PUREfrex RD: the unique antibody engineering method based on®
Ribosome Display with PUREfrex
Kanehisa Kojoh; Shizue Katoh; Kumiko Tsuihiji; Satoshi Murakami; Mikiko Nakamura;
Takashi Kanamori
GeneFrontier Corporation⑥ Optimization of synthesis step of H chain and L chain①② Our
Our cell-freecell-free protein
proteinsynthesis
synthesis system:
system: PUREfrex
PUREfrex

Library 1:EP
Library 2:CDR

RF mix (-)

Condition 2 :H and L chain mRNA
were separately translated in eachtube, then, both tubes were mixed.

H mRNA

H mRNA
L mRNA

L mRNA

RF mix (+)

RD Pull down

RF mix (-)

H mRNA+Ribosome+RF mix

Condition 3:L chain mRNA wastranslated in a tube, then, H chain
mRNA was added to L chain
expressed tube.

RD Pull down

L mRNA

L mRNA

RF mix (-)

RF mix(Release factor 1, 2, 3, and ribosome recycling factor)

clone A

Clone B

Points・Fully reconstituted.
・No cells, No bias.
・Fast, Easy, Pure.
・High throughput.
・Lower contaminants.

Translation factors

Best suited for
Ribosome display

Shimizu Y et al., Nat Biotechnol (2001) vol 19, p751-755

PURE system is a well-known reconstituted in vitro transcription and translation system
which consists of purified 36 proteins and E. coli ribosomes necessary for transcription,translation and energy recycling. It also contains amino acids, NTPs and E. coli tRNA, so thetarget protein can be synthesized just by addition of the template DNA to the reaction mixture.
PUREfrex is developed as highly purified PURE system , and it is clearly more suitable for
Ribosome Display.

In the case of Clone A, condition 3 showed the highest recovery of mRNA among
3 conditions. In contrast, the best condition for Clone B was Condition 2, while
Condition 3 was the worst. These results indicated the suitable condition forforming Fab displaying RD complex are different in each clone along with thecharacters such as expression level, stability, and solubility of Fab. Therefore, it
is important to optimize expressing conditions of Fab to fully utilize the effective
Fab displaying RD complex in the screening.

CDR1

CDR2

CLCDR1

Preparation of Library・Library with high diversity can not be
prepared because it is limited by theefficiency of transformation.
・Preparation of multiple-clones arevery difficult.

CDR2CDR3VL

Panning・Introducing mutations at every selection
round is very difficult.
CDR1

FR2

CDR2

CLFR3

CDR1

CDR2

CDR3

Total residues

1179Mutations (average)

1.57

1.00

1.07

CDR3

FR4

Hyper point mutation (12~13 a.a /clone)

Fab fragment
L chainLinker

CL

Link by Overlap-PCRRibosome
Sec M

L chain mRNA
VL

Amplify of CDRs by PCR

H chain

CHLinker

CSec M

In most cases of ribosome display, affinity maturation of antibody were applicable only tosinge-chain Fvs (scFvs), and could not be applied to Fab format. On the other hand, it wasreported that L chain shuffling method was highly effective on affinity maturation of Fabfragments with in vitro display such as phage display or yeast display. In 2012, we succeeded
the efficient presentation of Fab fragments on ribosome by using PUREfrex RD (PEGS 2012).
PUREfrex RD of Fab fragments is constituted from mutated L chain on ribosome and a H
chain expressed as a free-protein.

This table shows multiple alignment of amino acid sequences of selected binders
with higher affinity than the original clone. These binders were screened against
peptide antigen or antigen expressing cell. Rightmost column shows number ofmutations of each clone. Each color in table shows species of amino acids. The
average of mutations for all clones was 3.0 mutations/clone. The frequently mutatedregion is indicated in red, and the region which has diversity in mutations isindicated in blue at the two bottom lanes. These positions are candidates to explore
more mutations for creating higher affinity clones, and combination of thosemutations could result better affinity. This comprehensive information can be agood resource for generating more qualified Library for further affinity maturation.

