Poster
Achieving high affinity by affinity maturation
Utilization of Precise Mutagenesis Library (PML) and
High-Throughput (HTP) Fast Screening of Expression
Biophysical-properties Affinity (FASEBA) screening
platform to achieve high affinity by affinity maturation
Derek Chen, Yu Liang, Lindi Wang, Wenwan Fang, Jie Ma
ProBio Inc. USA
ABSTRACT
MATERIALS AND METHODS
Affinity maturation is known to improve the antibody-antigen binding kinetics for drug
optimization. Despite the popularity of NNK (degenerate primer, N= A/C/G/T, K= G/T)
mutagenesis and phage library, inherent limitations exist, such as unequal mutation distribution,
WT sequence enrichment, and limited saturation mutation coverage within the Comple-
mentarity-Determining Region (CDR). To address these limitations, we combined the
power of efficient, unbiased mutagenesis by PML technology and the high efficiency of the
HTP FASEBA affinity screening platform. In a case study of affinity maturation, we successfully
achieved an affinity improvement of 100+ fold, reaching a single-digit picomole level.
3.Two-step library screening and affinity ranking to obtain the AM variant
with desired affinity improvement
• K ranking and a combinatorial library construction
off
• Each saturation mutation library is subjected to SPR K ranking
• Beneficial mutations are built into another combinatoroifaf l library
• Combinatorial library is screened by SPR K ranking
off
RESULTS
INTRODUCTION
1. Sequencing saturation mutation library by NGS to verify distribution of
mutation
During affinity maturation (AM), the theoretical size of saturation mutation covering all CDR
is at the level of 20E60, which is difficult to build in display libraries. Instead of confining the
region of saturation mutation to very few residues in each CDR, we employed more powerful
tools, including Molecular Operating Environment (MOE) based structure modeling, PML,
E.coli. based HTP expression system, two-step mutation strategy, and a Surface Plasmon
Resonance (SPR) kinetics analytical platform (Biacore 8K) to cover all possible mutations
in each CDR.
MATERIALS AND METHODS
Figure 1. NGS disclosed that each CDR residue was mutated to the other 19 residues
(excluding itself) with similar frequency
1.Generation of saturation mutation library with similar frequency and 100%
coverage
2. Mutation libraries were screened by single concentration K ranking
off
• PML is created by on-chip synthesized Oligo to achieve controlled distribution of mutation
Figure 2. Koff ranking was applied for each saturation mutation library to screen the candi-
date for further library construction
3. Top candidate showed an affinity improvement greater than 163-fold,
reaching single digit picomole range
2. E.coli based library facilitates the HTP expression and FASEBA (Fast
Screening of Expression Biophysical-properties Affinity) screening
• Each saturation mutation library is transformed into FASEBA E.coli based expression system
Figure 3. Koff analysis revealed
that the affinity of one AM
variant was greatly improved
Rmax (RU)
Ligand
Analyte
Chi² (RU²)
ka (1/Ms)
kd (1/s)
KD (M)
< 1.00E-06
(LOD)
51.3
Target A
1.23E+00
1.97E+05
<5.08E-12
variant 2
64.7
WT
Target A
7.96E-01
1.40E+05 1.16E-04
8.29E-10
Table 1. The top candidate, variant 2, showed affinity improvement greater than 163-fold comparing to WT
CONCLUSIONS
In this report, we showed an application of the proprietary technology, PML-based CDR
mutagenesis, combined with the FASEBA soluble expression system, which allowed an
efficient introduction of mutations into antibody CDRs with unbiased frequency and distribution.
Followed by a two-step mutation library strategy and high throughput affinity ranking by SPR,
this comprehensive approach successfully improved the affinity of an engineered antibody
more than 100 fold. The success of the combination of PML and FASEBA demonstrates its
great power and potential compared to a conventional phage library-based AM approach.
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