigens; farnesoid X receptors; transforming growth factor receptors; transferrin receptors; hepatocyte growth factor receptors; or vasoactive intestinal polypeptide receptors 1 and 2.

6. The altered binary toxin system of claim 1 , wherein the non-toxin-associated receptor-binding ligand specific for the target cell is selected from an antibody or an affibody.

7. The altered binary toxin system of claim 6 , wherein the affibody is a HER2 affibody.

8. The altered binary toxin system of claim 7 , wherein the HER2 affibody is ZHER2.

9. The altered binary toxin system of claim 1 , wherein the therapeutic protein is the cytotoxic domain of shiga toxin, shiga-like toxin 1 and 2, ricin, ricin toxin A chain, abrin, gelonin, pokeweed antiviral protein, saporin, trichsanthin, pepcin, maize RIP, alpha-sarcin, Clostridium perfringens epsilon toxin, Botulinum neurotoxins, Staphylococcus enterotoxins, Clostridium difficile toxins, pertussis toxins, or pseudomonas exotoxin.

10. A kit for delivering bioactive molecules to a eukaryotic cell, comprising an altered binary toxin system of claim 1 and a pharmaceutically acceptable carrier.

11. A method for treating cancer comprising administering to a subject diagnosed with cancer the altered binary toxin system of claim 1 .

12. The method of claim 11 , wherein the altered binary toxin system comprises a receptor-redirected anthrax protective antigen.

13. The method of claim 12 , wherein the cancer is a HER2 positive cancer and the anthrax protective antigen is fused with a HER2 binding ligand.

14. The method of claim 13 , wherein the HER2 binding ligand is an antibody or an affibody.

Assignees

• Harvard College

Inventors

• Mechaly Adva
• Mccluskey Andrew J
• Collier R John

Classifications

• C07K16/2863

  against receptors for growth factors, growth regulators · CPC title

• C07K2318/00

  Antibody mimetics or scaffolds · CPC title

• C07K14/34

  from Corynebacterium (G) · CPC title

• C07K2317/73

  Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title