Method for rapid in vitro synthesis of glycoproteins via recombinant production of n-glycosylated proteins in prokaryotic cell lysates

Patent metadata

Field	Value
Publication number	US-2023279460-A1
Application number	US-202218065689-A
Country	US
Kind code	A1
Filing date	Dec 14, 2022
Priority date	Jul 14, 2016
Publication date	Sep 7, 2023
Grant date	—

Abstract

Disclosed are methods, systems, components, and compositions for cell-free synthesis of glycosylated proteins. The glycosylated proteins may be utilized in vaccines, including anti-bacterial vaccines. The glycosylated proteins may include a bacterial polysaccharide conjugated to a carrier, which may be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. The glycosylated proteins may be synthesized in cell-free glycoprotein synthesis (CFGpS) systems using prokaryote cell lysates that are enriched in components for glycoprotein synthesis such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) including OSTs and LLOs associated with synthesis of bacterial O antigens.

First claim

1 . A method for synthesizing an N-glycosylated carrier protein for a polysaccharide via coordinated transcription, translation, and N-glycosylation in vitro, the method comprising:
(i) transcribing and translating a carrier protein in a cell-free protein synthesis reaction, the carrier protein comprising an inserted consensus sequence, N-X-S/T, wherein X may be any natural or unnatural amino acid except proline; (ii) glycosylating the carrier protein in the cell-free protein synthesis reaction with at least one polysaccharide, wherein the at least one polysaccharide is at least one bacterial O-antigen.

2 . The method of claim 1 , wherein the bacterial O-antigen is from E. coli.

3 . The method of claim 1 , wherein the bacterial O-antigen is from Franciscella tularensis.

4 . The method of claim 1 , further comprising formulating the N-glycosylated carrier protein as an antigenic composition comprising the N-glycosylated carrier protein.

5 . The method of claim 1 , further comprising formulating the N-glycosylated carrier protein a vaccine composition comprising the N-glycosylated carrier protein.

5 . The method of claim 1 , wherein the vaccine composition further comprises an adjuvant.

6 . The method of claim 1 , wherein the carrier protein is an engineered variant of E. coli maltose binding protein (MBP).

7 . The method of claim 1 , wherein the carrier protein is selected from a detoxified variant of the toxin from Clostridium tetani , a detoxified variant of the toxin from Corynebacterium diphtheriae, Haemophilus influenzae protein D (PD) or a variant thereof, and Neisseria meningitidis porin protein (PorA) or a variant thereof.

8 . The method of claim 1 , wherein the glycosylating step utilizes an oligosaccharyltransferase (OST) which is a naturally occurring bacterial homolog of C. jejuni PglB.

9 . The method of claim 1 , wherein the glycosylating step utilizes an OST that is an engineered variant of C. jejuni PglB.

10 . The method of claim 1 , wherein the wherein the glycosylating step utilizes an OST that is a naturally occurring archaeal OST.

11 . The method of claim 1 , wherein the wherein the glycosylating step utilizes an OST which is a naturally occurring single-subunit eukaryotic OST.

12 . The method of claim 1 , wherein the cell-free protein synthesis reaction comprises a cell lysate prepared from an engineered bacterial strain that expresses a detoxified lipid A and/or reduced amounts of lipid A.

13 . The method of claim 1 , wherein the cell lysate has an endotoxin unit (EU) concentration of less than about 180,000 EU/ml.

14 . A kit for synthesizing a N-glycosylated carrier protein in vitro, the kit comprising as components:
(i) one or more cell lysates, wherein the one or more cell lysates comprise an orthogonal oligosaccharyltransferase (OST) and an O-antigen; (ii) a transcription template encoding a carrier protein and a polymerase for synthesizing an mRNA from the transcription template, the carrier protein comprising an inserted consensus sequence, N-X-S/T, wherein X may be any natural or unnatural amino acid except proline.

15 . The kit of claim 14 comprising one cell lysate prepared from a single source strain, wherein the orthogonal OST and the O-antigen are present in the one cell lysate.

16 . The kit of claim 14 comprising two cell lysates prepared from two source strains, wherein the orthogonal OST is present in one cell lysate of the two cell lysates and the O-antigen is present in the other cell lysate of the two cell lysates.

17 . The kit of claim 14 , wherein OST is produced from a source strain that overexpresses a gene encoding the orthogonal OST.

18 . The kit of claim 14 , wherein the O-antigen is produced from a source strain that overexpresses a synthetic glycosyltransferase pathway for producing the O-antigen.

19 . (canceled)

20 . A cell-free reaction mixture comprising an N-glycosylated carrier protein, wherein the N-glycosylated carrier protein is synthesized via a method comprising coordinated transcription, translation, and N-glycosylation in vitro, the method comprising:
(i) transcribing and translating a carrier protein in a cell-free protein synthesis reaction mixture, the carrier protein comprising an inserted consensus sequence, N-X-S/T, wherein X may be any natural or unnatural amino acid except proline; (ii) glycosylating the carrier protein in the cell-free protein synthesis reaction mixture with at least one polysaccharide, wherein the at least one polysaccharide is at least one bacterial O-antigen.

21 . A method of vaccinating a subject in need thereof against a bacterial O-antigen, the method comprising administering to the subject the cell-free reaction mixture of claim 20 and/or a purified N-glycosylated carrier protein therefrom.

Assignees

• Univ Northwestern
• Univ Cornell

Inventors

• Jewett Michael Christopher
• Stark Jessica Carol
• Delisa Matthew P
• Jaroentomeechai Thapakorn

Classifications

• C12P21/005 Primary

  Glycopeptides, glycoproteins · CPC title

• A61K38/04

  Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof ({enzyme inhibitors A61K38/005;} gastrins {A61K38/2207}, somatostatins A61K38/31, melanotropins A61K38/34 {; protease inhibitors A61K38/55}) · CPC title

• A61K38/16

  Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof {(enzyme inhibitors A61K38/005)} · CPC title

• C07K14/47

  from mammals · CPC title

• C12N9/10

  Transferases (2.) (ribonucleases C12N9/22) · CPC title