Engineering Escherichia coli for sialylation of therapeutic proteins

DESCRIPTION provided by applicant Glycoengineering is a clinically validated strategy to enhance the therapeutic properties of protein and peptide drugs This strategy involves the attachment and manipulation of carbohydrates i e glycans to improve the stability solubility serum half life and activity of these drugs A key factor in most glycoengineering is the inclusio of terminal sialic acid residues on glycans by a process known as sialylation Sialic acid is large and carries a negative charge which serves to improve stability decrease aggregation slow clearance and impede immune response Nearly all examples of glycoengineering require eukaryotic cell culture and or the in vitro conjugation of glycans Unfortunately eukaryotic cell culture can be expensive time consuming and can result in inconsistent and incomplete sialylation Although in vitro glycosylation can result in similar effects the process is expensiv difficult and has not been scalable to a commercial level Glycoengineering would be greatly improved if a simple host cell such as Escherichia coli was used for production of sialylated therapeutic proteins Glycobia specializes in genetically engineering bacteria for the bottom up glycoengineering BUG of therapeutic glycoproteins Since E coli lacks native protein glycosylation pathways of any kind BUG can produce tailored glycan structures that can be site specifically conjugated to target proteins The specific hypothesis behind this proposal is that glycoengineered E coli can produce enhanced therapeutic proteins by sialylation in a short single fermentation In Phase I of this project we engineered E coli to attach humanlike sialyl T glycans to recombinant proteins The sialyl T glycan is a sialylated Thomsen Friedenreich antigen that can be found on erythrocytes in the human body This type of glycosylation is simply not possible in any other known expression host We also show that bacterial glycosylation improves the in vitro stability of therapeutic proteins expressed in E coli We anticipate that our BUG expression platform will be capable of producing sialylated proteins in a controlled rapid cost effective manner The objective of this proposal is to synthesize and advance our first drug targets from glycoengineered E coli into preclinical testing by i expressing purifying and characterizing glycosylated drug candidates from E coli and ii testing stability pharmacokinetics and immunogenicity of these drug candidates in animal models We will compare their performance to aglycosylated and asialylated versions of these same drugs to isolate the effects of sialylation The benchmark of success for this project is the generation of positive preclinical validation data to further advance commercialization of this technology This bacterial expression platform represents a transformative solution to the unanswered biomedical challenge of generating cost effective glycoengineered protein drugs for both companies and patients PUBLIC HEALTH RELEVANCE Sialylation is a glycoengineering strategy to improve the therapeutic properties of protein and peptide drugs by the addition of sialic acid residues Sialic acid improves drug stability decreases aggregation slows clearance and hinders immune response The proposed studies focus on for the first time producing sialylated recombinant therapeutics in the simple bacterium Escherichia coli and advancing them into preclinical testing