Method for rapid in vitro synthesis of glycoproteins via recombinant production of N-glycosylated proteins in prokaryotic cell lysates
Inventors
Jewett, Michael Christopher • Stark, Jessica Carol • Delisa, Matthew P. • Jaroentomeechai, Thapakorn
Assignees
Cornell University • Northwestern University
Publication Number
US-12365930-B2
Publication Date
2025-07-22
Expiration Date
2037-07-14
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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.
Core Innovation
The invention discloses methods, systems, components, and compositions for cell-free synthesis of glycosylated proteins, particularly N-glycosylated recombinant protein carriers. These glycosylated proteins may include bacterial polysaccharides conjugated to a carrier protein, such as FDA-approved carrier proteins, for use in vaccines including antibacterial vaccines. The glycoproteins are synthesized in vitro in prokaryotic cell lysates enriched with components essential for glycoprotein synthesis, such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs), including those associated with bacterial O antigens.
The problem addressed is the limitations of current protein glycan coupling technology (PGCT) for bioconjugate vaccine production, which relies on living Escherichia coli cells for conjugation of polysaccharides to carrier proteins. PGCT is constrained by lengthy in vivo process development timelines and by the lack of demonstrated compatibility of FDA-approved carrier proteins, such as toxins from Clostridium tetani and Corynebacterium diphtheriae, with N-linked glycosylation in living E. coli. The invention solves these issues by employing a modular cell-free glycoprotein synthesis (CFGpS) technology that enables rapid, in vitro production of bioconjugate vaccines in prokaryotic cell lysates within 20-hour reactions.
The modularity of the CFGpS system allows for mixing lysates enriched separately with OSTs and bacterial O antigen LLOs, enabling user specification of carrier proteins and polysaccharide antigens for vaccine production. The system is amenable to lyophilization for storage and distribution, facilitating on-demand and portable vaccine synthesis, including in resource-limited settings. This approach reduces production time from weeks to days, addresses limitations affecting carrier protein compatibility, and enables rapid prototyping and production of novel bioconjugate vaccine candidates.
Claims Coverage
The patent claims 21 inventive features focused on a method for synthesizing N-glycosylated carrier proteins in vitro, the enriched lysates and reaction mixtures used, and kits comprising such components.
Coordinated cell-free synthesis of N-glycosylated carrier protein
A method comprising providing in a single vessel a cell-free transcription, translation, and glycosylation reaction mixture that contains a transcription template encoding a carrier protein with an inserted N-X-S/T consensus sequence (excluding proline at X), exogenous lipid-linked oligosaccharides (LLOs) with bacterial O-antigens, exogenous oligosaccharyltransferase (OST) enzymes transferring the O-antigen to the carrier protein, and lysates enriched in LLOs and OSTs produced from engineered Escherichia coli strains; followed by transcription, translation, and glycosylation of the carrier protein in vitro.
Use of bacterial O-antigens from pathogenic strains
The method specifies bacterial O-antigens as originating from Escherichia coli or Franciscella tularensis species for glycosylation of carrier proteins in vitro.
Formulation of glycosylated proteins as antigenic or vaccine compositions
Post-synthesis, the N-glycosylated carrier proteins can be formulated as antigenic compositions or vaccines, optionally including adjuvants to enhance immune response.
Selection of carrier proteins compatible with N-glycosylation
Carrier proteins include engineered variants of E. coli maltose binding protein, detoxified variants of toxins from Clostridium tetani and Corynebacterium diphtheriae, Haemophilus influenzae protein D, and Neisseria meningitidis porin protein PorA variants, each comprising the N-X-S/T consensus sequence for glycosylation.
Diverse oligosaccharyltransferase (OST) usage
The glycosylation step can use a naturally occurring bacterial homolog of Campylobacter jejuni PglB, engineered variants thereof, archaeal OSTs, or naturally occurring single-subunit eukaryotic OSTs, facilitating flexibility in enzymatic glycosylation.
Engineering of E. coli strains for optimized lysates
The engineered E. coli strains producing lysates possess inactivating modifications or deletions in endogenous genes such as waaL to enrich for LLOs and OSTs and reduce undesired ligase activity, improving cell-free synthesis efficiency.
Control of endotoxin content in lysates
Cell lysates used have controlled endotoxin levels, with an endotoxin unit concentration below approximately 180,000 EU/ml, relevant for vaccine safety and regulatory compliance.
Provision of kits containing enriched lysates and templates
Kits comprise one or more cell lysates enriched in orthogonal OSTs and O-antigens and a transcription template encoding carrier proteins with N-X-S/T consensus sequences, enabling in vitro synthesis of glycosylated proteins.
Preparation of lysates from single or multiple source strains
Kits may include either a single lysate containing both OSTs and O-antigens from the same source strain or multiple lysates from different strains separately enriched in OSTs or O-antigens for mixing during synthesis.
Overexpression of glycosylation pathway components in source strains
Source strains used for producing lysates overexpress genes encoding orthogonal OSTs and/or synthetic glycosyltransferase pathways to produce O-antigens, enhancing lysate capacity for glycoprotein synthesis.
Cell-free reaction mixtures containing synthesized N-glycosylated proteins
The claims cover cell-free reaction mixtures comprising the synthesized N-glycosylated carrier proteins produced in vitro by coordinated transcription, translation, and glycosylation using enriched lysates from engineered E. coli strains.
Vaccination methods using synthesized glycoproteins
Methods of vaccinating subjects against bacterial O-antigens through administration of such cell-free reaction mixtures or purified glycosylated carrier proteins are claimed, optionally including vaccine formulations with adjuvants.
The claims collectively cover modular methods and compositions for rapid, cell-free in vitro synthesis of N-glycosylated carrier proteins using enriched prokaryotic lysates from engineered E. coli strains, kits for performing such synthesis, and the use of resulting glycoproteins in vaccine formulations and vaccination protocols.
Stated Advantages
Enables rapid production and prototyping of bioconjugate vaccines, reducing development time from weeks to days.
Overcomes limitations of in vivo glycosylation such as compatibility issues with FDA-approved carrier proteins and lengthy cell growth timelines.
Modular design allows flexible mixing of lysates enriched with different glycosylation components for user-specified glycan and protein combinations.
Potential for portable, on-demand vaccine production facilitated by lyophilization and room temperature storage of reaction components.
Reduces metabolic burden related to glycosylation in living cells by performing glycoprotein synthesis in vitro.
Enables production of antibacterial vaccines for various pathogenic strains using known surface antigen gene clusters.
Documented Applications
Production of bioconjugate vaccines against pathogenic bacterial strains including Escherichia coli and Franciscella tularensis.
Rapid prototyping and development of novel bioconjugate vaccine candidates with various carrier proteins and glycan antigens.
On-demand, portable vaccine production in resource-poor or remote settings via lyophilized cell-free glycoprotein synthesis systems.
Use of synthesized N-glycosylated proteins as immunogens formulated into vaccines, optionally combined with adjuvants.
Vaccination of subjects in need using cell-free produced bioconjugate vaccines targeting bacterial O-antigens.
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