Methods of producing aggregate-free monomeric diphtheria toxin fusion proteins and therapeutic uses

Patent metadata

Field	Value
Publication number	US-11965009-B2
Application number	US-202117549845-A
Country	US
Kind code	B2
Filing date	Dec 13, 2021
Priority date	Mar 10, 2016
Publication date	Apr 23, 2024
Grant date	Apr 23, 2024

Abstract

The present invention is a DNA expression vector comprising: a toxP: a mutant toxO that blocks Fe-mediated regulation of gene expression; and a DNA sequence encoding a protein, wherein the toxP and the mutant toxO regulate expression of the DNA segment encoding the protein. It is preferred that DNA expression vectors of the present invention include DNA sequences encoding a signal peptide so that a protein expressed is attached to the signal peptide prior to processing. Novel proteins are produced off of the DNA expression vector of the present invention.

First claim

What is claimed is:

1. A method for producing aggregate-free, full-length, monomeric diphtheria toxin fusion proteins comprising:
(a) transforming bacteria with a DNA expression vector comprising:
(i) a toxP;
(ii) a mutant toxO that blocks Fe-mediated regulation of gene expression; and
(iii) a DNA sequence encoding a protein,
wherein the toxP and the mutant toxO regulate expression of the DNA sequence encoding the protein;

(b) incubating the transformed bacteria in a culture medium to allow expression of the protein that is secreted into the culture medium; and
(c) purifying the protein from the culture medium wherein the diphtheria toxin fusion protein is selected from the any one of SEQ ID NO:12, SEQ ID NO: 14 and SEQ ID NO:15.

2. The method of claim 1 , wherein the bacteria is Corynebacterium diphtheria.

3. A method for producing aggregate-free, full-length, monomeric diphtheria toxin fusion proteins comprising:
(a) transforming Corynebacterium diphtheria strain with a DNA vector comprising:
(i) a toxP;
(ii) a mutant toxO that blocks Fe-mediated regulation of gene expression;
(iii) a DNA sequence encoding a protein comprising:
a) a signal peptide;
b) diphtheria receptor binding domain or has a non-functional diphtheria toxin receptor binding domain; and
c) an IL-2 target receptor binding domain,
wherein the toxP and the mutant toxO regulate expression of the DNA sequence encoding the protein;

(b) incubating the transformed bacteria in a culture medium to allow expression of the protein and that is secreted into the culture medium; and
(c) purifying the diphtheria toxin fusion protein from the culture medium,
wherein the diphtheria toxin fusion protein is selected from the any one of SEQ ID NO:12, SEQ ID NO: 14 and SEQ ID NO:15.

4. The method of claim 3 , wherein the Corynebacterium diphtheria strain is Corynebacterium C7 beta (−), tox (−).

5. A method of making a protein comprising:
(a) providing a DNA expression vector comprising:
(i) a toxP;
(ii) a mutant toxO that blocks Fe-mediated regulation of gene expression;
(iii) a signal sequence, and
a DNA sequence encoding a protein;

(b) transforming a bacteria strain with the DNA expression vector to form a transformant;
(c) incubating the transformant in a culture medium for a period of time to allow expression of a protein that is secreted into the culture medium; and
(d) purifying the protein from the culture medium,
wherein the protein is selected from any one of SEQ ID NO:12, SEQ ID NO: 14 and SEQ ID NO:15.

6. A method for producing an aggregate-free, full-length, monomeric diphtheria toxin fusion proteins comprising:
(a) transforming Cornyebacterium diptheria strain with a DNA expression vector comprising:
(i) a toxP;
(ii) a mutant toxO that blocks Fe-mediated regulation of gene expression; and
(iii) a DNA sequence encoding a protein comprising:
a) signal peptide;
b) diptheria receptor binding domain or has a non-functional diptheria receptor binding domain, and
c) a target receptor binding domain selected from the group comprising IL-3, IL-4, IL-6, IL-7, IL-15, EGF, FGF, substance P, CD4, αMSH, GRP, TT fragment C, GCSF, heregulin β1, TNFα, TGFβ, a functional part thereof, or a combination thereof;
wherein the toxP and the mutant toxO regulate expression of the DNA sequence encoding the protein;

(b) incubating the transformed bacteria in a culture medium to allow expression of the protein that is secreted into the culture medium; and
(c) purifying the diphtheria toxin fusion protein from the culture medium,
wherein the diphtheria toxin fusion protein is selected from the any one of SEQ ID NO:12, SEQ ID NO: 14 and SEQ ID NO:15.

7. The method of claim 6 , wherein the Corynebacterium diphtheria strain is Corynebacterium diphtheria C7 beta (−), tox (−).

8. The method of claim 6 , wherein the target receptor binding domain is IL-3 or a functional part thereof.

9. The method of claim 6 , wherein the target receptor binding domain is IL-4 or a functional part thereof.

10. The method of claim 6 , wherein the target receptor binding domain is IL-6 or a functional part thereof.

11. The method of claim 6 , wherein the target receptor binding domain is IL-7 or a functional part thereof.

12. The method of claim 6 , wherein the target receptor binding domain is IL-15 or a functional part thereof.

13. The method of claim 6 , wherein the target receptor binding domain is EGF or a functional part thereof.

14. The method of claim 6 , wherein the target receptor binding domain is FGF or a functional part thereof.

15. The method of claim 6 , wherein the target receptor binding domain is substance P or a functional part thereof.

16. The method of claim 6 , wherein the target receptor binding domain is CD4 or a functional part thereof.

17. The method of claim 6 , wherein the target receptor binding domain is αMSH, GRP, TT fragment C, GCSF, heregulin β1, a functional part thereof, or a combination thereof.

18. The method of claim 6 , wherein the target receptor binding domain is TNFα or a functional part thereof.

19. The method of claim 6 , wherein the target receptor binding domain is TGFβ or a functional part thereof.

Assignees

• Univ Johns Hopkins
• Univ Boston

Inventors

• Bishai William R
• Murphy John R
• Cheung Laurene
• Gupta Shashank
• Bullen Cynthia K

Classifications

• C07K14/55 Primary

  IL-2 · CPC title

• C07K14/34

  from Corynebacterium (G) · CPC title

• C12N15/77 Primary

  for Corynebacterium; for Brevibacterium · 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

• C07K2319/02

  containing a signal sequence · CPC title