The efficacy of injected therapeutic proteins is often compromised by their rapid clearance from the circulation. This means that doses must be administered more often, impacting both patient compliance and cost of treatment. A major search is underway in academic and industrial laboratories to discover ways to modify protein drugs in such a way that their residence time in the body is enhanced. Approaches include chemical modification of the protein therapeutic, or creation of protein drugs that contain stretches of additional amino acids, or indeed, of whole proteins, designed to promote increased half-life. The long-term objective of the present proposal is to provide a means that is applicable in principle to all therapeutic proteins for extending their residence time in the body, thus making them easier to administer and lower in cost to the patient. In this application we propose to explore the introduction of peptide sequences, sequence tags that will allow for limited glycosylation of the protein therapeutic during its recombinant production in a mammalian cell line. The hypothesis is that this vehicle for enhancing glycosylation will protect the protein drug from rapid elimination. Our novel sequence tags are derived from regions in a human protein that are known to undergo glycosylation and that should allow for incorporation of carbohydrate to enhance the lifetime of the protein drug in the body. The specific aims of the proposal are 1. To clone and express in HEK-293 cells, N- and C-terminally extended variants of a well-studied therapeutic protein in which the extensions comprise human protein sequences, varying in length and known to undergo glycosylation; 2. To purify the proteins to homogeneity and verify that they contain levels of carbohydrate that correspond to the length of the adduct; 3. To show that the N- and/or C-terminal extensions have not abolished biological activity; and 4. To test these tagged proteins in animal models to see if they increase serum half-life relative to the native parent protein drug. The research plan will be designed to address these specific aims and will combine expertise with methods of molecular and cell biology for production and expression of the new gene constructs in HEK-293 cells, together with expertise in protein purification and characterization to produce viable proteins for testing.
Public Health Relevance: Project Narrative :The complexities of many unsolved and unmet medical needs such as cancer, Alzheimer's disease, ALS, and other neurodegenerative disorders has increased focus on development of proteins as drugs to treat these maladies. Protein drugs are expensive due to high cost of manufacture, and they tend to be rapidly eliminated from the body, thus increasing the dose regimen and adding to the price of treatment. The present proposal provides a novel method, applicable in principle to any protein drug, for producing variants with increased duration of activity (fewer doses), and reduced cost.
Thesaurus Terms: "address; Alzheimer; Alzheimer Disease; Alzheimer Sclerosis; Alzheimer Syndrome; Alzheimer's; Alzheimer's Disease; Alzheimers Dementia; Alzheimers Disease; Amino Acid Sequence; Amino Acid Sequence Analysis; Amino Acids; Animal Model; Animal Models And Related Studies; Animals; Assay; Bioassay; Biologic Assays; Biological; Biological Assay; Blood Circulation; Blood Serum; Bloodstream; C-Terminal; Ca-15-3 Antigen; California; Cancers; Carbohydrates; Cell Growth In Number; Cell Line; Cell Lines, Strains; Cell Multiplication; Cell Proliferation; Cellline; Cells; Cellular Proliferation; Cellular Biology; Chemicals; Circulation; Common Rat Strains; Compliance Behavior; Contractor; Df3 Antigen; Degenerative Diseases, Nervous System; Degenerative Neurologic Disorders; Dementia, Alzheimer Type; Dementia, Primary Senile Degenerative; Dementia, Senile; Development; Dose; Drugs; Elisa; Enzyme-Linked Immunosorbent Assay; Episialin; Epithelial Membrane Antigen; Genes; Glycopeptides; Glycosylated Muc-1; Goals; Half-Life; Half-Lifes; Hour; Human; Human Growth Hormone; Human, General; Laboratories; Length; Link; Muc-1; Muc-1 Antigen; Malignant Neoplasms; Malignant Tumor; Mammalian Cell; Mammals, Rats; Man (Taxonomy); Man, Modern; Medical; Medication; Metabolic Glycosylation; Methods; Modeling; Molecular; Molecular Biology, Protein Sequencing; Monitor; Muc1 Mucin; Mucin Peptide Muc-1; Mucin/Peptide; Mucins; Mucus Glycoprotein; Neurodegenerative Diseases; Neurodegenerative Disorders; Neurologic Degenerative Conditions; Neurologic Diseases, Degenerative; Outsourcing; Parents; Patient Compliance; Patient Cooperation; Patients; Peptide Sequence Analysis; Peptide Sequence Determination; Peptides; Pharmaceutic Preparations; Pharmaceutical Preparations; Pharmacology; Polymorphic Epithelial Mucin; Post-Translational Modifications; Post-Translational Protein Processing; Posttranslational Modifications; Preparation; Price; Primary Senile Degenerative Dementia; Production; Protein Glycosylation; Protein Modification; Protein Modification, Post-Translational; Protein Processing, Post-Translational; Protein Processing, Posttranslational; Protein Sequence Analysis; Protein Sequencing; Protein Structure, Primary; Protein/Amino Acid Biochemistry, Post-Translational Modification; Proteins; Rat; Rattus; Recombinants; Regimen; Relative; Relative (Related Person); Research; Spase Type I; Sampling; Sequence Analyses, Amino Acid; Sequence Analyses, Peptide; Sequence Analyses, Protein; Sequence Analysis, Protein; Sequence Determinations, Amino Acid; Sequence Determinations, Protein; Serum; Somatotropin (Human); Somatropin; Somatropin (Human); Stretching; Study Of Serum; Testing; Time; Transfection; Treatment Compliance; Treatment Cost; Universities; Variant; Variation; Abstracting; Adduct; Aminoacid; Aminoacid Sequence Of Peptide; Aminoacid Sequence Of Protein; Bacterial Leader Peptidase I; Cell Biology; Compliance Cooperation; Cost; Cultured Cell Line; Dementia Of The Alzheimer Type; Design; Designing; Drug/Agent; Experience; Experiment; Experimental Research; Experimental Study; Expression Cloning; Gene Product; Glycosylation; Hgh; Hgh (Human Growth Hormone); Interest; Leader Peptidase; Leader Peptidase I; Malignancy; Model Organism; Neoplasm/Cancer; Neurodegenerative Illness; Novel; Patient Adherence; Peptide Sequence; Phage-Procoat-Leader Peptidase; Pricing; Primary Degenerative Dementia; Procoat Protein Signal Peptidase; Protein Aminoacid Sequence; Protein Purification; Protein Sequence; Public Health Relevance; Research Study; Residence; Senile Dementia Of The Alzheimer Type; Serology; Signal Peptidase; Signal Peptidase I; Signalase; Therapeutic Protein; Therapy Compliance; Therapy Cooperation; Type I Signal Peptidase; Vector"
