ITHACA, NY, United States
ITHACA, NY, United States

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Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 197.76K | Year: 2013

DESCRIPTION: 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 13 billion of annual pharmaceutical salesand are part of a growing sector of the biopharmaceutical market. Unfortunately, therapeutic peptides typically suffer from poor stability and short half-lives in the human body, which limits their value. The requirement for high dosing and frequent injections follows, which can be inconvenient, expensive, and dangerous for patients. While there have been methods developed to address these issues, they either: (i) hinge on 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 oligosaccharides that are nonimmunogenic in the human body. Several therapeutic peptides (e.g., Exenatide, Glucagon-like peptide 1) 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. Glycobia has developed a transformative solution to this growing, unsolved problem by engineering bacteria as a platform for the biosynthesis of therapeutic glycopeptides. These novel strains of Escherichia coli are useful for the expression of recombinant peptides conjugated to nonimmunogenic, human-like oligosaccharides. The hypothesis to be tested here is that non-pathogenic, glycoengineered strains of E. coli can produce affordable recombinant peptide drugs with improved stability, biocompatibility, and prolonged half-life in serum. The main objective of this Phase I project is to identify and characterize peptide drug candidates for animal and preclinical studies in Phase II of this project. This will be accomplished through the following specific aims: (1) express a panel of therapeutic peptide glycoconjugates and screen for expression and glycosylation efficiency; and (2) screen glycoconjugate drug candidates for biophysical properties and in vitro activity. The panel of peptides to be studied here includes seven FDA- approved therapeutic peptides that will be evaluated for expression in the glycoengineered E. coli system. Of these seven peptides, five are currently produced using recombinant expression systems and account for over 2.6 billion in annual sales. Each of the targets will be assayed for solubility, stability, and in vitro activity. This information will be considered along with the commercial potential of each peptide, leading to the identification of candidate peptides for Phase II of this project. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: Therapeutic peptides account for 13 billion of pharmaceutical sales annually and are used to treat human diseases ranging from HIV to diabetes. Many approved and emerging peptide drugs suffer from poor stability and have short half-lives in the humanbody, which can be problematic and dangerous for patients. The goal of this work is to produce stable, soluble therapeutic peptides in glycoengineered Escherichia coli.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.61M | Year: 2011

DESCRIPTION (provided by applicant): Escherichia coli was the host organism for production of the first approved recombinant protein therapeutic in 1982. We now know that most therapeutic proteins require N-linked protein glycosylation to achieve their full clinical efficacy. Since E. coli has not been capable of protein glycosylation, the majority of approved therapeutic proteins are now expressed in mammalian host cells. While mammalian cells can express N-linked glycoproteins, they can have several drawbacks including: (i) slow growth, (ii) expensive media, (iii) long development timelines, (iv) low volumetric productivity, (v) susceptibility to viral contamination, and (vi) product heterogeneity. This problem has not gone unnoticed by the scientific community, and several eukaryotic organisms have been re-engineered for expression of therapeutic glycoproteins. Unfortunately, all eukaryotic hosts - including Chinese hamster ovary cells, plant cells, insect cells, or even genetically engineered yeast - introduce nonhuman glycoforms that arise from native glycosylation pathways. Glycobia specializes in glycoengineering bacteria as a platform for the stereospecific biosynthesis of therapeutic glycoproteins. The specific hypothesis of these proposed studies isthat glycoengineered E. coli can be used to express therapeutic glycoproteins. In Phase I of this project, we engineered E. coli capable of glycosylating proteins with the eukaryotic core glycan (Man3GlcNAc2) that is the predominant glycan in both plant and insect cells. In Phase II of this project, we propose to further engineer E. coli to enable glycosylation of therapeutic proteins with terminally sialylated human glycans. Specifically, we propose to engineer E. coli to glycosylate therapeutic proteins with eukaryotic N-glycans by screening enzymes to: (i) preferentially glycosylate N-X-S/T glycosylation motifs and (ii) efficiently glycosylate therapeutic target proteins with eukaryotic glycans. Further, we propose to engineer E. coli to synthesize and transfer complex terminally sialylated N-glycans by: (i) extending the Man3GlcNAc2 biosynthetic pathway for the biosynthesis of terminally sialylated glycans and (ii) screening enzymes for their ability to transfer the complex human N-glycan to target proteins. The benchmark of success for this project is expression of a commercial glycoprotein in E. coli. This bacterial expression platform represents a transformative solution to the unanswered biomedical challenge of generating cost-effective glycoproteins for both companies and patients. PUBLIC HEALTH RELEVANCE: Most approved therapeutic proteins require posttranslational N-linked protein glycosylation and, as a consequence, are expressed in eukaryotic host cells that can be expensive, susceptible toviral contamination, and prone to product heterogeneity. The outcomes are low profit margins for biotechnology and pharmaceutical companies and prices that are prohibitive to the healthcare consumer. The proposed studies focus on expressing safe, affordable, and controlled complex human glycoproteins in the simple bacterium Escherichia coli.


Grant
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.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 999.90K | Year: 2015

Not Available


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 299.45K | Year: 2013

Glycans are directly involved in the pathology of many major diseases. Glycosylated biomolecules are involved in biological functions including cell signaling, cell growth, immunity, three-dimensional protein folding and target interaction. However, medical benefits resulting from glycobiology are largely untapped because glycans are not well understood. The creation of a comprehensive glycoscience toolkit - including glycan libraries - is essential to drive biomedical research in this area. Such libraries would address the paucity of glycans available for high-throughput screening, substrates for enzymology studies, analytical standards, materials for structural studies, and monitoring in therapeutic manufacturing QA/QC. Glycobia Inc. proposes to useits novel bottom-up glycoengineering technology to produce a variety of functional, chemically defined glycan species. Glycobia shall use engineered Escherichia coli to synthesize the building blocks for many human glycans, and further modify them with glycosyltransferases in vitro. In this project, we shall develop our bottom-up glycoengineering technology could then be used to produce glycans for biomedical applications. PUBLIC HEALTH RELEVANCE


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 1.40M | 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


Disclosed are methods and compositions to produce various oligosaccharide compositions and glycoproteins. Prokaryotic hosts cells are cultured under conditions effective to produce human-like e.g., high-mannose, hybrid and complex glycosylation patterns by introducing glycosylation pathways into the host cells.


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.


Disclosed are methods and compositions to produce various oligosaccharide compositions and glycoproteins. Prokaryotic hosts cells are cultured under conditions effective to produce human-like e.g., high-mannose, hybrid and complex glycosylation patterns by introducing glycosylation pathways into the host cells.


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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