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ITHACA, NY, United States

Ollis A.A.,Cornell University | Zhang S.,Cornell University | Fisher A.C.,Glycobia, Inc. | Delisa M.P.,Cornell University
Nature Chemical Biology | Year: 2014

The Campylobacter jejuni protein glycosylation locus (pgl) encodes machinery for asparagine-linked (N-linked) glycosylation and serves as the archetype for bacterial N-linked glycosylation. This machinery has been functionally transferred into Escherichia coli, enabling convenient mechanistic dissection of the N-linked glycosylation process in this genetically tractable host. Here we sought to identify sequence determinants in the oligosaccharyltransferase PglB that restrict its specificity to only those glycan acceptor sites containing a negatively charged residue at the â '2 position relative to asparagine. This involved creation of a genetic assay, glycosylation of secreted N-linked acceptor proteins (glycoSNAP), that facilitates high-throughput screening of glycophenotypes in E. coli. Using this assay, we isolated several C. jejuni PglB variants that could glycosylate an array of noncanonical acceptor sequences, including one in a eukaryotic N-glycoprotein. These results underscore the utility of glycoSNAP for shedding light on poorly understood aspects of N-linked glycosylation and for engineering designer N-linked glycosylation biocatalysts. © 2014 Nature America, Inc. All rights reserved. Source

Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 636.91K | Year: 2015

DESCRIPTION provided by applicant Therapeutic peptides are used to treat human diseases ranging from HIV to diabetes and have some of the best features of small molecule and recombinant protein drugs Therapeutic peptides account for $ billion of annual pharmaceutical sales and are part of a growing sector of the biopharmaceutical market Unfortunately therapeutic peptides suffer from poor stability and short half lives in the human body which limits their value The requirement for high dosing and frequent injections can be inconvenient expensive and dangerous for patients While there have been methods developed to address these issues they either i hinge on the in vitro or recombinant attachment of a large polymer chain which dramatically impacts peptide activity or ii require in vitro processing steps which increase manufacturing costs and complicate purification It is now well established that the stability and half life of peptide drugs can be greatly improved by conjugation to humanlike oligosaccharides Several therapeutic peptides e g Exenatide Glucagon like peptide have benefitted significantly from glycosylation with small human like glycans by increasing protease resistance prolonging activity and improving biodistribution However this requires multiple complicated in vitro reactions and purifications which have kept this promising concept from reaching the industrial scale At Glycobia we have developed novel strains of Escherichia coli for the expression of recombinant peptides conjugated to humanlike oligosaccharides In Phase I of this project we applied our glycoengineered bacteria as a platform for the biosynthesis of therapeutic glycopeptides by expressing a panel of therapeutic peptide glycoconjugates and screening for glycosylation efficiency and screening glycoconjugate drug candidates for physical properties and in vitro activity We show proof of concept of several recombinant peptides with improved stability and or activity when modified with glycosylation Now having identified lead candidates for Phase II of this project the objective of this proposal is to synthesize and advance our first drug targets from glycoengineered E coli into preclinical testing by expressing scaling up purifying and characterizing a glycosylated human peptide drug from in E coli and testing pharmacology calcemic response and pharmacokinetics of a glycosylated human peptide drug in animal models We will attach two different humanlike glycans to the drug and compare performance to an aglycosylated version of the drug The benchmark of success for this project is the generation of positive preclinical validation data to further advance commercialization of this glycoengineering technology Our bacterial expression platform represents a transformative solution to the unanswered biomedical challenge of producing improved therapeutic peptides for patients PUBLIC HEALTH RELEVANCE Therapeutic peptides account for over $ billion of pharmaceutical sales and are part of a rapidly growing market with at least peptide drug candidates in clinical testing or development Many approved and emerging peptide drugs suffer from poor stability and have short half lives in the human body which can be problematic and dangerous for patients The proposed studies focus on for the first time producing stable longer lasting glycosylated therapeutic peptides in the simple bacterium Escherichia coli and advancing them into preclinical testing

