OprF/I fusion proteins, their preparation and use

Patent metadata

Field	Value
Publication number	US-9359412-B2
Application number	US-201214005579-A
Country	US
Kind code	B2
Filing date	Mar 19, 2012
Priority date	Mar 18, 2011
Publication date	Jun 7, 2016
Grant date	Jun 7, 2016

Abstract

The present invention relates to a novel trimeric OprF/I fusion protein comprising a portion of the Pseudomonas aeruginosa outer membrane protein F which is fused with its carboxy terminal end to a portion of the amino terminal end of the Pseudomonas aeruginosa out membrane protein I, wherein said portion of the Pseudomonas aeruginosa outer membrane protein F comprises the amino acids 190-342 of SEQ ID NO: 1 and wherein said portion of the Pseudomonas aeruginosa outer membrane protein I comprises the amino acids 21-83 of SEQ ID NO: 2, and further to a novel Opr F/I fusion protein which contains a disulphide bond pattern, preferably selected from the group consisting of (a) Cys18-Cys27-bond, (b) Cys18-Cys27-bond and Cys33-Cys47-bond, and (c) Cys18-Cys47 and Cys27-Cys33-bond, and to immunogenic variants thereof having at least 85% identity to the amino acid sequence of SEQ ID NO: 3. The present invention also relates to a novel method for producing said OprF/I fusion proteins and to their use for the preparation of a pharmaceutical composition and for the preparation of antibodies or antibody derivatives which specifically bind said novel OprF/I fusion proteins.

First claim

The invention claimed is:

1. A protein complex comprising a mixture of three OprF/I fusion proteins, wherein the OprF/I fusion proteins comprise
(a) the OprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond (SEQ ID NO: 9),
(b) the OprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID NO: 10), and
(c) the OprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11).

2. The complex of claim 1 , wherein at least 75% of the OprF/I fusion proteins in the mixture comprise (a) the OprF/I fusion protein with a Cys18-Cys27-bond (SEQ ID NO: 9), (b) the OprF/I fusion protein with a Cys18-Cys27-bond and a Cys33-Cys47-bond (SEQ ID NO: 10) and (c) the OprF/I fusion protein of with a Cys18-Cys47-bond and a Cys27-Cys33-bond (SEQ ID NO: 11).

3. A method for producing an OprF/I fusion protein, said method comprising the steps of
(a) reducing an OprF/I fusion protein with a reducing agent, and
(b) oxidizing the reduced OprF/I fusion protein with a redox agent, in the presence of the reducing agent;
wherein the OprF/I fusion protein is the OprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond (SEQ ID NO: 9), the OprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID NO: 10), or the OprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11).

4. The method according to claim 3 , wherein the concentration of the reducing agent of step (a) is from about 3 mM to about 10 mM and the reducing agent is dithiothreitol (DTT), dithioerythritol (DTE) or β-mercaptoethanol.

5. The method according to claim 3 , wherein the concentration of the redox agent of step (b) is from about 0.2 mM to about 4 mM; the redox agent is glutathione disulfide/glutathione or cystine/cysteine; the concentration of the reducing agent of step (b) is from about 0.375 mM to about 1.5 mM; and the reducing agent is dithiothreitol (DTT), dithioerythritol (DTE) or β-mercaptoethanol.

6. The method according to claim 3 , wherein the reaction temperature is from about 18° C. to about 25° C.

7. A pharmaceutical composition, comprising the protein complex according to claim 1 , and at least one additive or adjuvant.

8. The pharmaceutical composition according to claim 7 , wherein the additive or adjuvant is aluminium hydroxide.

9. The pharmaceutical composition according to claim 7 , wherein the pharmaceutical composition is formulated in an isotonic phosphate buffer saline solution at pH 7.4.

10. A vaccine, comprising the protein complex according to claim 1 and at least one additive or adjuvant.

11. The vaccine according to claim 10 , wherein the additive or adjuvant is aluminium hydroxide.

12. The vaccine according to claim 10 , wherein the vaccine is formulated in an isotonic phosphate buffer saline solution at pH 7.4.

Assignees

• Schlegl Robert
• Valneva Austria Gmbh

Inventors

• Schlegl Robert

Classifications

• C07K16/1214 Primary

  Pseudomonadaceae (F) · CPC title

• C07K14/21 Primary

  from Pseudomonadaceae (F) · CPC title

• C07K2319/02

  containing a signal sequence · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C07K2319/21

  containing a His-tag · CPC title