Selection and affinity maturation of cyclic peptide against
CTLA-4 with PUREfrexRD〇Kumiko Tsuihiji; Shizue Katoh; Mikiko Nakamura; Satoshi Murakami; Kanehisa Kojoh

GeneFrontier Corporation, Chiba, Japan (https://www.genefrontier.com/en/)
Abstract④In vitro selection by PUREfrexRD⑨Evaluation of amino acid sequence and ELISA in MBPfusion format after affinity maturation by PUREfrexRD[Background]
Ribosome display (RD) is the simplest method among existing in vitro selection
methods. We developed RD by optimizing the composition of PUREfrex, for instance,removing release factors from the system, adjusting the concentration of metal ions andribosome, etc. We named it as PUREfrexRD. We have already reported that a lot of cyclic
peptides and antibodies were successfully selected simply and effectively by
PUREfrexRD. This time, we carried out in vitro selection using PUREfrexRD to selectcyclic peptides binding to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) with
inhibitory activity to the interaction between CTLA-4 and CD80, which can be one of thenew modality as an immune check point inhibitor.
[Methods and Results]
Cyclic peptide library having 12mer random sequences (Cys - X12 - Cys) was screened
against a biotinylated CTLA-4-Fc fusion protein. After 3 rounds of selection, enriched
mRNAs were subcloned, and a lot of enriched clones with variety were obtained fromsequence analysis. The enriched clones were examined for their binding activity toCTLA-4 by RD pull down assay, and 3-02 clone showed specific binding activity to CTLA4. To evaluate the affinity to CTLA-4 and the inhibitory activity to the interaction between
CTLA-4 and CD80, 3-02 clone was expressed as MBP-fusion protein in E.coli and purified.
Purified 3-02 clone showed very low affinity and very low inhibitory activity, so affinity
maturation of 3-02 clone by off-rate selection using PUREfrexRD was performed toimprove its affinity and inhibitory activity. Mutated library against 3-02 clone wasprepared by error prone PCR. After 3 rounds of off-rate selection over several hours (1stround; 2 hours, 2nd round; 19 hours, 3rd round; 67 hours), mutated clones wereexpressed in E.coli, and their binding activities were evaluated by ELISA. The purified
clones showed the higher activity in ELISA, and each EC 50 was determined forcomparison in specific activity of each clone to the original 3-02 clone. Finally, we haveobtained several high affinity clones by approximately 50-fold more than the original 3-02
clone.

Clones from affinity
maturation library

Streptavidin

Cyclic peptide library having 12mer random sequences (Cys X12 - Cys) was screened against biotinylated CTLA-4-Fc
protein. Recovered mRNA increased along with the progress ofselection round.
⑤Sequencing after 3rd round selection

It seems that mutating of position 6 residue was very
important to have the higher binding activity, and position
2,3,5,9,11,12 residues would be essential for specific binding toCTLA-4.
Some of enrichment were observed from the sequencing (94clones). Each color represents specific amino acid.

⑥Confirmation of binding activity by RD pull down assay

DNA or mRNA

Ribosome
Translation factors

MBP-fusion peptide

CTLA-4[Conclusion]
PUREfrexRD will be applied to simple and effective in vitro selection of cyclic peptideand will contribute to the development of cyclic peptide based drugs.

①Best reconstituted cell-free protein synthesis system for invitro selection having the lowest level of RNase contamination:PUREfrex

15th Annual PEGS BOSTON (April 8‑12, 2019)⑩Measurement of affinity (EC50) as MBP-fusion peptide
E.coli expression host : BL21(DE3)
Culture condition: 30℃℃
Induction: final 0.1 mM IPTG
Purification : His tag purification

Points・Fully reconstituted.
・No cells, No bias.
・Fast, Easy, Pure.
・High throughput.
・Lower contaminants.
Very low level of RNase,patented as JP4931135

CTLA‑4

Human IgG

FcFc

SA

SA:Streptavidin

Conc.(µg/mL)

Best suited for Ribosome
display and mRNA display

The high affinity clones (27 clones) from ELISA were purified,and EC50 of the each were measured. As a result, all clones
except No.27 showed the higher EC50 than original. In particular,
No.19, 10, 24 (red square) showed about 50 times higher
affinities than the original.

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

PUREfrex is the reconstituted in vitro transcription and translation
system w hich consists of purified 36 proteins and E. coliribosomes 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 addition of thetemplate DNA to the reaction mixture.

