SBIR-STTR Award

Metachromatic leukodystrophy (MLD) is an genetic disease caused by mutations in the gene encoding the lysosomal enzyme, arylsulfatase A (ASA). Symptoms including neurodegeneration and mental retardation appear during infancy or childhood; and early death can occur due to organ damage in the brain. Enzyme replacement therapy (ERT) cannot treat the brain, since recombinant ASA does not cross the blood-brain barrier (BBB). Accordingly, clinical trials of children with MLD and recombinant ASA have been abandoned. The present work will re-engineer human ASA to enable transport across the BBB using a molecular Trojan horse technology. A molecular Trojan horse is a genetically engineered peptidomimetic monoclonal antibody (MAb) against an endogenous BBB peptide receptor, such as the human insulin receptor (HIR). The human ASA is fused to the heavy chain of the HIRMAb to create a new chemical entity, called the HIRMAb-ASA fusion protein. Feasibility studies with the HIRMAb-ASA fusion protein were enabled following the cloning of a high producing, stably transfected host cell line. The HIRMAb-ASA fusion protein retains high ASA enzyme activity and high binding to the HIR. This phase I SBIR work will further validate the pharmacologic activity of the HIRMAb-ASA fusion protein in MLD fibroblasts, using ASA enzyme activity assays and confocal microscopy. The HIRMAb-ASA fusion protein penetration of the BBB in vivo will be confirmed in the Rhesus monkey. This work provides the rationale for future phase II studies that provide the bridge to subsequent GMP/GLP work that supports an IND for treatment of MLD with the HIRMAb-ASA fusion protein.

Public Health Relevance:
Metachromatic leukodystrophy (MLD) is an genetic disease caused by mutations in the gene encoding the lysosomal enzyme, arylsulfatase A (ASA). Enzyme replacement therapy cannot treat the brain, since recombinant ASA does not cross the blood-brain barrier. The present work will re- engineer human ASA to enable transport across the BBB using a molecular Trojan horse technology.

Public Health Relevance Statement:
Metachromatic leukodystrophy (MLD) is an genetic disease caused by mutations in the gene encoding the lysosomal enzyme, arylsulfatase A (ASA). Enzyme replacement therapy cannot treat the brain, since recombinant ASA does not cross the blood-brain barrier. The present work will re- engineer human ASA to enable transport across the BBB using a molecular Trojan horse technology.

NIH Spending Category:
Biotechnology; Brain Disorders; Neurodegenerative; Neurosciences; Pediatric

Project Terms:
Address; Adolescent; Adult; Affinity; Age; Animal Testing; Animals; Arylsulfatases; base; Binding (Molecular Function); Biological Assay; Bioreactors; Blindness; Blood; Blood - brain barrier anatomy; Blood capillaries; Blood Vessels; Brain; brain cell; capillary; Cell Culture Techniques; Cell Line; Cell membrane; Cells; Cessation of life; Chemicals; Child; Childhood; Chimeric Proteins; Chinese Hamster; Chinese Hamster Ovary Cell; Chronic; Clinical Trials; Cloning; Coma; Confocal Microscopy; Convulsions; Deglutition; Dementia; Developmental Delay Disorders; Disease; disease-causing mutation; Dose; drug development; Drug Kinetics; Engineering; enzyme activity; enzyme replacement therapy; Enzymes; Evaluation; Feasibility Studies; Fibroblasts; Future; Genes; Hereditary Disease; Human; Human Engineering; human INSR protein; Immunoglobulin G; In Vitro; in vivo; infancy; Inherited; Insulin Receptor; Label; Macaca mulatta; Medical; Mental Retardation; Metachromatic Leukodystrophy; Methodology; Methods; Modeling; molecular trojan horse; Monoclonal Antibodies; Muscle; Muscle Rigidity; Mutation; Nerve Degeneration; Neuraxis; Neurons; Organ; Ovary; Paralysed; Penetration; Peptide Receptor; Peptide Signal Sequences; peptidomimetics; Peripheral; Pharmaceutical Preparations; Phase; phase 1 study; phase 2 study; Plasma; Primates; Property; Propionates; protein structure; Proteins; Radio; Recombinant Fusion Proteins; Recombinants; Research; response; Rodent; Scientist; Small Business Innovation Research Grant; Spinal Cord; Symptoms; Time; Toxic effect; Triage; uptake; wasting; Work

