(690b) Ironing out the Wrinkles for Botox Therapeutic Applications | AIChE

(690b) Ironing out the Wrinkles for Botox Therapeutic Applications


Griswold, K. E. - Presenter, Dartmouth College
Bailey-Kellogg, C., Dartmouth
Fang, Y., Dartmouth
Eszterhas, S., Dartmouth
Miyashita, S. I., Boston Children's Hospital
Verma, D., Dartmouth College
Choi, Y., Dartmouth
Dong, M., Boston Children's Hospital
Botulinum neurotoxin (BoNT), commonly known as Botox, is widely used to reduce facial wrinkles, but its potent paralytic activity has diverse and important applications that range far beyond its $4 billion global cosmetic market. This exceptionally potent drug has the potential to address a long list of unmet medical needs, such as cervical dystonia, and could additionally revolutionize treatments in critical medical spaces such as acute and chronic pain management. Unfortunately, the development of anti-BoNT antibodies can limit many of these therapeutic uses. To fully capitalize on BoNT’s therapeutic potential, therefore, the immunogenicity of this bacterial toxin in human patients must be addressed. Unfortunately, BoNT deimmunization has been an elusive goal for the biotech industry, due in large part to the drug’s structural and functional complexity. We have solved this challenge through the combined application of state-of-the-art computational protein design tools and a sophisticated ultra-high throughput functional screen. We designed and constructed a combinatorial library of 300 million deimmunized BoNT light chain candidates, in which the entire library ensemble was computationally optimized for both reduced T cell epitope content and maintenance of stability and activity. From this large, diverse population of molecules, we isolated active variants using a customized high-speed flow cytometric screen for BoNT enzymatic activity. The kinetics of engineered variants were analyzed in vitro, and promising constructs were subsequently tested for immunogenicity in humanized HLA transgenic mice. Ultimately, the best variants were incorporated into full-length toxin and their paralytic activity was evaluated in a murine model of flaccid paralysis. In total, our molecular engineering efforts have created a large panel of deimmunized BoNT candidates with functional, immunological, and pharmacokinetic profiles that are tailored for therapeutic applications.

From this presentation audience members will gain an:

-Introduction to high-impact medical applications of Botox, beyond cosmetics

-Overview of causes and consequences of biotherapeutic immunogenicity

-Discussion of T cell epitope deletion as a strategy to deimmunize biotherapeutics

-Demonstration of state-of-the-art computational design tools for engineering deimmunized biotherapies

-Disclosure of an innovative ultra-high throughput screen for BoNT enzymatic activity

-Presentation of immunological and functional characterization of next-generation BoNT biotherapies

-Example of how humanized HLA transgenic mice can enable pre-clinical immunogenicity analysis of protein drugs