⑪Cell based binding assay with the synthetic peptide (FACS)⑤②Simple selection with PUREfrexRD for cyclic peptide③⑨⑧<Instrument for measurement>
・BLItz system (FORTE Bio)
<Condition>
・Sample・Antigen・Biosensor

SecM
c-Myc

⑤SecMLinker

3-02

3.18X103

kd (1/s)

KD (M)

1.23X10-2

3.88X10-6

3-02 binder was expressed as MBP-fusion protein in E.coli andpurified by affinity resin. Binding kinetics to CTLA-4-Fc wasdetermined using BLItz system. Biotinylated CTLA-4-Fc wasimmobilized on a SA Biosensor according to the standard
method.

CB7(CD80/86））

PUREfrexRD

Washing time
1st round : 2h
2nd round : 19h
3rd round : 67h
Mutation :Every round

CCTLA-4

CD28

Negative signal

Activated T cell

APC

CD28

MHCTCR

When CTLA-4 bind to B7, T-cells are led to anergy. Anergic T-cells
have limited effector function. Anti-CTLA-4 neutralizing cyclic
peptides inhibit CTLA-4 binding to B7 and promote T-cellactivation.

SACTLA-4CD80

Cyclic peptide

B

CTLA‑4CTLA‑4CTLA‑4CTLA‑4Peptide

MBP-fusion

Original

No.19

No.19

Activity %Original

Streptavidin-HRP

Conc.[µM]

IC50 = 11.63 µM

Conc.[µM]

IC50 = 116.4 µM

No.19 binder showed the inhibitory activity against interaction
between CD80 and CTLA-4 in both MBP-fusion and fully
synthetic peptide format, but original clone (3-02) showed very
low activity. However, the inhibitory activity of the synthetic
peptide was lower than MBP-fusion. It seemed that the activity ofsynthetic peptide was influenced by the characters of peptide
such as solubility, isoelectric point and conformationalfluctuation.

Summary

T CellInactivated T cell

MBP-fusion
peptideActivity %⑧Affinity maturation by PUREfrexRD (Off-rate selection)

In vitro translation
by PUREfrex

Cyclic peptide

T CellBiotinylated CD80
HRP

③Immune checkpoint via CTLA-4TCR

EC50 = 0.59 µM

No.19 binder as synthetic peptide (N-term biotinylated) boundto CTLA-4 on the cells.

In vitrotranscription

MHC④③②Luminescence

Cys

Positive signal

2nd antibody:
Streptavidin-iFluor 647: Cyclic peptide- MBP-fusion protien: Biotinylated CTLA-4-Fc protein: SAka (1/Ms)

When the arrest sequence of SecM at 3''terminus is translated inRUREfrex, a ribosome can be fixed strongly on mRNA. Also,
release factors (RF1, RF2, RF3, RRF) were removed from PUREfrex,
and oxidized glutathione (GSSG) and disulfide isomerase fromE.coli (DsbC) in optimized concentration to form disulfide bondwere added into PUREfrex. As a result, RD complex become highly
stable, and cyclized peptide is displayed on ribosome with highefficiency.

APC⑥⑫Measurement of inhibition (IC50)

CmRNA

B7(CD80/86））⑦log (µM)

N

Cys⑨⑦Affinity measurement of 3-02 binder by BLItz system

Cyclicpeptide

FLAG

⑧⑦

①

⑥①(NNS)12②

Synthetic peptide:
N-term Biotinylated peptide

Only 3-02 binder which was the most enriched clone bound tothe CTLA-4 region in CTLA-4-Fc protein specifically (redsquare), and another clones bound to the Fc region.

Ribosome Display complex with cyclic peptide④CTLA-4 Cell line:
1X 105 cells/reaction, 100 µL

In off-rate selection, RD complexes bound to CTLA-4immobilized streptavidin beads were washed with large excess
of free CTLA-4 to prevent the rebinding of RD complexes to thebeads. The higher-affinity binders can be retained on the beads
in a longer washing time. Library was prepared by error prone
PCR using 3-02 clone as template DNA.

1, Functional cyclic peptides against CTLA-4 were selectedeasily and rapidly by PUREfrexRD.
2, In affinity maturation of CTLA-4 binders, we succeeded toselect lead cyclic peptide with good EC50/IC50.
E-mail: contactus01@genefrontier.com