Metachromatic Leukodystrophy or MLD, is a genetic disease that affects the lysosomal enzyme, arylsulfatase A (ASA). People born with MLD develop extensive lysosomal storage product accumulation in tissues, including the brain. Children with MLD develop multiple brain disorders including mental retardation and hydrocephalus early in life. The current therapy in clinical trials for MLD is Enzyme Replacement Therapy (ERT) with the recombinant human enzyme, ASA. However, ERT does not treat the brain of MLD, because ASA does not cross the blood-brain barrier (BBB). The present work will continue work on the development of a new treatment of the brain of MLD, which is a genetically engineered IgG-enzyme fusion protein. The ASA enzyme is fused to a BBB molecular Trojan horse, which is a genetically engineered peptidomimetic monoclonal antibody (MAb) against an endogenous BBB peptide receptor, the human insulin receptor (HIR). The human ASA is fused to the heavy chain of the HIRMAb to create a new biological entity, the HIRMAb-ASA fusion protein named AGT-183. Phase I studies show the HIRMAb-ASA fusion protein could be engineered and expressed by stably transfected host cells. The fusion protein retained high affinity binding to the HIR, retained high ASA enzyme activity, was triaged to the lysosomal compartment of MLD fibroblasts, and rapidly penetrated the BBB in rhesus monkeys after intravenous administration. The proposed phase II work will develop a manufacturing plan that can be replicated for future GMP manufacturing. The AGT-183 produced in phase II will be evaluated for safety, toxicology and pharmacokinetics in Rhesus monkeys. The completion of this work will enable entry of the AGT-183 drug development program into GLP toxicology and GMP manufacturing required for submission of an IND to begin treatment of the brain in patients with MLD.

Public Health Relevance Statement:


Public Health Relevance:
People born with the genetic disorder called Metachromatic Leukodystrophy, or MLD, develop severe abnormalities of the brain, owing to the inherited defect in the gene encoding the lysosomal enzyme, arylsulfatase A (ASA). Enzyme replacement therapy is not effective for the brain, because the ASA does not cross the blood-brain barrier (BBB). This research will develop a new treatment for the brain in patients with MLD, which involves the re-engineering of human ASA as a fusion protein that crosses the human BBB.

Project Terms:
Active Biological Transport; Adult; Affect; Affinity; Affinity Chromatography; Anions; Antibodies; Arylsulfatases; Binding (Molecular Function); Biochemical; Biological; Bioreactors; Blood; Blood - brain barrier anatomy; Brain; brain cell; Brain Diseases; Cations; Cell membrane; Cells; Cerebrospinal Fluid; Child; Chimeric Proteins; Chinese Hamster; Chinese Hamster Ovary Cell; Chromatography; Clinical Trials; Defect; design; Development; Disease; Dose; drug development; Drug Kinetics; Engineering; enzyme activity; enzyme replacement therapy; Enzyme-Linked Immunosorbent Assay; Enzymes; Exhibits; Fibroblasts; Filtration; Future; Genes; Genetic Engineering; Growth; Hereditary Disease; Human; Human Engineering; human INSR protein; Hydrocephalus; Immunoglobulin G; in vivo; Inherited; Insulin Receptor; intravenous administration; Life; Macaca mulatta; Measurement; Measures; Mediating; Membrane; Mental Retardation; Metachromatic Leukodystrophy; molecular trojan horse; Monoclonal Antibodies; Mutate; Mutation; Names; nano; Neuraxis; Neurons; neuropathology; Organ; Ovary; Patients; Peptide Receptor; peptidomimetics; Perfusion; Peripheral; Pharmaceutical Preparations; Phase; phase 1 study; Plasma; Primates; Process; Production; programs; Progress Reports; Protein Binding; Proteins; public health relevance; receptor; Recombinants; Research; Safety; Small Business Innovation Research Grant; Structure; Sulfoglycosphingolipids; Temperature; Testing; Tissues; Toxicology; Triage; uptake; Validation; Work