Affinity Maturation of the D1.3 Antibody Using Yeast Surface Display and Flow Cytometry

Different methodologies were explored for improving YSD affinity maturation of anti-protein antibodies, using scFv D1.3. Using equilibrium sorted YSD with the D1.3/M3 antibodies, a single-pass enrichment factor of 125-fold (+/-65-fold) was achieved, indicating excellent differentiation between clones of only slightly different affinity. Optimal equilibrium affinity screening of a randomly mutated D1.3 library was performed with and without an osmotic stressor present in the binding reactions. Osmotic stress yielded a more diverse set of mutant clones from the screen, and recombination of selected mutations using site-directed mutagenesis produced a four-fold higher affinity mutant (MEC1). MEC1 was randomly mutagenized and screened by kinetic selection, identifying a large number of improved mutants. In both screens, library size was approximately 5 x 106, and mutagenesis was by error-prone PCR of the entire scFv gene. This affinity compares favorably to the highest affinities attained previously with phage display for anti-protein antibodies, without the necessity for large libraries or site-directed saturation mutagenesis.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemical Engineering

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• VanAntwerp, Jennifer Jewett

Contributors dc:contributor

• Wittrup, K. Dane

• Biology, Molecular

Language dc:language

eng

Identifier

(MiAaPQ)AAI9953167

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/82476