The potential impact of monoclonal antibodies on human health through the treatment of a wide range of human diseases has led to accelerated development of these molecules for clinical use. However, current manufacturing of therapeutic antibodies is limited to mammalian cell-culture systems that, despite intense and continuing efforts, suffer from safety concerns, product heterogeneity and high costs of production. This application seeks to address each of these concerns by creating an alternative microbial expression platform for the production of monoclonal antibodies in the common pond water ciliate, Tetrahymena thermophila. Successful development of this platform will allow Tetragenetics Inc to make this alternative manufacturing platform widely available to the pharmaceutical and contract manufacturing industry for the production of this increasingly important class of therapeutics. Compared to mammalian expression systems, the production of therapeutic recombinant proteins in microbes offers higher levels of safety due to growth in animal-free media and significant reductions in production costs due to higher growth rates and the economies of scale. Despite these advantages, the application of these systems for the production of full-length monoclonal antibodies has not been adopted due to inefficient folding and assembly of these tetrameric molecules in bacteria, and the addition of non-human, potentially antigenic, N-linked glycans in yeast. Neither of these drawbacks applies to Tetrahymena, which enjoys all the advantages common to microbial expression systems and additionally are able to efficiently assemble multi-subunit eukaryotic proteins that may be modified with primitive N-glycans that comprise the trimannosyl core of human N-linked carbohydrates and are therefore unlikely to be antigenic. In the current application Tetragenetics Inc will apply it's current expression technology to the production of human subtypes of the most therapeutically relevant immunoglobulin family, IgG. Recent expression of a murine IgG1 antibody resulted in abundant expression of a correctly folded and functional antibody with an unanticipated lack of glycosylation. Aglycosylated antibodies comprise an increasingly important subset of therapeutic antibodies where target cell depletion is not needed and indeed may be deleterious to a treatment regimen. Therefore, the first set of experiments will determine the broad applicability of the current expression strategy for the production of aglycosylated IgG subtype antibodies. Secondly, a method will be developed that results in rational N-linked glycosylation of recombinant IgG molecules. Therefore, native IgG molecules will be able to be produced with or without N-glycan addition depending on the clinical indication. Thirdly, an industry first production method utilizing the regulated secretion pathway of Tetrahymena will be applied to the production of IgG molecules. The direct link between biomass and product accumulation implicit in this process may result in a substantial positive impact on the cost of production and therefore treatment.
Public Health Relevance: Therapeutic antibodies represent the fastest growing class of protein-based drugs, and are effective in the treatment of many types of human disease ranging from autoimmune and infectious disease to multiple forms of cancer. This project will develop novel expression technology for the production of monoclonal antibodies that will substantially increase product safety and efficacy and decrease cost of goods sold compared to current production methods.
Thesaurus Terms: "address; Adopted; Animals; Antibodies; Antibody Formation; Antibody Fragments; Antibody Production; Antibody Response; Autoimmune; Autoimmune Process; Bacteria; Biomass; Biosynthetic Proteins; Cancers; Carbohydrates; Cell Culture System; Cells; Clinical; Communicable Diseases; Contracting Opportunities; Contracts; Development; Drugs; Family; Gamma Globulin, 7s; Generalized Growth; Glycans; Growth; Health; Heterogeneity; Human; Human, General; Hydrogen Oxide; Igg; Igg1; Immune Globulins; Immunoglobulin Fragments; Immunoglobulin G; Immunoglobulins; Immunoglobulins / Antibodies; Industry; Infectious Disease Pathway; Infectious Diseases; Infectious Diseases And Manifestations; Infectious Disorder; Laboratories; Leader Peptide; Length; Light; Link; Maintenance; Maintenances; Malignant Neoplasms; Malignant Tumor; Mammalian Cell; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Medication; Metabolic Glycosylation; Methods; Mice; Microbe; Moab, Clinical Treatment; Monoclonal Antibodies; Murine; Mus; Mutate; Pathway Interactions; Pattern; Peptide Leader Sequences; Peptide Library; Peptide Signal Sequences; Pharmaceutic Preparations; Pharmaceutical Agent; Pharmaceutical Preparations; Pharmaceuticals; Pharmacologic Substance; Pharmacological Substance; Photoradiation; Polysaccharides; Process; Production; Proteins; Protocols, Treatment; Rgm; Recombinant Antibody; Recombinant Proteins; Recombinants; Regimen; Reliance; Safety; Signal Peptide; Signal Peptide, Leader; Signal Sequences; Signal Sequences, Peptide; Stimulus; System; System, Loinc Axis 4; Technology; Testing; Tetrahymena; Tetrahymena Thermophila; Therapeutic; Therapeutic Antibodies; Tissue Growth; Treatment Protocols; Treatment Regimen; Treatment Schedule; Water; Yeasts; Antibody Biosynthesis; Base; Cost; Drug/Agent; Experiment; Experimental Research; Experimental Study; Gene Product; Glycosylation; Human Disease; Immunoglobulin Biosynthesis; Malignancy; Microbial; Monoclonal Antibody Production; Neoplasm/Cancer; Novel; Ontogeny; Pathway; Protein Signal Sequence; Public Health Relevance; Research Study; Success; Tissue/Cell Culture; Tool"
