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

Patent metadata

Field	Value
Publication number	US-2020157166-A1
Application number	US-201716611243-A
Country	US
Kind code	A1
Filing date	Sep 11, 2017
Priority date	Mar 10, 2016
Publication date	May 21, 2020
Grant date	—

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

1 .- 39 . (canceled)

40 . A pharmaceutical composition comprising a polypeptide selected from the group consisting of SEQ ID NOs: 12-15, 30, 38-40, 42-43, 45-46, and 58, and a combination thereof, or a polypeptide encoded by a nucleic acid selected from the group consisting of SEQ ID NOs: 31, 41, 44, and 59, and a combination thereof, and a pharmaceutically acceptable carrier.

41 . The pharmaceutical composition of claim 40 , wherein the composition comprises greater than about 80% purity of said polypeptide.

42 . The pharmaceutical composition of claim 41 , wherein the composition comprises greater than about 85% purity of said polypeptide.

43 . The pharmaceutical composition of claim 42 , wherein the composition comprises greater than about 90% purity of said polypeptide.

44 . The pharmaceutical composition of claim 43 , wherein the composition comprises greater than about 97% purity of said polypeptide.

45 . The pharmaceutical composition of claim 40 , wherein the composition comprises greater than about 80% aggregate-free, full-length, monomeric polypeptide.

46 . The pharmaceutical composition of claim 45 , wherein the composition comprises greater than about 85% aggregate-free, full-length, monomeric polypeptide.

47 . The pharmaceutical composition of claim 46 , wherein the composition comprises greater than about 90% aggregate-free, full-length, monomeric polypeptide.

48 . The pharmaceutical composition of claim 47 , wherein the composition comprises greater than about 97% aggregate-free, full-length, monomeric polypeptide.

49 . A method of making a pharmaceutical composition comprising the following steps:
a. transforming Cornebacterium 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 polypeptide selected from the group consisting of SEQ ID NOs: 12-15, 30, 38-40, 42-43, 45-46, and 58, and a combination thereof, or a polypeptide encoded by a nucleic acid selected from the group consisting of SEQ ID NOs: 31, 41, 44, and 59, and a combination thereof;
b. forming a transformant; c. incubating the transformant in a culture medium to allow expression of the protein and that is secreted into the culture; d. purifying the diphtheria toxin fusion protein from the culture medium; and e. forming a pharmaceutical composition.

50 . The method of claim 49 , wherein the pharmaceutical composition comprises greater than about 80% purity of said polypeptide.

51 . The method of claim 49 , wherein the pharmaceutical composition comprises greater than about 85% purity of said polypeptide.

52 . The method of claim 49 , wherein the pharmaceutical composition comprises greater than about 90% purity of said polypeptide.

53 . The method of claim 49 , wherein the pharmaceutical composition comprises greater than about 97% purity of said polypeptide.

54 . The method of claim 49 , wherein the pharmaceutical composition comprises greater than about 80% aggregate-free, full-length, monomeric polypeptide.

55 . The method of claim 49 , wherein the pharmaceutical composition comprises greater than about 85% aggregate-free, full-length, monomeric polypeptide.

Assignees

• Univ Johns Hopkins
• Univ Boston

Inventors

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

Classifications

• C07K2319/21

  containing a His-tag · CPC title

• C07K2319/034

  containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved · CPC title

• C07K14/34 Primary

  from Corynebacterium (G) · 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

• C07K14/55 Primary

  IL-2 · CPC title