Affinity tuning of antibodies using yeast surface display

Yeast surface display of I-Ani1: Two epitope tags, One “antigen” binding site

Yeast surface display of I-Ani1: single bp resolution of binding specificity

1 bp change 2 bp change 3 bp change

1 bp change 2 bp change

Strategies for generating variants & libraries

Rational & Computational

Design

Error Prone PCR Combinatorial Libraries & Gene

Shuffling

Jim, Summer, Justin, Audrey, Umut , and

others?

Optimal specific S/N ratio at physiological osmolarity

75 mM KCl 100 mM KCl 125 mM KCl 150 mM KCl 175 mM KCl 200 mM KCl341 mOsm 472 mOsm405 mOsm316 mOsm262 mOsm222 mOsm

4

6

8

10

12

14

16

18

200 250 300 350 400 450 500

Sign

al/N

ois

e

mOsm

Analysis of computationally designed I-Ani1 variant: K24N T29R E31L R72S

JH&ST-Ani combinatorial Lib1.0

-ve MACS Fraction

+ve MACS Fraction

Targeting STS3 and STS4 to generate a high-affinity I-Ani1 variant

Kd = 65nM

Kd = 8 nMKd = 20 nM

ReoAni

STS3/4 Lib1.0.A2

Yeast surface display of I-Ani1: ionic effects on HE specificity

Standard conditions: 150mM KCl, 10mM NaCl, 5mM CaCl2, 10mM HEPES, 0.2% BSA, 0.1% Galactose, pH 7.5