Author ORCID Identifier
https://orcid.org/0000-0001-8329-7214
Date of Award
2023
Document Type
Thesis (Ph.D.)
Department or Program
Engineering Sciences
First Advisor
Karl Griswold
Second Advisor
Margaret Ackerman
Third Advisor
Chris Bailey-Kellogg
Abstract
High throughput cell-based screening methodologies have become core development tools for the generation and engineering of various biotherapeutics. For example, SpCas9 variants for use in CRISPR-Cas9 therapeutics have been engineered for enhanced specificity using a high throughput directed evolution approach in E. coli. Additionally, antibodies are routinely discovered and engineered for high affinity binding using yeast surface display. These high throughput techniques typically involve generation of large protein libraries which require innovative screening strategies depending on the target protein’s function. In this thesis work, we describe high throughput protein library screening approaches to address recombinant biologic aggregation and immunogenicity in the context of biotherapeutic development.
First, an in vivo aggregation biosensor is developed to engineer improved solubility characteristics for highly aggregation-prone biologics. This system enables high throughput fluorescent-based screening of enhanced-solubility protein variants, and is demonstrated by screening an aggregation-prone ecGP123 library for soluble green fluorescent protein mutants. Here, we are able to select for variants that achieve up to 40% solubility improvement over wild-type. Next, a yeast surface display library screening approach is proposed to engineer deimmunized thyroid stimulating hormone receptor variants for the treatment of Grave’s Disease. In this study, we show that glycosylation considerations or engineering are necessary to generate and screen large receptor libraries for targeted pathogenic antibody binding. Finally, we detail development of a deimmunized botulinum toxin therapeutic that utilizes combinatorial protein library generation and functional screening using a catalytic Förster resonance energy transfer sensor. These studies represent unique strategies for screening large recombinant biotherapeutic libraries in order to advance development of biologic treatment options for various clinical needs.
Original Citation
Fang, Yongliang, et al. "Functional Deimmunization of Botulinum Neurotoxin Protease Domain via Computationally Driven Library Design and Ultrahigh-Throughput Screening." ACS Synthetic Biology (2023).
Recommended Citation
Chang, Andrew Y., "High Throughput Cellular Screening Technologies for Engineering Enhanced-Solubility and Deimmunized Biologics" (2023). Dartmouth College Ph.D Dissertations. 155.
https://digitalcommons.dartmouth.edu/dissertations/155
