In vitro selection and affinity maturation of CTLA-4 binding cyclic peptide withPUREfrexRD, and conversion of the peptide to small molecules.
PUREfrexRDにによるCTLA-4結結合環状ペプチドの試験管内選択と親和性向上および環状ペプチドの低分子化合物への変換
對比地 久美子1、加藤 静恵1、中村 美紀子1、村上 智史1、高島 一2、河西政次2、毛利 睦2、吉森篤史3、〇古城 周久1
1ジーンフロンティア（株）、2（株）
PRISM BioLab、、3理論創薬研究所
Abstract④In vitro selection by PUREfrexRD⑨Measurement of inhibition (IC50) by inhibitory ELISA[Background]
LuminescenceIn the drug development, small molecule drugs have a lot of advantage over
peptides in terms of oral administration and intracellular migration. However,since diversity of small molecule library and selection methods are limited, it isdifficult to get the promising drugs that has the inhibitory activity for proteinprotein interactions. On the other hands, we have reported that the functional
cyclic peptides can be easily selected / screened by the ribosome display
method (PUREfrexRD) using PUREfrex. Here, we report selection and affinity
maturation of functional cyclic peptide that inhibited the binding between CD80and CTLA-4, and conversion of the cyclic peptide to small molecules.

Biotinylated CD80
Streptavidin-HRP

HRP

MBP-fusion
peptideSACTLA-4

CD80

Cyclic peptide

B

CTLA‑4CTLA‑4CTLA‑4CTLA‑4

PeptideMBP-fusion

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

Activity %A lot of cyclic peptides that specifically binds to CTLA-4 was obtained using
PUREfrexRD (3 rounds selection) from cyclic peptide library consisting of arandom sequence of 12 residues. Next, a cyclic peptide having inhibitory activity
was selected and applied for affinity maturation (off-rate selection) by
PUREfrexRD. As a result, a high affinity cyclic peptide having an improved
affinity of about 630 times was obtained. Furthermore, the comprehensive
information related to the correlation between amino acid sequence and binding
activity was obtained from a series of experiments such as identification of
essential amino acids for binding by alanine scanning, exhaustive expression ofrecombinant mutants and ELISA. Based on these data, the 3D-structure of thecyclic peptide was predicted in silico, and about 350 small molecules similar tothe simulated cyclic peptide structure were picked out from PRISM BioLab s
small molecule library, and binding inhibition activity were estimated by ELISA.
As a result, a several small molecules having inhibitory activity could beobtained.

Activity %[Method and Results]⑩Designing small molecules based on cyclic peptides

Ribosome
Translation factors

N

O‑ ‑H ‑NHNX
ORi+1

Some of enrichment were observed from the sequencing (94clones). Each color represents specific amino acid.

H---NOO--Ri+2

β -turn peptide-mimetic

β -turn

J.Med.Chem., 45, 1396 (2002)

Cys

32

154

6789101211(Expression)
E.coli host : BL21(DE3)
Culture condition : 30℃℃

EC50 µM(Purification)
His tag purification

XY

3

4

Scaffold
322GZ

Scaffold

Scaffold

34

Small molecules mimicking β -turn derived from cyclic peptides

Three amino acids are introduced into the R position (redsquare) of the β -turn peptide-mimetic scaffold in order from theN-terminus of the cyclic peptide.

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.

⑪Inhibitor Screening assay and Measurement of IC50Conc.(µg/mL)

Inhibitor screening assay

Measurement of IC50 for Pm2

Activity %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 higher EC50 than the original (3-02). In
particular, No.19, 10, 24 (red square) showed about 50 times
higher affinities than the original.

Conc.[µM]⑦Identification of amino acids required for binding byalanine scanning

CyclicpeptideCys(Off-rate slection)
Library:Error prone mutants
Wash time:1st-2H, 2nd-19H, 3rd-67H

Best suited for Ribosome
display and mRNA display

Ribosome Display complex with cyclic peptide

Ri+3N

Ri+1

Ri+2

O

1②Simple selection with PUREfrexRD for cyclic peptide

ORi+3

Ri

⑥Measurement of affinity (EC50) as MBP-fusion peptide
after affinity maturation (off-rate selection) by PUREfrexRD
Points・Fully reconstituted.
・No cells, No bias.
・Fast, Easy, Pure.
・High throughput.
・Lower contaminants.
Very low level of RNase,patented as JP4931135

ORiHN

DNA or mRNA

IC50 = 11.6 µM

Conc.[µM]

No.19 binder showed the inhibitory activity against interaction
between CD80 and CTLA-4 in both MBP-fusion and fully
synthetic peptide format. The inhibitory activity of the synthetic
peptide was lower than MBP-fusion.

It indicates that PUREfrexRD is an effective way for obtaining functional cyclic
peptides and their structure-activity relationship information, and conversion
from cyclic peptides to small molecules.

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

IC50 = 1.16 µM

Conc.[µM](Construct)
MBP-fusion peptide

NIC50 = 295 µM

Several small molecules with inhibition activity were obtained.
IC50 of Pm2 with the highest activity showed about 1/20 of thesynthetic peptide.
⑫Comparison of affinity among Pm2 analogs

SecMCmRNA
FLAG
Cys(NNS)12c-Myc

Cys

SecM

LinkerCyclic peptide
CB7(CD80/86））

45

Scaffold

6789101112Cys

Essential AA for binding

Pm2
IC50=295 µM

Activated T cell

T Cell⑤④③②⑦①⑧⑨CTLA-4 Cell line:
1X 105 cells/reaction, 100 µL
Synthetic peptide:
N-term Biotinylated peptide⑧⑦⑥⑥T Cell

Inactivated T cell

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-cell
activation.

Pm2+X
Weaker than Pm2

10Ala
Pm2-AlaNo activity

Summary

CTLA-4TCR

11⑧Cell based binding assay with the synthetic peptide(FACS)

CD28MHC

Scaffold

Amino acids of position 2, 3, 5, 6, 9, 11, 12 (red circle) werenecessary for binding to CTLA-4.

⑨TCR

1012Pm2 was designed from 3 amino acids including essential 2amino acids for binding at C-terminal of peptide. Two analogs
with adduct or substitution of 1 amino acid showed thedecrease in IC50.

Negative signal

APC

12X

Scaffold
1110C

CD28
Positive signal

MHC

312③Immune checkpoint via CTLA-4

APC

2Cys

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) are removed from PUREfrex,
and oxidized glutathione (GSSG) and disulfide isomerase from
E.coli (DsbC) in optimized concentration to form disulfide bond are
added into PUREfrex. As a result, RD complex become highly
stable, and cyclized peptide is displayed on ribosome with highefficiency.

B7(CD80/86））

1

2nd antibody:
Streptavidin-iFluor 647

⑤
①

②

③

1, Functional cyclic peptides against CTLA-4 were selectedeasily and rapidly by PUREfrexRD.
2, In affinity maturation of CTLA-4 binder, we succeeded toselect lead cyclic peptide with good EC50/IC50.
3, Essential amino acids for binding were indentified by alanine
scanning.

④log (µM)

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

4, Based on structure-activity relationship information, peptidemimetic small molecules were designed, and successfully
selected with good inhibitory activity.
E-mail: contactus01@genefrontier.com