⑧ Protocol of off-rate selection with PUREfrex RD<Material>
Target : membrane protein
Original clone: KD=10-8 M (humanized antibody, epitope identified)Panning antigen: Biotinylated Peptide (epitope peptide)
Free-antigen: Peptide (epitope peptide)mRNA Libraries （diversity:1.2××1012)↓

In vitro translation with PUREfrex (customized, 200 µL )⑤ In vitro affinity maturation in PUREfrex RD

KD of the high affinity clones from 2 nd screening ELISA w ere determined.
Approximately, a lot of clones showed more than 10-fold lower KD than that of theoriginal clone. EP1-EP4 clones indicated low EC50, however, they showed the same
degree of KD of the original clone. From these results, Library 2 (CDR) seemed better
than Library 1 (EP) to obtain higher affinity binders.

⑪ Alignment of amino acid sequence of binders

VL domain④ Affinity maturation of Fab antibody using PUREfrex RDVH

C
DR
5C
DR
3
C
DR
8C
DR
1C
DR
2C
DR
9C
DR
6Ori ginal

CDR3VLFR1H chain mRNA

CDR
7CDR4EP4

Library 1 (EP):PCR-based DNA shuffling L chain Library (Nat.Biotechnol., 16. 258)

Library 2 (CDR):CDR mutated L chain LibraryPanning・Introducing mutation at every selection
round is easy.

Instrument; BIACORE 3000
Sensor Tip; SA Tip
Immobilized antigen(biotinylated-peptide); 126 RU
Flow Rate; 30 μL/min
Injection time; 3 min
Dissociation time;5 minConcentration of sample;100 nM, 50 nM, 25 nM, 12.5 nM⑦ Construction of mutated L chain Library

4.4 a.a mutations / clone (average)Preparation of Library・Library with high diversity can beprepared easily and quickly by only PCR.
・Preparation for multiple-clones aredoable in parallel.

5⑩ Determination of KD by BIACORE③ Advantage of affinity maturation using Ribosome Display
Ribosome DisplayEP

The high affinity clones from 2nd screening ELISA were purified, and EC50 of each
was measured for peptide antigen. EC50 of each clone was shown as relative ratio tooriginal clone. Clones from Library 1 (EP series, orange bars) tended to have higher
affinity. EC50 of the original clone was 7.49 µg/mL, and that of EP3 was the lowest
with 0.12 µg/mL.

DNA or mRNARibosome

1Fab sample
Clone Aaffinity; KD = 10-10 M
Clone Baffinity; KD = 10-9 M

EPRD pull down
PUREfrex (customized)↓translation (Condition 1, 2, 3), volume;20 µL↓binding , washing↓recovery of mRNA↓quantitative RT-PCR

2

Because removal of RF mix from PUREfrex is effective for sticking ribosome onmRNA, the efficiency of constructed L chain displaying RD complex has been
increased in Condition 1 above. However, a part of H chain mRNA also constructed
H chain displaying RD complex in this condition, and it resulted in the reduction ofwhole Fab displaying RD complex. Therefore, we investigated the suitable process
for forming Fab displaying RD complex, which greatly influenced on the screening
efficiency. Accordingly, we found that several conditions, such as the amount ofribosome to add into the system, or synthesizing H and L chain at different timing,
have huge impact on the screening efficiency, so we optimized these conditions.