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 391.73K | Year: 2010

DESCRIPTION (provided by applicant): Omalizumab (XolairTM) is a recombinant monoclonal anti-IgE antibody used in the fight against severe allergic asthma that generates 500 million per year. However, 2 out of every 1,000 patients treated with omalizumab suffer from anaphylaxis, a severe allergic reaction to the therapeutic. Moreover, omalizumab immunotherapy is prohibitively expensive, reaching nearly 30,000 per year. Thus, there is a great unmet need for an improved omalizumab composition that is affordable to health care consumers and tolerated in circulation. Genentech currently produces omalizumab in mammalian cell culture, which is expensive and susceptible to viral contamination. Alternatively, Escherichia coli culture is inexpensive, well-characterized, fast-growing, and not susceptible to viral contamination. However, E. coli is not typically used for full-length antibody production and instead is relegated to expression of smaller, engineered antibodies such as antigen binding fragments (Fabs). Fabs bind equally or better to target antigens, but their persistence in the human body can be limited. In spite of these shortcomings, therapeutic antibody fragments are regularly produced in E. coli including Genentech's ranibizumab (LucentisTM), a Fab designed for intraocular use. An emerging solution to prolong the half-life of Fabs in circulation is the covalent attachment of human-type oligosaccharides. Lipid-linked oligosaccharides identical to human blood group ABO O-type antigens are naturally synthesized in E. coli strains of serogroup O86. The hypothesis of this proposal is that non-pathogenic strains of E. coli can be engineered to produce and transfer O-type antigens to specific sites in recombinant Fabs. To test this hypothesis, the objective of this proposal is to generate anti-IgE recombinant antibodies with improved serum tolerance in by: (i) cloning and expressing the biosynthetic machinery for the human blood group O-type oligosaccharide in E. coli K12, (ii) cloning and expressing a recombinant anti-IgE Fab in E. coli, and (iii) conjugating blood group type O-type antigens to anti-IgE Fabs in glycoengineered E. coli. It is anticipated that these studies will result in an efficient E. coli expression platform for the production of anti-IgE Fabs linked to human blood group O-type oligosaccharides in a controlled, rapid, and cost-effective manner. These studies are significant because they explore a paradigm-shifting technology for the production of therapeutic Fabs for the treatment of asthma and other immunological diseases. PUBLIC HEALTH RELEVANCE: Monoclonal antibodies are prominent therapeutics in the fight against many immunological diseases including severe allergic asthma. Unfortunately, the production process for monoclonal antibodies is expensive, resulting in a cost of therapy that is unaffordable for the healthcare consumer. The proposed studies focus on producing well-tolerated therapeutic antibody conjugates in Escherichia coli fermentation without the need for costly mammalian cell culture or in vitro chemical modification.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 175.86K | Year: 2011

DESCRIPTION (provided by applicant): Gaucher's disease is the most common lysosomal storage disease in humans resulting in the harmful accumulation of fatty glucocerebroside in the spleen, liver, lungs, bone marrow, and brain. Gaucher patients exhibit a hereditary deficiency of glucocerebrosidase (GCase), but effective enzyme replacement therapy is available for most patients with Gaucher's disease. Recombinant GCase currently generates over 1 billion in annual revenue, but production of GCase is expensive. The resulting cost leaves many patients unable to afford treatment and health carriers reluctant to underwrite lifelong treatment. Currently, the glycoprotein GCase is expressed in Chinese Hamster ovary (CHO) cells and further processed in vitro to expose requisite terminal mannose residues for biological uptake in human patients. In addition to being expensive, mammalian cell culture is susceptible to viral contamination. In fact, viral contamination has resulted in a severe shortage of GCase that has set back revenues and endangered patients who depend on regular intravenous administration of the drug. This has opened the market for alternative industrial scale GCase expression platforms that are well-characterized, not susceptible to viral contamination, and do not require intricate in vitro chemical modification. Glycobia specializes in glycoengineering bacteria as a platform for the stereospecific biosynthesis of therapeutic glycoproteins. The specific hypothesis of these proposed studies is that glycoengineered Escherichia coli can be used to produce active recombinant GCase without the need for in vitro chemical modification. The advantage of E. coli as a host for GCase expression is that - unlike yeast, CHO, plant or all other eukaryote cells - thereare no native glycosylation pathways to result in uncontrolled glycoforms. We anticipate that an E. coli expression platform will be capable of producing active GCase in a controlled, rapid, and cost-effective manner. The objective of this proposal is togenerate GCase by cloning and expressing the genetic machinery for mannose oligosaccharide synthesis in E. coli (Aim 1) and expressing active GCase in glycoengineered E. coli (Aim 2). This bacterial expression platform represents a transformative solutionto the unanswered biomedical challenge of delivering a cost-effective GCase enzyme replacement therapy to patients. PUBLIC HEALTH RELEVANCE: Glucocerebrosidase enzyme replacement therapy has revolutionized the clinical treatment of Gaucher's disease, but inefficiencies in the production platform have resulted in prohibitive costs to the healthcare consumer. Recombinant glucocerebrosidase is expressed in mammalian cell culture making the process expensive, susceptible to viral contamination, and subject to further in vitro processing of uncontrollable glycoforms. The proposed studies focus on producing active glucocerebrosidase in Escherichia coli fermentation without the need for mammalian cell culture or in vitro chemical modification.

The invention described herein generally relates to glycoengineering host cells for the production of glycoproteins for therapeutic use. Host cells are modified to express biosynthetic glycosylation pathways. Novel prokaryotic host cells are engineered to produce N-linked glycoproteins wherein the glycoproteins comprise polysialic acid or blood group antigens.

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