Humanizing Alginate Depolymerase: New Strategies for De-immunizing Enzyme Therapies

Junior Investigator and Project Leader

Karl E. Griswold, Ph.D.
Assistant Professor
Thayer School of Engineering
Dartmouth College

Advances in the field of biotechnology have produced a wealth of cellular and molecular tools for a wide variety of practical applications. One of the most impactful achievements has been the development of therapeutic proteins useful in treating human disease. Biotherapeutics can be particularly powerful drugs, but these molecules suffer from their own unique limitations including their propensity to illicit an immune response in human patients. Because of its direct relationship to safety and efficacy, addressing the immunogenicity of therapeutic proteins is a critical aspect of their development. Conventional approaches to de-immunizing biotherapeutics at the molecular level focus on covalently masking or genetically altering specific antigenic sequences. An alternative approach, termed humanization, addresses immunogenicity on a more global scale by engineering proteins to “blend in” with the human proteome rendering them less likely to be recognized as foreign agents by the human immune system. While conventional humanization has been widely successful with therapeutic antibodies, it is not applicable to more diverse classes of proteins, such as enzymes. To deimmunize therapeutic enzymes by humanization will require the development of advanced biomolecular engineering technologies, and patients suffering from a variety of diseases would benefit from such a breakthrough.

Premature mortality in cystic fibrosis typically results from chronic P. aeruginosa infection of the patient's airways. The mucoid strains of P. aeruginosa associated with cystic fibrosis are characterized by their production of the exopolysaccharide alginate, a known pathogenic factor. Alginate lyases are enzymes that degrade the alginate polymer, and have shown great promise for symptomatic treatment of cystic fibrosis. However, the microbial origin of these biocatalysts predisposes them towards high level immunogenicity, and brings into question their utility in a clinical setting. A seemingly straightforward approach to ameliorating the immunogenicity of microbial alginate lyases would be to substitute a human ortholog for the foreign enzymes. Unfortunately, as is the case with numerous microbial biotherapeutic candidates, alginate lyases have no exact human counterpart. However, the human genome encodes numerous enzymes with similar catalytic functions, i.e. degradation of carbohydrate-based biopolymers. The existence of human enzymes exhibiting similar catalytic functions but different substrate selectivities suggests that the alginate lyase therapeutic activity could be humanized by engineering a human carbohydrate hydrolase to act on the alginate substrate.

We propose to engineer alginate degrading activity into a human enzyme template. We hypothesize that the human origins of the resulting biocatalyst will result in its tolerance by the human immune system. To accomplish our goal, we will generate large, highly diverse, recombinant enzyme libraries, and we will functionally screen those libraries in a high throughput fashion to identify enzymes that degrade alginate. The isolated candidates will subsequently be evaluated for potential immunogenicity.

The results of these studies will have direct implications not only for CF research, but will provide the ground work for developing humanized enzyme therapeutics with applicability to an array of bacterial infections with associated biofilm modes of growth. Additionally, insights into the extent to which molecular engineering can tune the substrate selectivity of carbohydrate degrading enzymes could impact the development of treatments for human diseases not associated with bacterial infections, such as mucopolysacchride storage disorders. Ultimately, we hope to establish a new paradigm for humanizing enzyme based biotherapeutics.

Students

Thomas C. Scanlon, Ph.D. - Postdoctoral Fellow
Tom's research is focused on development of high throughput alginate degrading screens, and their use for engineering human alginate depolymerases. He also works on development of enzyme based antibiotics for pulmonary infections.

Avinash Gill, Ph.D. – Postdoctoral Fellow
Avi's projects involve engineering enzymes to treat S. aureus infections. He also works on a collaborative project to computationally de-immunize therapeutic proteins.

John W. Lamppa – Graduate Student
John's research is focused on de-immunization of bacterial alginate lyases, and the development of engineered variants with enhanced capacity to disrupt bacterial biofilms.

Heather Jewell – Technician
Heather assists in the development novel proteins designed to efficiently kill drug-resistant pathogens.

Elizabeth Gray – Undergraduate
Elise has worked to optimize recombinant library construction techniques in the host S. cerevisiae, and is now examining the evolution of drug-resistance in bacterial pathogens.

Publications

Pubmed