EPRapid and Easy in vitro screening method basedon ribosome display for scFv, scaffold or FabRD Pull down

3

Fully reconstituted cell-free protein expression
systemH mRNA
L mRNAEP① Our unique solutions for protein based biologics

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

OD450 nmRecently, in vitro screening methods such as phage display or yeast display are commonlyused for engineering antibodies, while they have Pros/Cons. Standing on this point, thedevelopment of other screening methods will contribute to engineer and make it better
therapeutic antibodies. Ribosome display (RD) is expected to have several advantages overexisting in vitro screening methods, so we developed our own RD (PUREfrex RD) using very
effective fully reconstituted cell-free expression system (PUREfrex). We showed that ascaffold, a scFab format and a Fab format without conversion to scFv could be used onPUREfrex RD. That was the first report of a direct Fab fragment selection on RD (PEGS 2012).
Here we report another application of PUREfrex RD that can be utilized for further antibody
engineering such as affinity maturation on the Fab format. We applied PUREfrex RD to thelead therapeutic antibody for in vitro affinity maturation. We investigated the process forforming Fab displaying RD complex, which greatly influenced on the screening efficiency. Forinstance, we found that several conditions, such as the amount of ribosome to add into thesystem, or synthesizing H and L chain at different timing, had huge impact on the screening
efficiency. We prepared two kinds of randomly mutated L chain libraries for whole region or
only for CDRs of the L chain by PCR-based DNA shuffling. After three rounds of off-rate
selection over several days, the Fabs were expressed in E. coli and their binding affinities
were evaluated by ELISA assay. Finally, EC50 was determined for comparison in specificactivity of each Fabs to the original Fab. We have successfully improved the EC 50
approximately 60-fold better than the original Fab.
As such, PUREfrex RD is the unique screening method, and it could be customized forvarious use. We would like to discuss the potential applications of PUREfrex RD here.

⑨ Determination of EC50 by ELISA for peptide antigen

Library 2(CDR) Library 1(EP)Cell Peptide Cell Peptide

Abstract

Summary
1.. It is important to optimize expressing conditions of Fab to fully utilize the Fabdisplaying RD complex as much as possible in the screening, for example, theamount of ribosome to add into the system, and synthesizing H and L chain atdifferent timing.
2. KD of the higher affinity clones were determined, and a lot of clones showed
more than 10-fold lower KD than that of the original clone. Library 2 (CDR) seemed
better than Library 1 (EP) to obtain higher affinity binders.

↓Antigen peptide-immobilized streptavidin beads in TBST 200 µL↓4 ℃, 60 min
Washing five times with 500 µL TBST X 5 times

In vitro transcription
mRNA Library

DNA Library↓

In vitro translation
by PUREfrex

RT-RCR

Addition of free-antigen peptide (final 1 µM)↓4 ℃, incubation for long time (1st round:1 day, 2nd round:3 days, 3rdround:7 days)
Washing five times with 500 µL TBST X 10 times↓Isolation of mRNA

PUREfrex RD
Affinity Maturation(off-rate selection)

Elution of mRNA from RD complexes with 100 µL EDTA (50 mM)

3. Alignment of amino acid sequences of selected binders with higher affinity thanthe original clone indicated the region of frequently mutated or mutated in common,
which suggests important region to improve affinity, and exploration of thosemutations will lead to better affinity.

GFC pipline
We are engineering these antibodies with PUREfrex RD.

↓Binding
Streptavidin
beads

Biotin-Antigen

RT-PCR→→next round selection↓

Sub-cloning↓

Expression in E.coli and 1st screening ELISA with cell extract (380 clones),and pick up the higher affinity binders with 3-fold more than the original.
(ELISA antigen; peptide or antigen expressing cell)↓Screening unique clones by sequencing.
ElutionWashing with freeantigen for a longer time↓

Expression and Purification of unique clones in small scale (E.coli. 5 mLculture).
↓In off-rate selection, RD complexes bound to antigen-immobilized streptavidin beads were
washed with large excess of free antigen to prevent the rebinding of RD complexes to thebeads. The higher-affinity binders can be retained on the beads for a longer washing time.

2nd Screening ELISA of higher affinity clones than the original clone in 1stscreening ELISA.
↓Purification of higher affinity clones in large scale (E.coli 200 mL culture), andmeasurement of EC50 and KD values.

Contact
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E-mail:contactus@genefrontier.com