Conjugating amines

Patent metadata

Field	Value
Publication number	US-9616139-B2
Application number	US-201214128958-A
Country	US
Kind code	B2
Filing date	Jul 11, 2012
Priority date	Jul 12, 2011
Publication date	Apr 11, 2017
Grant date	Apr 11, 2017

Abstract

The disclosure provides directly conjugated polysaccharide vaccine molecules and methods related thereto.

First claim

What is claimed is:

1. A conjugate molecule comprising:

wherein P is a protein, PS is a polysaccharide from lipopolysaccharide comprising an O-polysaccharide (O-PS) and a core (O-PS-Core), and the conjugate molecule is prepared by a method comprising conjugation of the O-PS-Core directly to the alkyl squarate via a free amine inherently present in the O-PS-Core such that there are no intervening linkers between the P, squarate, and O-PS-Core.

2. The conjugate molecule of claim 1 , wherein PS is a bacterial polysaccharide or an antigenic fragment thereof comprising an O-PS and an O-PS-Core, and P is a carrier protein.

3. The conjugate molecule of claim 1 , wherein PS is a detoxified bacterial polysaccharide or a fragment thereof comprising an O-PS and an O-PS-Core.

4. The conjugate molecule of claim 1 , wherein PS comprises one or more of Escherichia coli O-PS-Core, Shigella O-PS-Core, Enterobacteriaceae O-PS-Core, Pseudomonas sp. O-PS-Core, P. aeruginosa O-PS-Core, Moraxella sp. O-PS-Core, Helicobacter O-PS-Core, Stenotrophomonas O-PS-Core, Bdellovibrio O-PS-Core, acetic acid bacteria O-PS-Core, Legionella O-PS-Core, Wolbachia O-PS-Core, cyanobacteria O-PS-Core, Spirochaetes O-PS-Core, green sulfur bacteria O-PS-Core, green non-sulfur bacteria O-PS-Core, Neisseria sp. O-PS-Core, N. gonorrhoeae O-PS-Core, Meningitis sp. O-PS-Core, N. meningitides O-PS-Core, Moraxella O-PS-Core, M. catarrhalis O-PS-Core, Hemophilus sp. O-PS-Core, H. influenza O-PS-Core, Klebsiella sp. O-PS-Core, K. pneumonia O-PS-Core, Legionella sp. O-PS-Core, L. pneumophila O-PS-Core, Proteus mirabilis O-PS-Core, Enterobacter cloacae O-PS-Core, Serratia marcescens O-PS-Core, Helicobacter sp. O-PS-Core, H. pylori O-PS-Core, Salmonella sp. O-PS-Core, S. enteritidis O-PS-Core, Salmonella typhi O-PS-Core, Acinetobacter baumannii O-PS-Core, V. cholera O-PS-Core, V. cholerae Inaba O-PS-Core, V. cholerae Ogawa O-PS-Core, an antigenic fragment comprising an O-PS and an O-PS-Core of the one or more polysaccharides, or any combination thereof.

5. The conjugate molecule of claim 1 , wherein PS is V. cholerae O-PS-Core or an antigenic fragment thereof comprising an O-PS and an O-PS-Core, V. cholerae Inaba O-PS-Core or an antigenic fragment thereof comprising an O-PS and an O-PS-Core, V. cholerae Ogawa O-PS-Core or an antigenic fragment thereof comprising an O-PS and an O-PS-Core, or any combination thereof.

6. A method of manufacturing the conjugate molecule of claim 1 , the method comprising:
(a) treating a polysaccharide from lipopolysaccharide comprising an O-polysaccharide (O-PS) and a core (O-PS-Core) having at least one accessible amine group with an alkyl squarate in the presence of a first buffer to manufacture a polysaccharide squarate monoester; and
(b) treating the polysaccharide squarate monoester with the protein in the presence of a second buffer, to thereby manufacture the conjugate molecule wherein there are no intervening linkers between the P, squarate, and O-PS-Core.

7. The method of claim 6 , wherein the first buffer comprises a buffer of about pH 7.0.

8. The method of claim 6 , wherein the second buffer comprises a buffer of about pH 9.0.

9. The method of claim 6 , wherein the first buffer comprises a phosphate buffer.

10. The method of claim 6 , wherein the second buffer comprises a borate buffer.

11. The method of claim 6 , wherein the alkyl squarate is selected from the group consisting of dimethyl squarate, diethyl squarate, dipropyl squarate, dibutyl squarate, and didecyl squarate.

12. The method of claim 6 , wherein the alkyl squarate is dimethyl squarate.

13. The method of claim 6 , wherein the molar ratio of polysaccharide to the alkyl squarate is between about 1:1 to about 50:1.

14. The method of claim 6 , wherein the molar ratio of polysaccharide squarate monoester to the protein is between about 1:1 to about 50:1.

15. A conjugate molecule comprising:

wherein P is tetanus toxin or a fragment thereof, PS is a V. cholerae O-PS-Core, and the conjugate molecule is prepared by a method comprising conjugation of the O-PS-Core directly to the alkyl squarate via a free amine inherently present in the O-PS-Core such that there are no intervening linkers between the P, squarate, and O-PS-Core.

16. The conjugate molecule of claim 15 , wherein P comprises Tetanus Toxin C-fragment.

17. The conjugate molecule of claim 15 , wherein PS comprises V. cholerae Inaba O-PS-Core, and/or V. cholerae Ogawa O-PS-Core.

18. The conjugate molecule of claim 15 , wherein PS comprises V. cholerae O1 Ogawa O-PS-Core.

Assignees

• Ryan Edward T
• Kovac Pavol
• Qadri Firdausi
• Xu Peng
• Calderwood Stephen B
• Vann Willie Frank
• Peterson Dwight Christopher
• Massachusetts Gen Hospital
• The United States As Represented By The Secretary Dept Of Health And Human Services
• Int Centre For Diarrhoeal Disease Res Bangladesh

Inventors

• Ryan Edward T
• Kovac Pavol
• Qadri Firdausi
• Xu Peng
• Calderwood Stephen B
• Vann Willie Frank
• Peterson Dwight Christopher

Classifications

• C08H1/00

  Macromolecular products derived from proteins (food proteins A23; glue, gelatine C09H) · CPC title

• A61K2039/627

  characterised by the linker · CPC title

• A61P31/00

  Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics · CPC title

• C08L89/00

  Compositions of proteins; Compositions of derivatives thereof (foodstuff preparations A23J3/00) · CPC title

• A61K2039/6037

  Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT] · CPC title