(664a) Tuning Phage Display and Yeast Surface Display for Panning for Novel Ligands on Tumor Cells

Tuning Phage Display and Yeast Surface
Display for Panning for Novel Ligands on Tumor Cells

Lawrence A. Stern, Ian A. Schrack, Aakash
Deshpande, Sadie M. Johnson, and Benjamin J. Hackel

Yeast surface display and phage
display have proven to be effective tools in the evolution and screening of
ligands with novel binding activity. 
Generally, selections for binding activity are carried out using immobilized
recombinant domains of target molecules. 
While this method often provides ligands with apparent high affinity and
specificity for the target of interest, translatability to true cellular target
in vitro is a common concern.  Direct selections on mammalian cell surfaces
can be carried out either exclusively or in combination with soluble
target-based selections to further direct toward evolution of ligands with affinity
for true cellular target.  Further, this
method can be applied to simultaneous discovery of molecular targets and new
binding ligands using poorly characterized cell types.  This study aims to optimize direct cellular
selections using multiple formats of phage display and yeast surface display by
using an epidermal growth factor receptor (EGFR) model system that encompasses
multiple tiers of affinity and cellular target expression, and to directly
compare these methods for applicability to binder selection from mock
libraries.  Cell-based selections with
standard yeast surface display can be successful with up to mid-nanomolar
affinity, yielding 15-30% of inputted binders with enrichment ratios reaching 96
± 84.  However, this level of enrichment
requires at least one million targets per cell, with yields and enrichment
ratios decreasing dramatically for a 10-fold decrease in targets per cell.  One hypothesis for this decrease in
enrichment is a lack of accessibility of relevant epitopes due to steric
hindrance by glycosylation and other membrane-bound proteins on cell surfaces.
The impacts of displayed protein linker length, receptor epitope, and receptor
identity will be discussed. Mathematical modeling of phage display selections
highlights the importance of (a) binding strength, which is being evaluated
with various intrinsic affinities and four modes of valency
? monovalent pIII, bivalent pIII
using a previously validated antibody hinge - leucine zipper fusion approach
(Lee, et al., J. Immun. Meth. 2004),
avid pIII from phage vector, and multivalent pVIII via pVIII engineering ? and
(b) fraction of phage displaying ligand, which is being evaluated using the
aforementioned display approaches. Western blotting techniques are used to
quantify displaying fraction. Mock library selections are used to quantify
enrichment. Optimal conditions, and their consistency with mathematical
modeling, will be discussed as well as ongoing efforts in target-ligand
co-discovery.