Prodrug activation in cancer cells using molecular switches

Patent metadata

Field	Value
Publication number	US-9469841-B2
Application number	US-201414291925-A
Country	US
Kind code	B2
Filing date	May 30, 2014
Priority date	Aug 13, 2008
Publication date	Oct 18, 2016
Grant date	Oct 18, 2016

Abstract

The present invention features a novel protein engineering strategy by combining the domains of two independent proteins into a molecular switch. The invention features polypeptides comprising a prodrug activating enzyme and a protein that binds a cancer specific marker, polynucleotides encoding the polypeptides, and molecular switches for converting a prodrug into a toxin, comprising the polypeptides. The invention also features methods for converting a prodrug into a toxin, methods for treating cancer, and methods for making the molecular switches, as well as kits.

First claim

What is claimed is:

1. A method to convert a prodrug into a toxin in a cell that expresses HIF-1a comprising:
expressing a polypeptide comprising (A) a prodrug activating enzyme selected from the group consisting of cytosine deaminase, thymidine kinase, nitroreductase, carboxypeptidase A, cytochrome P450, beta-glucosidase and beta-lactamase and (B) a peptide comprising a CH1 domain of a p300, the amino acid residues 9-102 of SEQ ID NO: 1, or the amino acid residues 9-100 of SEQ ID NO: 2 in the cell; and
treating the cells with a prodrug selected from the group consisting of: fluorocytosine (5-FC), ganciclovir, 5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954), methotrexate-alanine, ifosfamide, amygdalin and cephalosporin-derivatized prodrugs,

wherein the peptide binds to HIF-1a in the cell and activates the prodrug activating enzyme, thereby converting the prodrug into a toxin.

2. The method of claim 1 , wherein the prodrug activating enzyme is cytosine deaminase and the prodrug is fluorocytosine (5-FC).

3. The method of claim 1 , wherein the CH1 domain of the p300 protein comprises the amino acid residues 9-102 of SEQ ID NO: 1.

4. The method of claim 1 , wherein the CH1 domain of the p300 protein comprises the amino acid residues 9-100 of SEQ ID NO: 2.

5. The method of claim 1 , wherein said polypeptide is expressed from a vector in said cell.

6. A method to convert fluorocytosine (5-FC) into 5-fluorouracil (5-FU) in a cell that expresses a cancer specific marker, wherein the marker is HIF-1a, comprising:
expressing a polypeptide comprising a cytosine deaminase (CD) and a CH1 domain of a p300 in a cell, wherein CH1 domain of a p300 comprises the amino acid residues 9-102 of SEQ ID NO: 1 in the cell; and
treating the cells with fluorocytosine (5-FC),

wherein the CH1 domain of p300 binds to HIF-1a and activates cytosine deaminase in the cells, thereby converting the fluorocytosine (5-FC) into 5-fluorouracil (5-FU).

7. A method to convert 5-FC into 5-fluorouracil (5-FU) in a cell that expresses a cancer specific marker, wherein the marker is HIF-1a, comprising:
expressing a polypeptide comprising a cytosine deaminase (CD) and CH1 domain of a p300 in a cell, wherein the CH1 domain of a p300 comprises the amino acid residues 9-100 of SEQ ID NO: 2 in the cell; and
treating the cells with 5-FC,

wherein the CH1 domain binds to HIF-1a and activates cytosine deaminase in the cells, thereby converting 5-FC into 5-FU.

Assignees

• Univ Johns Hopkins

Inventors

• Ostermeier Marc A
• Wright Chapman M

Classifications

• C12N9/0077

  with a reduced iron-sulfur protein as one donor (1.14.15) · CPC title

• C12Y207/01021

  Thymidine kinase (2.7.1.21) · CPC title

• C12N9/78

  acting on carbon to nitrogen bonds other than peptide bonds (3.5) · CPC title

• C12N9/0036

  acting on NADH or NADPH (1.6) · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title