# US 2017/0283471 A1 · Modified microbial toxin receptor for delivering agents into cells

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- Number: US 2017/0283471 A1
- Document ID: US20170283471A1
- Status: Published application
- Kind: A1
- Application number: 15/621,159
- Filed: 2017-06-13
- Published: 2017-10-05
- Earliest claimed: 2012-02-23
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: Adva Mechaly; Andrew J. McCluskey; R. John Collier
- Web page: https://patentgrep.com/patent/US20170283471A1

## CPC

- C07K 14/32: from Bacillus (G)
- A61P 35/00: Antineoplastic agents
- C07K 14/21: from Pseudomonadaceae (F)
- C07K 14/25: Shigella (G)
- C07K 14/28: from Vibrionaceae (F)
- C07K 14/33: from Clostridium (G)
- C07K 14/34: from Corynebacterium (G)
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/32: against translation products of oncogenes
- C07K 16/40: against enzymes
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2318/00: Antibody mimetics or scaffolds
- C07K 2319/00: Fusion polypeptide
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C07K 2319/55: containing a fusion with a toxin, e.g. diphteria toxin
- C07K 2319/75: containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## 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.

## Claims

### Claim 1

An altered binary toxin system for delivery of an active molecule 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 a target cell, and
a complementary toxin unit capable of associating with the pore-forming toxin unit for delivery of a therapeutic protein to the cytosol of the target cell.

### Claim 2 (depends on 1)

The altered binary toxin system of claim 1, wherein the AB toxin is botulinum neurotoxin, anthrax toxin, diphtheria toxin, ricin, shiga toxin, shiga like toxin, exotoxin A, or cholera toxin.

### Claim 3 (depends on 2)

The altered binary toxin system of claim 2, wherein the receptor-ablated pore-forming unit is derived from the B unit of botulinum neurotoxin, diphtheria toxin, ricin, shiga toxin, shiga like toxin, exotoxin A, or cholera toxin.

### Claim 4 (depends on 3)

The altered binary toxin system of claim 3, wherein the complementary toxin unit is derived from the A unit of Botulinum neurotoxin, diphtheria toxin, ricin, shiga toxin, shiga like toxin, exotoxin A, or cholera toxin.

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 3)

The altered binary toxin system of claim 3, wherein the binary toxin is Clostridium perfringens toxins alpha, beta, epsilon or iota; Clostridium botulinum C2 toxin; or Clostridium spiroforme Iota-like toxin.

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 1)

The altered binary toxin system of claim 1, wherein the receptor-binding ligand 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.

### Claim 10 (depends on 1)

The altered binary toxin system of claim 1, wherein the receptor-binding ligand is selected from an antibody or an affibody.

### Claim 11 (depends on 10)

The altered binary toxin system of claim 10, wherein the affibody is a HER2 affibody.

### Claim 12 (depends on 11)

The altered binary toxin system of claim 11, wherein the HER2 affibody is ZHER2.

### Claim 13 (depends on 1)

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

### Claim 14 (depends on 1)

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

### Claim 15 (depends on 14)

The method of claim 14, wherein the altered binary toxin system comprises a receptor-redirected B unit of botulinum neurotoxin, diphtheria toxin, ricin, shiga toxin, shiga like toxin, exotoxin A, or cholera toxin.

### Claim 16 (depends on 15)

The method of claim 15, wherein the cancer is a HER2 positive cancer and the receptor-redirected B unit of botulinum neurotoxin, diphtheria toxin, ricin, shiga toxin, shiga like toxin, exotoxin A, or cholera toxin is fused with a HER2 binding ligand.

### Claim 17 (depends on 16)

The method of claim 16, wherein the HER2 binding ligand is an antibody or an affibody.

### Claim 18 (depends on 16)

The method of claim 16, wherein the HER2 affibody is ZHER2.

### Claim 19 (depends on 14)

The method of claim 14, wherein the altered binary toxin system comprises:
a therapeutic protein that 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, or
a therapeutic protein that is the catalytic domain of diphtheria toxin (DTA), ricin, shiga toxin, or Pseudomonas exotoxin A.

### Claim 20 (depends on 15)

The method of claim 15, wherein the altered binary toxin system comprises a complementary toxin unit is derived from the A unit of botulinum neurotoxin, diphtheria toxin, ricin, shiga toxin, shiga like toxin, exotoxin A, or cholera toxin.