In vitro affinity maturation based on Ribosome Display Systemwith PUREfrex
Kanehisa Kojoh; Kumiko Tsuihiji; Shizue Katoh; Takashi Kanamori
GeneFrontier Corporation
Abstract
The affinity maturation is one of the key technologies to distinguish the binder from theothers against the same target. For easy and feasible affinity maturation, ribosome display iswell known to give great advantages. Especially, ribosome display on the PURE system wasreported as a powerful method not only for in vitro selection of specific binders such as
antibodies or designed proteins like scaffolds, but also for in vitro affinity maturation.
The PURE system is one of the cell-free protein synthesis system reconstituted from thepurified components necessary for the translation in E. coli. Recently, we developed theupgraded PURE system (PUREfrex), in w hich the contaminants such as RNases,
lipopolysaccharides and other proteins unnecessary for the translation are extremelydecreased. We also developed the ribosome display system utilizing PUREfrex (PUREfrexRD).
The selection efficiency of PUREfrexRD is raised approximately 100-fold or 1000-fold higherthan ribosome display using the original PURE system or E.coli S-30 extract system.
In this conference last year, we reported in vitro selection of the binders to Erk2 protein fromour own designed protein scaffold library based on FHA domain of RNF8 (human E3 ubiquitinligase) by PUREfrexRD. This year, we report the application of PUREfrexRD to in vitro affinity
maturation by off-rate selection. We prepared two randomized RNF8 libraries based on thespecific binder to Erk2 by PCR-based DNA shuffling or grafting one of the loops of RNF8. After
three rounds of off-rate selection over several days, we successfully improved the affinity
approximately 35-fold more than the original binder in very simple steps.
On the other hand, the affinity maturation of Fab will give great advantages in terms of
seamless development for generating better antibody therapeutic. Therefore, we have applied
the PUREfrexRD to the affinity maturation of Fab fragments and have developed the protocols
to successfully form Fab fragments on ribosome.

①PUREfrex:Highly purified PURE system

PURE system is a reconstituted in vitro transcription and translation system which consistsof purified 36 proteinous factors and E. coli ribosome necessary for transcription,translation and energy recycling. It also contains amino acids, NTPs and E. coli tRNA, so
the target protein can be synthesized just by adding the template DNA to the reaction
mixture. PUREfrex is developed ashighly purified PURE system which has been welloptimized to ribosome display; Contamination of such as Lipopolysaccharide (LPS) in thesystem is very low.

②mRNA-ribosome-protein complex of PUREfrexRDmRNA
Library protein

RibosomeSec MCLinker

Library 1:PCR-based DNA shuffling library (ref:Zhao et al.1998. Nat.Biotechnol., 16. 258)

Sec M

Advantages
1)mRNA-ribosome-protein complex is highly stable in the PUREfrex because activies ofnucleases and other Inhibitory factors are very low in this system.
2)The mRNA encoding a scaffold protein with translation arrest sequence of SecM
remains stably attached to a ribosome in the PUREfrex.
3)PUREfrexRD have high selection efficiency than using normal PURE system RD.

③In vitro affinity maturation with PUREfrexRDLoop1

Loop4

Library 2:Loop4 grafting library

RNF8 Erk2-binder
Loop2
Loop3

Loop1

Library diversity: 1.2××1011

mRNA Libraries （1.2××1012 molecules)↓In vitro translation in 20 µL of PUREfrex↓4°C、、30 min
Mixing with Erk2 protein (0.1 nM)-immobilized streptavidin beads
in TBST 200 µL↓4°C、、120 min
Adding of free-Erk2 protein (final 1 µM)↓4°C、、incubation for long times(1st round:1 day, 2nd round:4 days, 3rd round:7 days)
Washing five times with 500 µL of TBST↓
Eluting mRNA from mRNA-ribosome-protein complexes with 100 µLof 50 mM EDTA↓
RT-PCR→→Next round selection↓
Subcloning↓
Selecting unique clones by sequencing↓
Screening higher affinity clones by competitive ELISA↓
Purifying the higher affinity clones and determining IC50

Last year, we reported in vitro selection of the binders to Erk2 protein from the designed
RNF8 scaffold library. The library was constructed by randomizing the 4 loops (red lines) toNNS. The Kd value of an obtained binder is 2.9 × 10-8. This binder was used for affinity
maturation.
RNF8 is a E3 ubiquitin ligase which plays a critical role in the early DNA-damage response via the ubiquitination ofhistone H2A and H2AX. The global fold of the FHA domain of RNF8 is an 11-stranded β -sandwich structure with thephosphopeptide-binding surface comprised of 4 loops.

H mRNA

L mRNA

30 minH mRNA
L mRNA

Pull down

H mRNA,Ribosome
30 min30 minL mRNA

L mRNA

Pull downmRNA↓PUREfrexSS↓translation (condition 1,2,3), 37°C
↓pull down (antigen ; TNF-αα)↓recovery mRNA↓quantitative PCR

3000 %

1500 %
570 %100 %0%Condition 1

Condition 2

Condition 3

The recovered mRNA from condition 2 and 3 was increased at 5-fold and 25-foldthan condition 1, respectively. These results indicate that H and L chain mRNAshould be expressed in separated tube (condition 2) or in the same tube
sequentially (condition 3). In translation of condition 1, because L chain mRNAs
formed the L chain mRNA-ribosome-L chain protein complex, active ribosome wasdecreased, and it result in reduction of H chain protein. Also when H chain mRNA
was independently translated, the synthesized H chain protein was easily caused toaggregation. Therefore, it seems that the recovered mRNA from condition 2 waslower than that from condition 3.

