Methods for making targeted protein toxins by sortase-mediated protein ligation

Patent metadata

Field	Value
Publication number	US-2016102332-A1
Application number	US-201514727017-A
Country	US
Kind code	A1
Filing date	Jun 1, 2015
Priority date	Dec 3, 2012
Publication date	Apr 14, 2016
Grant date	—

Abstract

We described novel methods for making targeted protein toxins by sortase-mediated protein ligation. The methods allow for a toxin and receptor-binding ligand to be ligated under mild conditions in vitro, following their expression and purification as single entities. The methods also provide a much more efficient way of making functional targeted fusion toxins compared to recombinant or chemical production of these structures.

First claim

We claim:

1 . A method for making a protein toxin comprising a receptor-binding ligand, the method comprising the steps of:
a. providing a protein toxin substrate and a C-terminal sortase-recognition motif optionally followed by an affinity epitope that is separated from the toxin by a linker; b. providing a targeting moiety comprising an N-terminal peptide; and c. contacting the protein toxin substrate of step (a) with the targeting moiety of step (b) with a sortase enzyme.

2 . The method of claim 1 , wherein the protein toxin substrate does not comprise its natural receptor binding domain or comprises a non-functional natural receptor binding domain.

3 . The method of claim 1 , wherein the targeting moiety is a receptor targeting ligand.

4 . The method of claim 1 , wherein the receptor is HER 2.

5 . The method of claim 3 , wherein the receptor targeting ligand is a HER2 antibody or HER2 AFFIBODY.

6 . The method of claim 1 , wherein the C-terminal sortase recognition motif is a sortase A recognition motif.

7 . The method of claim 6 , wherein the sortase A recognition motif is LPXTG (SEQ ID NO: 1), wherein X is any amino acid.

8 . The method of claim 7 , wherein the sortase A recognition motif is LPETGG (SEQ ID NO: 2).

9 . The method of claims 1 , wherein the C-terminal sortase recognition motif is a sortase B recognition motif.

10 . The method of claim 9 , wherein the sortase B recognition motif is NPQTN (SEQ ID NO: 3) or NPKTG (SEQ ID NO: 4).

11 . The method of claim 1 , wherein the affinity epitope is selected from a Histidine repeat (His 6 ) (SEQ ID NO: 5), maltose binding protein (MBP), protein A (ProtA), glutathione S-transferase (GST), calmodulin binding peptide (CBP), calmodulin, thioredoxin, Strep-tags, hemagglutinin, biotin, FLAG, V5, and c-myc.

12 . The method of claim 1 , wherein the linker comprises at least one Glycine-Serine repeat.

13 . The method of claim 12 , wherein the linker comprises 1-10 Glycine-Serine repeats (SEQ ID NO: 6).

14 . The method of claim 13 , wherein the linker comprises 3 or 4 Glycine-Serine repeats (SEQ ID NO: 7).

15 . The method of claim 1 , wherein the N-terminal peptide consists of more than two Glycine residues.

16 . The method of claim 15 , wherein the N-terminal peptide consists of 3-10 Glycine residues (SEQ ID NO: 8).

17 . The method of claim 16 , wherein the N-terminal peptide consists of five Glycine residues (SEQ ID NO: 9).

18 . The method of claim 1 , wherein the protein toxin is an anthrax toxin.

19 . The method of claim 1 , wherein the protein toxin is a diphtheria toxin.

20 . The method of claim 1 , further comprising the step of purifying the toxin that comprises a receptor-binding ligand.

21 . The method of claim 20 , wherein the step of purifying comprises sequential Ni 2+ -NTA affinity and size exclusion chromatography.

Assignees

• Harvard College

Inventors

• Collier R John
• Mccluskey Andrew J
• Pentelute Bradley L

Classifications

• C12P21/00 Primary

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• A61K47/6817

  Toxins · CPC title

• C07K2319/01

  containing a localisation/targetting motif · CPC title

• C07K2319/20

  containing a tag with affinity for a non-protein ligand · CPC title

• C07K2319/55

  containing a fusion with a toxin, e.g. diphteria toxin · CPC title