Loop2

Loop3

CLOff-rate selection by PUREfrex RD

IC50 of the each purified clone was determined, 4 clones from library 1, and 7
clones from library 2 showed more than 10-fold higher affinity than the original
RNF8 Erk2-binder. The most improved clone was Lib2-1 from Library 2 ( 35fold ). As a whole, the higher-affinity mutants were easily obtained from Library
2 than library 1.

⑫Selection of Fab with higher affinity by ELISA
263
136

185

L chainLibrary: L chain random mutagenesis library
Antigen: protein X (Soluble Domain of membrane protein)
Kd value of original antibody (IgG) :4.8××10-8

Sec MmRNAVLRibosome

CLLinker

CSec M

In almost the cases using cell-free display methods such as ribosome display,affinity maturation of antibody are only applicable to singe-chain Fvs (scFvs), butcannot applied to Fab fragment. On the other hand, it was reported that L chain
shuffling method is highly effective on affinity maturation of Fab fragments with invitro display method such as phage display and yeast display. Therefore, we havebeen developed the optimum representation of Fab fragments on ribosome. InPUREfrexRD of Fab fragments, the randomized light chain (L chain) was presented
on ribosome and the heavy chain (H chain) was translated as a free-protein.

⑨Optimization of PUREfrex for Fab synthesis (PUREfrexSS)

PUREfrex↓+/- GSSG,DsbC↓+/- H chain DNA (+FLAG)+/- L chain DNA↓incubation at 37°C for 4h↓→Ag-coated well↓+anti-FLAG/HRP↓+TMB1.50.5

H chain DNA
L chain DNA
GSSG
DsbC

Relative specific binding activity(Original Fab = 1)

<11-10

1-100

100<

Number of clones

632

18

3

The figure shows the comparison of a specific binding activity of each clone. The
specific binding activity is calculated by binding activity/protein. The activity andprotein concentration were determined by ELISA and Western blotting, respectively,
using cell extracts of E.coli expressed each clone after the three round selection.
The higher activity clone with 10-fold more than the original clone (yellow) indicatesin red bar.

Summary
1, We successfully demonstrated the affinity maturation with PUREfrexRD. Bymeans of the off-rate selection from DNA shuffling and loop grafting libraries, theaffinity of mutated RNF8-binder was improved ~ 35-fold without affecting thespecificity.

3, We were obtained a lot of the high affinity clones by PUREfrexRD optimized Fabdisplay, and three of clone showed >100-fold of specific activity than original.

1.00

Result2, We developed the optimum display method of Fab fragments on ribosome basedon PUREfrexRD. In the optimum condition (Condition 3), functional Fab fragment
was presented by 25-fold more better than in the conventional condition (Condition1).

peptideNative structure of RNF8-peptide complex(side-view & top-view)

30 minVL

Synthesis of Fab fragment using PUREfrex with oxidized glutathione (GSSG) andDsbC protein that catalyzed the formation of correct disulfide bounds.

peptidePull down

2785 %

Loop1

Biotin-Antigen

Feature of RNF8 Erk2-binder
・MW：：18kDa
・structure：：β sandwich
・Loop: 4 loop
・Kd :2.9××10-8・antigen: Erk2 protein

60 minPCR-based DNA shuffling library (ref:Zhao et al.1998. Nat.Biotechnol., 16. 258)

Binding④Affinity maturation of RNF8 Erk2-binder

H mRNA
L mRNA⑪Construction of mutated L chain LibraryLinker

In off-rate selection, mRNA-ribosome-protein complexes bound to antigen-immobilized
streptavidin beads are washed with a large excess of free antigen to prevent the rebindingof mRNA-ribosome-protein complexes to the beads. The higher-affinity binders can beretained on the beads for a longer washing time.

Stop
codon

SecM

Stop
codon
FLAGCHCondition 3:L chain mRNA wastranslated in a tube, after 30 min, Hchain mRNA was added to L chain
expressed tube.

⑦Comparison of IC50 of selected higher affinity clones

H chainElution

VH

Condition 2 :H and L chain mRNA
were separately translated in eachtube, after 30 min, both tubes weremixed.

⑥Protocol of off-rate selection with PUREfrexRD

Fab fragment

Washing with free
antigen for a longer time

RD Linker

Condition 1 :H and L chain mRNA
were translated in a tube at thesame time

Loop4 (NNS)⑧Affinity maturation of Fab fragment with PUREfrexRDStreptavidin
beads

CLError rate was 0.53%: 2.3 AA /a clone

In vitro translation
by PUREfrex

Isolation of mRNA

VL

5'UTR

mRNA Library

PUREfrexRD
Affinity Maturation(off-rate selection)

5'UTR

H chain

PUREfrexRD format to Fab fragment

RT-RCR

L chainc-Myc

In vitro transcription
DNA Library

RNF8 Erk2-binder
Loop2
Loop3

Relative binding activity

Linker

⑩Optimization of synthesis step of H chain and L chain
fragment

Selected clone IC50/ Original IC50

NLibrary protein⑤Construction of mutated RNF8 Erk2-binder Library- + - ++++- - + +++++ + + + - + + + + + - - +Fab fragment was synthesized using PUREfrex supplemented with 3 mM GSSG
and 23 µg/mL DsbC protein. When both of GSSG and DsbC were added in thesystem, functional Fab fragment with good binding activity was synthesized.

In conclusion, PUREfrexRD is expected to be applied to rapid and convenientaffinity maturation for developing high-affinity binders of such as antibodies orscaffold proteins, especially for therapeutic and diagnostic applications.

Contact
URL:www.genefrontier.com
E-mail:kojoh@genefrontier.com
contactus@genefrontier.com